GlpR Regulates the Glyoxylate Pathway and Virulence Factor Production by Pseudomonas aeruginosa by Jessica Anne Scoffield A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 7, 2012 Keywords: Pseudomonas aeruginosa, cystic fibrosis, glyoxylate pathway, glycerol metabolism, gene regulation Approved by Laura Silo-Suh, Chair, Assistant Professor of Biological Sciences Sang-Jin Suh, Co-Chair, Associate Professor of Biological Sciences James Barbaree, Professor of Biological Sciences Holly Ellis, Associate Professor of Chemistry and Biochemistry Stuart Price, Associate Professor of Pathobiology ii Abstract Pseudomonas aeruginosa infections are the leading cause of morbidity and mortality for cystic fibrosis (CF) patients. P. aeruginosa establishes life-long infection in the CF lung by utilizing various adaptation strategies to cause a chronic infection including alterations in central metabolic activities. The glyoxylate pathway is utilized by bacteria to grow on acetate or fatty acids as a sole carbon source to replenish intermediates of the tricarboxylic acid cycle and it appears to play a role in P. aeruginosa persistence in the CF lung. Son et. al. (2007) demonstrated that the genes encoding for the glyoxylate pathway enzymes, aceA and glcB which encode for isocitrate lyase (ICL) and malate synthase respectively, are upregulated in P. aeruginosa growing in the CF lung. In addition, we determined that this pathway becomes permanently upregulated in some CF adapted isolates of P. aeruginosa (Hagins et. al. 2010, 2011; Lindsey et. al. 2008). The occurrence of these isolates suggests deregulation of the glyoxylate pathway may benefit P. aeruginosa growing within the CF lung. However, the mechanism(s) responsible for alterations in the pathway have yet to be elucidated. GlpR is a transcriptional repressor that regulates the genes responsible for glycerol metabolism in P. aeruginosa. I demonstrate in this body of work that GlpR also regulates the glyoxylate pathway. To date, regulators of the glyoxylate pathway in P. aeruginosa have not been identified with the exception of RpoN, which plays an indirect role in the regulation of this pathway. GlpR?s role in the regulation of the glyoxylate pathway provides a novel perspective into the interplay between fatty acid and glycerol metabolism in P. aeruginosa. Finally, I show that glycerol metabolism is iii altered in a CF adapted isolate of P. aeruginosa compared to an acute isolate and that production of alginate is influenced by growth on glycerol. Alginate is an important virulence determinant produced by P. aeruginosa during infection of the CF lung. These results suggest the carbon sources present in the CF infection environment impact virulence factor production by P. aeruginosa. iv Acknowledgements I would like to give a heartfelt thanks to Dr. Laura Silo-Suh for providing me with the opportunity to be a graduate student in her laboratory. Her guidance, support, advice and patience have been invaluable and greatly appreciated. I would also like to thank my co-advisor Dr. Sang-Jin Suh for his support, ideas and useful advice. Thanks to my advisory committee members and Dr. John Murphy for their support, time, and creative ideas. I want to give special thanks to Dr. Narendra Singh for all of his support and helpful advice. I also want to thank Jessica Hagins, Paul Dawson, Tamishia Lindsey, Yi Liu, Suihan Wu, and Zhou Tong for their help in the lab as well as their support. I am also thankful to all of the members of the Silo-Suh and Suh labs for their friendship and support. I want to thank my parents, grandfather, my sister Lindsay, best friend Dana, and godparents for supporting me throughout my graduate career. v Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements........................................................................................................................ iv List of Tables ...........................................................................................................................................viii List of Figures.................................................................................................................................ix Chapter 1 Literature Review......................................................................................................................1 1.01. Introduction..............................................................................................................1 1.02. Pseudomonas aeruginosa.......................................................................................2 1.03. Cystic fibrosis????? ???... ?.................. .................................................4 1.04. Microbial colonization of the CF lung.................................................................4 1.05. Pseudomonas and cystic fibrosis ????.. .......................................................5 1.06. Adaptations of P. aeruginosa to the cystic fibrosis lung?................... ............6 1.07. The glyoxylate pathway?????.???? ??....... .....................................8 1.08. The role of the glyoxylate pathway in pathogenesis..........................................9 1.09. Regulation of aceA and glcB in P. aeruginosa..?????....... ....................10 1.10. Glycerol metabolism and regulation.?? ????...................... ...................11 1.11. Nutrient acquisition via the glyoxylate pathway and glp regulon..................12 1.12. Summary ...............................................................................................................14 1.13. References..............................................................................................................16 vi Chapter 2 The Role of GlpR in the Regulation of the Glyoxylate Pathway ??????.........22 2.01. Abstract ..................................................................................................................22 2.02. Introduction............................................................................................................23 2.03. Materials and Methods.........................................................................................25 2.04. Results....................................................................................................................31 2.05. Discussion .............................................................................................................43 2.06. References..............................................................................................................46 Chapter 3 The Chronic Cystic Fibrosis Isolate, FRD1, is Enhanced for Growth on Glycerol ???????????????????...??????? .............49 3.01. Abstract ..................................................................................................................49 3.02. Introduction............................................................................................................50 3.03. Materials and Methods.........................................................................................52 3.04. Results....................................................................................................................53 3.05. Discussion..............................................................................................................64 3.06. References..............................................................................................................67 Chapter 4 GlpR is Required for Virulence in a Chronic Isolate of P. aeruginosa ........................69 4.01. Abstract ..................................................................................................................69 4.02. Introduction............................................................................................................70 4.03. Materials and Methods.........................................................................................71 4.04. Results....................................................................................................................73 4.05. Discussion..............................................................................................................81 4.06. References..............................................................................................................84 Chapter 5 Published and Unpublished Miscellaneous Results ???? .?. ???..???.?.? 88 5.01. Abstract ..................................................................................................................88 vii 5.02. Introduction...............................................................................................................88 5.03. Materials and Methods............................................................................................89 5.04. Results........................................................................................................................91 5.05. Discussion.............................................................................................................. 103 5.06. References...............................................................................................................105 Chapter 6 Conclusions and Future Directions ...................................................................................108 6.01. Conclusions............................................................................................................108 6.02. Future Directions ...................................................................................................110 6.03. References...............................................................................................................112 viii List of Tables Table 2.1. Bacterial strains and plasmids?? ???? ??????????...???? ....30 Table 3.1. Bacterial strains and plasmids.......................................................................................53 Table 4.1. Bacterial strains and plasmids.......................................................................................73 Table 4.2. GlpR is required for optimal infection of CF isolates on alfalfa seedlings and alginate is increased in the FRD1 glpR mutant??? ???. ?........ ......................78 Table 5.1. Bacterial strains and plasmids.......................................................................................90 ix List of Figures Figure 1.1. The Glyoxylate Pathway ?? ?..???? ????????????????.. 9 Figure 1.2. Organization of the glp regulon in P. aeruginosa???????...? ...???? 12 Figure 1.3. Degradation of phophatidylethanolamine to yield glycerol and fatty acids via the action of P. aeruginosa phospholipases and lipases??.????.??.. 14 Figure 2.1. Effect of glpR mutation on ICL activity in PAO1?? ?.??????. ?? .?.. 32 Figure 2.2. Effect of glpR mutation on ICL activity in PAO1?? ?.. ???????? .?.. 33 Figure 2.3a. Effect of glpR mutation on aceA expression in PAO1? .....? ????...?? ?.. 34 Figure 2.3b. Effect of glpR mutation on glcB expression in PAO1?.?? ..? ..?. ??? ?... ..34 Figure 2.4. Expression of glpR in PAO1 and FRD1??????.??? ?... ??. ..??. ......36 Figure 2.5. Expression of glpD in PAO1 and FRD1.....................................................................36 Figure 2.6. Growth of PAO1 on glycerol-3-phosphate................................................................38 Figure 2.7. aceA and glcB expression in PAO1 in LB vs. glycerol?? ? ????? .?. ......38 Figure 2.8. Comparison of putative glpR binding sites ???? ..???? ??..?. ?.??.. 40 Figure 2.9a. Overexpression of the 28 kDa protein GlpR in E. coli?? .???? .............?? 41 Figure 2.9b. Purification of the 28kDa protein GlpR in E. coli???? ? ????..??.?.. 41 Figure 2.10. Gel shift assays using the putative GlpR binding sites ????... ....????..... 42 Figure 3.1. FRD1 displays a growth advantage on glycerol........................................................54 Figure 3.2a. FRD1 grown on 0.1% Casamino Acids.......................................................................55 Figure 3.2b. PAO1 grown on 0.1% Casamino Acids......................................................................55 Figure 3.3. Survey of glycerol utilization in P. aeruginosa isolates...........................................57 x Figure 3.4. Survey of glycerol utilization in sequential P. aeruginosa CF isolates.............................................................................................................................58 Figure 3.5. algD and algT are required by FRD1 for optimal growth on glycerol???? ????????????????????????? ...?.. 60 Figure 3.6. PAO1 mucA mutant and wild-type PAO1 on glycerol? ? ???.. ??..?......... 60 Figure 3.7. Glycerol increases alginate production in FRD1?????...?... ??????. 62 Figure 3.8. Expression of algD in FRD1???????...?? ...?????..??.??...? 62 Figure 3.9. Glycerol increases alginate production in PAO1????...??? ?. ..????. 63 Figure 3.10. Expression of algD in PAO1??????..????..??? ...????.........? 63 Figure 3.11. Pathway for alginate biosynthesis from glycerol?? ??????.????.? 64 Figure 4.1. The effect of glpR on pyoverdine production in FRD1 and PAO1?????... ..75 Figure 4.2. The effect of glpR on pyocyanin production in FRD1 and PAO1????? ?. .75 Figure 4.3. The effect of glpR on rhamnolipid production in FRD1 and PAO1.?..??... .....76 Figure 4.4. The effect of glpR on hydrogen cyanide production in FRD1 and PAO1?? .....76 Figure 4.5. Rhamnolipid biosynthesis from glycerol????????..?????......?? 77 Figure 4.6. The effect of glpR on persister cell formation in FRD1 and PAO1?? ?... ??. 80 Figure 4.7. Summary of virulence phenotypes regulated by GlpR and glycerol catabolism??????? ?? ??????? ...???????. 81 Figure 5.1. Expression of an rpoN::lacZ transcriptional fusion??? .??????..? ........92 Figure 5.2. glcB::lacZ expression in FRD1 and PAO1?????????? ???. ?...?. 94 Figure 5.3. Effect of various carbon sources on glcB::lacZ expression in FRD1 and PAO1??????????? ?. ??????????????? 96 Figure 5.4. Malate synthase is required for hydrogen cyanide production by Pseudomonas aeruginosa??????? ?. ..????????????...?? 98 Figure 5.5. Malate synthase is not required for alginate production by FRD1?? ?? ?... ...99 Figure 5.6. Effect of various mutations on aceA::lacZ expression in P. aeruginosa? ..........101 xi Figure 5.7. Effect of a mutation in the lrp gene on ICL activity in PAO1????? ??. 102 1 Chapter 1 Literature Review Introduction Some bacterial pathogens are able to establish life-long chronic infections subsequent to an initial acute infection. The ability of many bacterial pathogens to survive during chronic infections is contingent upon their ability to adapt to the environment within the host. Bacteria capable of establishing persistent infections have developed unique strategies that allow them to overcome environmental stress caused by antibiotics, the immune response, limited nutrients, or oxygen availability. The mechanisms used by bacteria to cause chronic infections differ from those used during acute infections, however little is known about the mechanisms used by bacteria to persist during long-term infections. In order to treat chronic infections we need a better understanding of how persistent infections are established and maintained by these organisms. Pseudomonas aeruginosa is a bacterium that is notorious for establishing decade long infections in cystic fibrosis (CF) patients and it is also an important component of some persistent wound infections. Therefore, P. aeruginosa can establish persistent infections at multiple infection sites and it appears to utilize multiple strategies for this process (Kukavica- Ibrulj et. al. 2008; Mathee et. al. 2008). Hence, P. aeruginosa is a good model system for studying chronic infection mechanisms. In addition, it is easy to grow and to genetically 2 manipulate in the laboratory. Our overall goal is to use P. aeruginosa as a model system to identify global bacterial persistent strategies. One strategy that may be used by P. aeruginosa to persist during chronic infection is upregulation of the glyoxylate pathway. This pathway is required by many microorganisms for pathogenesis and is also required for some bacteria to cause persistent infections. It has been implicated in the ability of P. aeruginosa to cause persistent infections, and therefore, is the focus of my dissertation research. My initial studies centered on understanding regulation of the glyoxylate pathway by the alternative sigma factor RpoN (Hagins 2009, 2011). However, the bulk of my dissertation is characterization of GlpR as an additional regulator of the glyoxylate pathway. GlpR is a transcriptional regulator that represses the genes responsible for glycerol metabolism in P. aeruginosa. During the course of my studies, I discovered that some CF isolates, including the paradigm of chronic CF isolate FRD1, are able to utilize glycerol more efficiently as a carbon source than non-CF isolates. This suggests that this phenotype evolved in the CF lung and may provide some benefit to P. aeruginosa. Glycerol in the CF lung is likely derived from hydrolysis of host cell membranes by P. aeruginosa produced phopholipases that liberates fatty acids and glycerol from phopholipids. The concomitant availability of both fatty acids and glycerol in the CF lung suggests that P. aeruginosa may regulate metabolism of both carbon sources in a coordinate manner. The specific goals of this study were to determine whether GlpR regulates the glyoxylate pathway in P. aeruginosa and characterize glycerol utilization by both CF and non-CF isolates of P. aeruginosa. Pseudomonas aeruginosa Pseudomonas aeruginosa is a facultative anaerobic, gram negative, rod-shaped bacterium that is ubiquitously distributed in the environment (Lee et. al. 2006; Stover et. al. 2000). The 3 genome of P. aeruginosa contains a large number of genes devoted to regulation, catabolism, transport, and efflux of organic compounds which enable this bacterium to quickly respond and adapt to a wide range of environmental conditions (Lee et. al. 2006; Stover et. al. 2000). P. aeruginosa is commonly found in soil and water but has a broad environmental range. Its diverse repertoire of genes involved in metabolism gives it the flexibility to colonize and infect different hosts, including plants, insects, nematodes, fungi, and animals (Kukavica-Ibruli et. al. 2008). In humans, P. aeruginosa is an opportunistic pathogen and is the leading gram-negative bacterial cause of nosocomial infections (Kukavica-Ibruli et. al. 2008). P. aeruginosa causes many acute infections, including nosocomial pneumonia, respiratory infections, urinary tract infections from catheter use, infections in burn patients, and is the dominant cause of microbial keratitis (Twining et. al. 1993; Willcox et. al. 2008). P. aeruginosa is also responsible for causing devastating, chronic respiratory infections in cystic fibrosis (CF) patients. One reason P. aeruginosa has become an incredibly successful pathogen is because it is equipped with an arsenal of both cell-associated and extracellular virulence factors (Lee et. al. 2006). Cell-associated virulence factors include fimbriae, flagellae, lipopolysaccharide, a type III secretion system, and alginate (Choi et. al. 2002). Exoproducts such as exotoxin A, exoenzyme S, elastase, and alkaline protease production have been shown to be important in establishing P. aeruginosa-associated infections (Delden and Iglewski, 1998). These exoproducts degrade complement components and interfere with other innate defenses like interleukin 1 and 2, natural killer cells, polymorphonuclear leukocyte chemotaxis, and tumor necrosis factor (Twining et. al. 1993). Exoenzyme S, a cytotoxin secreted by P. aeruginosa?s type III secretion system, contributes to tissue damage, and exoenzyme A inhibits protein synthesis by inhibiting elongation factor 2 (Sadikot et. al. 2004; Twining et. al. 1993). 4 P. aeruginosa also produces several diffusible pigments: pyocyanin, pyoverdine, and pyochelin (Govan et. al. 1996; Sokol and Woods, 1998). During infection iron can be a limiting factor for bacteria (Sadikot et. al. 2004). Pyochelin and pyoverdine are siderophores that acquire iron from the host and transport it to the bacteria (Sadikot et. al. 2004). Pyocyanin is a blue- green, redox-active phenazine compound produced by P. aeruginosa (Fothergill, 2007; Lau, 2004). This pigment generates reactive oxygen species and induces apoptosis of neutrophils in host cells (Fothergill, 2007; Lau, 2004). In mice, pyocyanin mediates damage and necrosis in lung epithelial tissue and is required for airway infection (Lau, 2004). All of the virulence factors produced by P. aeruginosa contribute to establishing infection at one or more sites (Kukavica-Ibrulj et. al. 2008; Lindsey et. al. 2008; and Mathee et. al. 2008). Some of these defense strategies allow P. aeruginosa to cause life threatening infections in cystic fibrosis patients and allow this organism to evade clearance by the host immune system, resist treatment with antibiotics, and persist in the lung during the patient?s lifetime. Cystic fibrosis Cystic fibrosis (CF) is an inherited, multi-system, autosomal recessive disorder. Most cases of CF are caused by a deletion of phenylalanine at position 508 (?F508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein (Welsh et. al. 1996 and 2001). Normally, CFTR functions in the transport of small molecules in epithelial cells (Welsh et. al. 1996 and 2001). Defective transport of small molecules caused by altered CFTRs produces multiple symptoms in CF patients including salty sweat, pancreatic insufficiency, intestinal obstruction, male infertility and severe pulmonary disease (Welsh et. al. 1996 and 2001). 5 Consequently CF life expectancy is limited due to a progressive loss of lung function caused by chronic microbial colonization. Microbial colonization of the CF lung During early childhood most CF patients are colonized by a mixture of microbes, including various bacterial pathogens (Lyczak 2002). Defects in the CFTR protein inhibit proper mucociliary clearance of microbes from lung, and as a result, these organisms are able to colonize and establish infections within the lung (Lyczak 2002). Some of the microbes persist in the CF lung throughout the patient?s entire life (Kukavica et. al. 2008; Mahenthiralingam et. al. 1996; Smith et. al. 2006). The most common microbes detected during early infection of the CF lung include Burkholderia cepacia, Staphylococcus aureus, Haemophilus influenza, and Streptococcus pneumonia. Co-infections from two or more bacterial species are extremely common in the CF lung (Gilligan 1991; Harrison 2007). The CF lung represents a diverse community of bacterial species that coexist and interact both synergistically and antagonistically (Gilligan, 1991; Harrison, 2007). Aggressive antibiotic therapy has been effective in clearing or delaying some of these infections. However, most CF patients are eventually colonized with recalcitrant variants of P. aeruginosa. P. aeruginosa and cystic fibrosis P. aeruginosa is the most common and clinically important pathogen in patients with CF. Chronic lung infections caused by P. aeruginosa are the leading cause of lung deterioration and mortality for cystic fibrosis (CF) patients (Chamber et. al. 2005). P. aeruginosa is acquired from the environment and is maintained permanently within the CF lung. During colonization, P. aeruginosa converts to the mucoid phenotype, which is caused by the overproduction of the 6 exopolysaccahride alginate (Smith et. al. 2006). The mucoid phenotype contributes to the formation of biofilms by P. aeruginosa, prevents the penetration of antibiotics, inhibits phagocytosis and inhibits the activation of complement (Govan and Deretic, 1996). As a result, P. aeruginosa is able to resist clearing by therapeutic measures and the host immune system (Govan and Deretic, 1996; Kukavica et. al. 2008; Mahenthiralingam et. al. 1996; Smith et. al. 2006). Eventually, copious amount of alginate accumulate in CF sputum, which contributes to airway blockage, chronic lung infections and a decline of pulmonary functions (Kukavica et. al. 2008; Mahenthiralingam et. al. 1996; Smith et. al. 2006). Unfortunately, an effective P. aeruginosa vaccine is currently unavailable. Adaptations of P. aeruginosa to the CF lung Very few studies have addressed the microevolution of P. aeruginosa within the CF lung and the mechanisms it uses to persist in this environment. The study of P. aeruginosa is complicated by genotypic alterations this organism undergoes over the course of chronic infection in the lungs of CF individuals (Mena et. al. 2008). The harsh environment of the CF lung triggers mutations in the P. aeruginosa genome and selects for variants that are better equipped to survive (Ciofu et. al. 2010; D?Argenio et. al. 2007; Hoffman et. al. 2010). Consequently, the P. aeruginosa population over time differs genotypically and phenotypically from the initial infecting strain (Kukavica et. al. 2008; Mahenthiralingam et. al. 1996; Smith et. al. 2006). P. aeruginosa strains isolated during the course of chronic lung infection in patients with CF are altered for a variety of phenotypes including motility, acquisition of the mucoid phenotype, reduced virulence, antibiotic resistance, and mutations in lasR, a major transcriptional activator of virulence genes and biofilm formation (Bragonzi et. al. 2009; Hogardt and Hessemann, 2010; Mena et. al. 2008; Oliver, 2011; Smith et. al. 2006). One of the most 7 significant alterations that occur is the conversion to the mucoid phenotype which results from the overproduction of alginate (Schobert and Jahn, 2006; Xie et al. 1996). Mucoid strains of P. aeruginosa assist P. aeruginosa in improved biofilm formation in the CF lung (Stapper et. al. 2004). The biofilm communities found within the lung have increased resistance to antimicrobials (Spoering and Lewis, 2004). Hydrogen cyanide has also been detected in CF sputum where P. aeruginosa bacterial communities reside (Ryall et. al. 2008). Hydrogen cyanide is a poisonous gas that interferes with cellular respiration (Ryall et. al. 2008). Mucoid strains of P. aerugionsa have been shown to upregulate genes responsible for hydrogen cyanide and produce seven-fold more hydrogen cyanide than nonmucoid strains (Carterson et. al. 2004; Firoved and Deretic, 2003). This suggests that hydrogen cyanide is an important virulence factor for P. aeruginosa growing within the CF lung. Many of the phenotypic variations detected in CF P. aeruginosa are the result of hypermutations within the genome. The high mutation rate is largely caused by oxidative stress encountered by P. aeruginosa during chronic CF infection (Ciofu et. al. 2005). Hypermutable variants selected for within in the lung are thought to play a role in supporting persistence and antibiotic resistance (Mena et. al. 2008; Oliver et. al. 2000). P. aeruginosa also undergoes extensive metabolic changes during chronic respiratory infection that can include the constitutive expression or deregulation of genes responsible for carbon catabolism. Amino acid auxotrophs of P. aeruginosa commonly arise in the lung (Barth and Pitt, 1995). The selection for auxotrophic variants suggests that some amino acid biosynthetic genes may no longer be required because amino acids are freely available in the lung and appear to be a key carbon source for the bacterium (Barth and Pitt, 1996). The loss of several other regulatory mechanisms has been reported in chronic isolates. These alterations can include the upregulation of zwf, which encodes for glucose-6-phosphate dehydrogenase and is required for optimal 8 alginate production (Silo-Suh et. al. 2005). High expression of zwf could possibly be important for the survival of P. aeruginosa growing within the CF lung (Silo-Suh et. al. 2005). A transcriptome study by Son et. al. in 2007 revealed that some of the genes constitutively expressed during chronic infection are involved in central metabolic pathways including fatty acid and amino acid degradation and glycerol metabolism (Son et. al. 2007). Constitutive expression of these genes is likely due to the high availability of fatty acids and amino acids present in the lung which can serve as nutrients to fuel P. aeruginosa growth (Son et. al. 2007). Carbon sources utilized by P. aeruginosa during CF infection include lipids obtained from lung surfactant, fatty and amino acids (Son et. al. 2007). Long chain fatty acids are generated when lipases hydrolyze phosphatidylcholine (PC) from host lipid membranes and are eventually metabolized via ?-oxidation. As a result, these fatty acids can become one the most abundant nutrients in the CF lung. Two genes that are induced by the presence of fatty acids and are constitutively upregulated in CF P. aeruginosa are aceA and glcB. These two genes are unique to the glyoxylate pathway and they encode for isocitrate lyase (ICL) and malate synthase (MS), respectively (Hagins et. al. 2010, 2011). The glyoxylate pathway The glyoxylate shunt is an anabolic pathway of the tricarboxylic acid (TCA) cycle that allows for growth on C2 compounds by bypassing the CO2-generating steps of the TCA cycle (Figure 1) (Dunn et. al. 2009). The first unique step of the glyoxylate cycle is catalyzed by ICL and involves the cleavage of isocitrate to glyoxylate and succinate (Dunn et. al. 2009; Gui et. al. 1996). During the next step, glyoxylate condenses with acetyl-CoA to form malate. This conversion is catalyzed by MS. The end result of the glyoxylate pathway is the production of 9 TCA cycle intermediates that can be used for gluconeogenesis or other biosynthetic reactions (Dunn et. al. 2009; Gui et. al. 1996). The glyoxylate pathway is induced by growth on acetate or fatty acids that are degraded by ?-oxidation (Dunn et. al. 2009; Gui et. al. 1996). Isocitrate lyase and isocitrate dehydrogenase utilize the same substrate, isocitrate. When TCA cycle intermediates are present in the growth medium, isocitrate dehydrogenase becomes dephosphorylated and isocitrate is directed towards the TCA cycle (Dunn et. al. 2009; Gui et. al. 1996). When acetate or fatty acids are present in high concentrations in the growth medium, isocitrate dehydrogenase is inactivated by phosphorylation and the glyoxylate pathway is induced (Dunn et. al. 2009; Gui et. al. 1996). The glyoxylate pathway occurs in many microorganisms and plants, but is absent in humans, which makes it an attractive target for therapy (Dunn et. al. 2009; Gui et. al. 1996; Hagins et. al. 2010 and 2011). Understanding the mechanisms by which pathogens use ICL and MS to infect a host will provide better insight on the role these enzymes play in pathogenesis. Figure 1.1 The Glyoxylate Pathway is marked by bold arrows 10 The role of the glyoxylate pathway in pathogenesis. The glyoxylate pathway is required by diverse microbes to cause disease in a variety of hosts (Dunn et. al. 2009). Mutations in genes that encode for isocitrate lyase and malate synthase lead to reduced virulence in microorganisms such as P. aeruginosa, Mycobacterium tuberculosis, Cryptococcus neoformans, Rhodococcus equi, Salmonella enterica, and Candida albicans (Cramer et. al. 2007; Dunn et. al. 2009; Hagins et. al. 2010; Lindsey et. al. 2008; Munoz-Elias et. al. 2005; Wall et. al. 2005). More importantly, bacterial pathogens including M. tuberculosis, Salmonella enterica serovar Typhimurium and Burkholderia pseudomallei also rely on the glyoxylate pathway for persistence in animal models of infection (Fang et. al. 2005; McKinney et. al. 2000; Schaik et. al. 2009). In B. pseudomallei, mutations in aceA prevent the bacterium from entering the persistent mode of infection and force it to remain in an acute state, which is much easier to treat with antibiotics (Schaik et. al. 2009). Furthermore, in M. tuberculosis, loss of aceA results in the build-up of a toxic intermediate, which is lethal to the bacterium (McKinney et. al. 2000). We previously showed that isocitrate lyase (ICL) is required for an acute isolate of P. aeruginosa to cause disease in the rat lung model of infection (Lindsey et. al. 2008). Moreover, ICL is required for optimal production of two important virulence determinants, hydrogen cyanide and alginate, by a chronic isolate of P. aeruginosa (Hagins et. al. 2009; Lindsey et. al. 2008). These data suggest that P. aeruginosa benefits from high ICL activity in the CF lung either by utilization of certain compounds as carbon sources or for optimal production of virulence determinants. 11 Regulation of aceA and glcB in P. aeruginosa. Few studies have examined the regulation of aceA and glcB in P. aeruginosa. Based on observations from other bacteria, regulators of aceA and glcB in P. aeruginosa are predicted to be encoded by genes that respond to acetate, fatty acids and possibly amino acids in the growth medium. In Corynebacterium glutamicum, aceA is positively and negatively regulated by RamA and RamB, respectively, and is dependent on acetate availability (Cramer et. al. 2007). In Escherichia coli, aceA is negatively regulated by FadR and IclR and responds to the presence of acetate or fatty acids (Gui et. al. 1996). However, these genes do not regulate aceA or glcB in P. aeruginosa (Hagins et. al. 2010). We previously demonstrated that in an acute isolate of P. aeruginosa, aceA and glcB are negatively regulated by RpoN (Hagins et. al. 2010 and unpublished data). RpoN is an alternative sigma factor that regulates several virulence genes in P. aeruginosa, and therefore, is required for virulence under certain conditions (Hendrickson et. al. 2001). However, in chronic CF isolates of P. aeruginosa, RpoN does not appear to regulate aceA and glcB (Hagins et. al. 2010 and unpublished data) probably because both genes are permanently upregulated in this isolate. Previous predictive and non-predictive approaches have failed to identify transcriptional regulators of aceA and glcB in P. aeruginosa suggesting the presence of novel regulators of these genes. In an effort to better understand aceA and glcB regulation, we characterized gene expression in response to various carbon sources (Hagins 2010, Hagins 2011). For example, aceA and glcB have been shown to be induced in P. aeruginosa cultures grown on leucine. However, the regulatory mechanisms involved in these processes have yet to be identified (Hagins et. al. 2010; Diaz Perez et. al. 2007). Due to the predominance of nutrients in the CF lung that require both the glyoxylate pathway and the glp 12 regulon for catabolism, we questioned whether glycerol metabolism might overlap with fatty acid metabolism in P. aeruginosa in the regulation of the glyoxylate pathway. Glycerol metabolism and regulation In P. aeruginosa, glycerol metabolism is controlled by GlpR, a negative transcriptional regulator that controls expression of the genes in the glp regulon (Schweizer et. al. 1996). The glp regulon encodes for a membrane-associated glycerol diffusion facilitator (GlpF), a glycerol kinase (GlpK), a membrane protein involved in alginate biosynthesis (GlpM), and a glycerol-3- phosphate dehydrogenase (GlpD) (Figure 2). Glycerol is transported into the cell via GlpF and is phosphorylated to glycerol 3-phosphate (G3P) by GlpK, whereas exogenous glycerol 3- phosphate is transported into the cell via the GlpT transporter system (Schweizer et. al. 1996 and 1997). Importantly, G3P induces the glp regulon by binding to GlpR (Schweizer et. al. 1996). Ultimately, the products of glycerol metabolism can be used by the cell as a source of energy or for the synthesis of alginate (Schweizer et. al. 1996 and 1997). Glycerol metabolism in P. aeruginosa is similar to other proteobacteria. However, in E. coli the glp genes are arranged as operons in three different loci on the chromosome: glpTQ/glpABC, glpEGR/glpD and glpFKX. These operons are all controlled by the glp repressor, GlpR (Danilova et. al. 2003; Zeng et. al. 1996). In E. coli G3P can be oxidized anaerobically or aerobically the products of glpA or glpD, respectively (Iuchi et. al. 1990). Interestingly, two of the most abundant products generated from the microaerobic metabolism of glycerol in E. coli are ethanol and acetate (Durnin et. al. 2009), which would require induction of the glyoxylate pathway. 13 Figure 1.2. Organization of the glp regulon in P. aeruginosa. Nutrient acquisition and metabolism via the glyoxylate pathway and the glp regulon in P. aeruginosa During lung infection, P. aeruginosa secretes phospholipases that cleave host lipid membranes (e.g. phosphatidylcholine and phosphatidylethanolamine) yielding free fatty acids and glycerol, which become available to P. aeruginosa for use as carbon sources (Figure 3). Phosphatidylcholine (PC) derived nutrients require the glyoxylate pathway and glp regulon for metabolism. In vivo expression studies reveal that P. aeruginosa recovered from the CF lung show higher constitutive expression of genes that encode for lipases and phospholipases compared to non-CF P. aeruginosa (Son et. al. 2007). In addition, two genes involved in glycerol metabolism, glpD and glpK, are also constitutively expressed under the same conditions in P. aeruginosa (Son et. al. 2007). These results correlate upregulation of genes involved in fatty acid and glycerol metabolism in P. aeruginosa during infection of the CF lung. Furthermore, the entire glp regulon is induced in an acute isolate of P. aerugionsa when grown on PC (Son et. al. 2007), and it is likely that this regulon also responds to fatty acids derived from PC. Another study revealed that the expression of the glyoxylate pathway genes, glcB and aceA, is induced two and five-fold, respectively, during chemotaxis towards the membrane lipid, phosphatidylethanolomine (Miller et. al. 2008). In the same study, expression of glpF and glpK 14 was also induced, as well as the transcriptional regulator, GlpR (Miller et. al. 2008). Taken together, the glyoxylate pathway and the glp regulon appear to play a crucial role in acquiring nutrients for P.aeruginosa growing within the CF lung. Our current understanding of how the glyoxylate pathway and the glp regulon might contribute to the ability of P. aeruginosa to cause chronic disease is extremely limited. It is possible that both pathways share some regulatory element, particularly because both respond to nutrients found within the CF lung. Unfortunately, little is known about the regulation of both pathways in P. aeruginosa and the relationship between the glyoxylate pathway and glp regulon has never been examined. A better understanding of the glyoxylate pathway, as well as the glp regulon, in chronic CF isolates of P. aeruginosa will provide valuable insight into the CF lung environment. Figure 1.3. Degradation of phophatidylethanolamine to yield glycerol and fatty acids via the action of P. aeruginosa phospholipases and lipases. 15 Summary The constitutive expression of genes responsible for glycerol and fatty acid metabolism in the CF lung likely provides an advantage to P. aeruginosa. Efficient catabolism of these carbon sources may require coordination of the glyoxylate and glycerol pathways. Therefore my thesis project focused on the role of GlpR in regulating the glyoxylate pathway and characterized glycerol catabolism by a CF isolate of P. aeruginosa. This research provides novel insight into the regulatory interchange involved in glycerol and fatty acid metabolism in P. aeruginosa, and promotes our understanding of how these two networks enable P. aeruginosa to establish and maintain chronic infections. Understanding the regulation of genes specific to the glyoxylate pathway will advance our knowledge of how bacterial pathogens cause disease and provide clues on how to treat CF lung infection, as well as move us a step closer to understanding how P. aeruginosa adapts to the CF lung. The glyoxylate pathway is an attractive therapeutic target because it is not present in humans. 16 References Bals, R., P. Hiemstra. 2004. Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. European Respiratory Journal 23: 327?333. Barth, a L., and T. L. Pitt. 1995. Auxotrophic variants of Pseudomonas aeruginosa are selected from prototrophic wild-type strains in respiratory infections in patients with cystic fibrosis. Journal of Clinical Microbiology 33: 37-40. Barth, a L., and T. L. Pitt. 1996. The high amino-acid content of sputum from cystic fibrosis patients promotes growth of auxotrophic Pseudomonas aeruginosa. Journal of Medical Microbiology 45: 110-119. Bragonzi, A., M. Paroni, A. Nonis, N. Cramer, S. Montanari, J. Rejman, C. Di Serio, G. D?ring, and B. T?mmler. 2009. Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence. American Journal of Respiratory and Critical Care Medicine 180: 138-145. Carterson, A. J., L. A. Morici, D. W. Jackson, A. Frisk, S. E. Lizewski, R. Jupiter, K. Simpson, D. A. Kunz, S. H. Davis, J. R. Schurr, D. J. Hassett, and M. J. Schurr. 2004. The Transcriptional Regulator AlgR Controls Cyanide Production in Pseudomonas aeruginosa. Journal of Bacteriology 186: 6837-6844. Chambers, D., F. Scott, R. Bangur, R. Davies, a Lim, S. Walters, G. Smith, T. Pitt, D. Stableforth, and D. Honeybourne. 2005. Factors associated with infection by Pseudomonas aeruginosa in adult cystic fibrosis. The European Respiratory Journal: Official Journal of the European Society for Clinical Respiratory Physiology 26:651-656. Choi, J. Y., C. D. Sifri, B. C. Goumnerov, L. G. Rahme, F. M. Ausubel, and S. B. Calderwood. 2002. Identification of Virulence Genes in a Pathogenic Strain of Pseudomonas aeruginosa by Representational Difference Analysis. Journal of Baceriology. 184: 952-961. Ciofu, O., L. F. Mandsberg, T. Bjarnsholt, T. Wassermann, and N. H?iby. 2010. Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants. Microbiology (Reading, England) 156: 1108-1119. Cramer, A., M. Auchter, J. Frunzke, M. Bott, and B. Eikmanns. 2007. RamB, the Transcriptional Regulator of Acetate Metabolism in Corynebacterium glutamicum, is Subject to Regulationby RamA and RamB. Journal of Bacteriology. 189: 1145?1149. Danilova, L. V., M. S. Gelfand, V. A. Lyubetsky, and O. N. Laikova. 2003. Computer- Assisted Analysis of Regulation of the Glycerol-3-Phosphate Metabolism in Genomes of Proteobacteria. Molecular Biology 37:843-849. 17 D?Argenio, D. a, M. Wu, L. R. Hoffman, H. D. Kulasekara, E. D?ziel, E. E. Smith, H. Nguyen, R. K. Ernst, T. J. Larson Freeman, D. H. Spencer, M. Brittnacher, H. S. Hayden, S. Selgrade, M. Klausen, D. R. Goodlett, J. L. Burns, B. W. Ramsey, and S. I. Miller. 2007. Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients. Molecular Microbiology 64: 512-533. D?az-P?rez, A. L., C. Rom?n-Doval, C. D?az-P?rez, C. Cervantes, C. R. Sosa-Aguirre, J. E. L?pez-Meza, and J. Campos-Garc?a. 2007. Identification of the aceA gene encoding isocitrate lyase required for the growth of Pseudomonas aeruginosa on acetate, acyclic terpenes and leucine. FEMS microbiology letters 269: 309-316. Dunn, M. F., J. a Ram?rez-Trujillo, and I. Hern?ndez-Lucas. 2009. Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology (Reading, England) 155: 3166-3175. Fang, F.C., Libby, S.J., Castor, M.E., and Fung, A.M. 2005. Isocitrate lyase (AceA) is required for Salmonella persistence but not for acute lethal infection in mice. Infect. Immun. 73: 2547-2549. Fisher, E., and U. Sauer. 2003. A novel metabolic cycle catalyzes glucose oxidation and anaplerosis in hungry Esherichia coli. J. Biol. Chem. 278: 46446-46451. Firoved, A. M., and V. Deretic. 2003. Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa. J. Bacteriol. 185:1071?1081. Gilligan, P. 1991. Microbiology of airway disease in patients with cystic fibrosis. Clinical Microbiology Reviews 4: 35?51. Govan, J. R., and V. Deretic. 1996. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiological reviews 60:539-574. Grimek, T. L. and J. Escalante-Semerena. 2004. The acnD genes of Shewenella oneidensis and Vibrio cholerae encode a new Fe/Sdependent 2-methylcitrate dehydratase enzyme that requires prpF function in vivo. J. of Bacteriology 186: 454?462. Gui, L., A. Sunnarborg, and D. LaPorte. 1996. Regulated Expression of a Repressor Protein:FadR Activates iclR. Journal of Bacteriology. 178: 4704?4709. Hagins. J., R. Locy and L. Silo-Suh. 2009. Isocitrate lyase supplies precursors for hydrogen cyanide production in a cystic fibrosis isolate of Pseudomonas aeruginosa. Journal of Bacteriology. 191: 6335-6339. Hagins, J., J. Scoffield, S. Suh and L. Silo-Suh. 2010. Influence of RpoN on isocitrate lyase activity in Pseudomonas aeruginosa. Microbiology. 156: 1201-1210. 18 Hagins, J., J. Scoffield, S. Suh and L. Silo-Suh. 2011. Malate synthase expression is deregulated in the cystic fibrosis isolate FRD1. Canadian Journal of Microbiology. 195:186- 195. Harrison, F. 2007. Microbial ecology of the cystic fibrosis lung. Microbiology (Reading, England) 153: 917-23. Hendrickson, E., J. Plotnikova, S. Mahajan-Miklos, L. Rahme, F. Ausubel. 2001 Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice. J Bacteriology. 183: 7126-7134. Henry, R., L. Mellis, L. Petrovic. 1992. Mucoid Pseudomonas aeruginosa is a marker of poor survival in cystic fibrosis. Pediatr Pulmonol 12: 158?161. Hoffman, L. R., A. R. Richardson, L. S. Houston, H. D. Kulasekara, W. Martens-Habbena, M. Klausen, J. L. Burns, D. a Stahl, D. J. Hassett, F. C. Fang, and S. I. Miller. 2010. Nutrient availability as a mechanism for selection of antibiotic tolerant Pseudomonas aeruginosa within the CF airway. PLoS pathogens 6:e1000712. Hogardt, M., and J. Heesemann. 2010. Adaptation of Pseudomonas aeruginosa during persistence in the cystic fibrosis lung. International Journal of Medical Microbiology: IJMM. Elsevier GmbH. 300:557-62. Iuchi, S., S. T. Cole, and E. C. Lin. 1990. Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control. Journal of Bacteriology. 172:179-184. Kukavica, I., and R. Levesque. 2008. Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies. Laboratory Animals. 42: 389- 412. Lau, G. W., D. J. Hassett, H. Ran, and F. Kong. 2004. The role of pyocyanin in Pseudomonas aeruginosa infection. Trends in Molecular Medicine 10: 599-606. Lee, D. G., J. M. Urbach, G. Wu, N. T. Liberati, R. L. Feinbaum, S. Miyata, L. T. Diggins, J. He, M. Saucier, E. D?ziel, L. Friedman, L. Li, G. Grills, K. Montgomery, R. Kucherlapati, L. G. Rahme, and F. M. Ausubel. 2006. Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Genome Biology 7:R90. Lindsey, T. L., J. Hagins, P. Sokol, and L. Silo-Suh. 2008. Virulence determinants from a Cystic Fibrosis isolates of Pseudomonas aeruginosa include isocitrate lyase. Microbiology 154:1616-1627. Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2002. Lung Infections Associated with Cystic Fibrosis. J. Bacteriology. 15:194-222. 19 Mahenthiralingam, E., Campbell, E. Foster, J. Lam, D. Speert. 1996. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol 34: 1129?1135. Mathee, K., G. Narasimhan, C. Valdes, X. Qiu, J. Matewish, M. Koehrsen, A. Rokas, C. Yandava, R. Engels, E. Zeng, R. Olavarietta, M. Doud, R. Smith, P. Montgomery, J. White, P. Godfrey, C. Kodira, B. Birren, J. Galagan, and S. Lory. 2008. Dynamics of Pseudomonas aeruginosa genome evolution. Proc. Natl. Acad. Sci. USA 105: 3100?3105. Mena, A., E. E. Smith, J. L. Burns, D. P. Speert, S. M. Moskowitz, J. L. Perez, and a Oliver. 2008. Genetic adaptation of Pseudomonas aeruginosa to the airways of cystic fibrosis patients is catalyzed by hypermutation. Journal of Bacteriology 190:7910-7917. Munoz-Elias, E.J., and McKinney, J.D. 2005. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11: 638?644. Oliver, A. 2011. High Frequency of Hypermutable Pseudomonas aeruginosa in Cystic Fibrosis Lung Infection. Science. 288:1251-1253. Roucourt, B., Minnebo, N., Augustijns, P., Hertveldt, K., Volckaert, G., and Lavigne, R. 2009. Biochemical characterization of malate synthase G of P. aeruginosa. BMC Biochem. 10(1):20. Sadikot, R. T., T. S. Blackwell, J. W. Christman, and A. S. Prince. 2005. Pathogen-host interactions in Pseudomonas aeruginosa pneumonia. American Journal of Respiratory and Critical Care Medicine 171:1209-1223. Schaik, E. J. V., M. Tom, and D. E. Woods. 2009. Burkholderia pseudomallei Isocitrate Lyase Is a Persistence Factor in Pulmonary Melioidosis?: Implications for the Development of Isocitrate Lyase Inhibitors as Novel Antimicrobials. Journal of Bacteriology. 77: 4275-4283. Schobert, M., and D. Jahn. 2010. Anaerobic physiology of Pseudomonas aeruginosa in the cystic fibrosis lung. International Journal of Medical Microbiology?: IJMM. 300:549-556. Schweizer, H. and C. Po. 1996. Regulation of Glycerol Metabolism in Pseudomonas aeruginosa: Characterization of the glpR Repressor Gene. Journal of Bacteriology. 178: 5215? 5221. Schweizer, H., R. Jump and C. Po. 1997. Structure and gene-polypeptide relationships of the region encoding glycerol diffusion facilitator (glpF) and glycerol kinase (glpK) of Pseudornonas aeruginosa. Microbiology. 143: 1287-1297. Silo-Suh, L., Suh, S. J., Sokol, P. A. & Ohman, D. E. 2002. A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma- 22) and RhlR contribute to pathogenesis. Proc Natl Acad Sci. 99: 15699?15704. 20 Silo-Suh, L., Suh, S. J., Phibbs, P. V. & Ohman, D. E. 2005. Adaptations of Pseudomonas aeruginosa to the cystic fibrosis lung environment can include deregulation of zwf, encoding glucose-6- phosphate dehydrogenase. J Bacteriol 187: 7561?7568. Smith, E., E.Buckley, D. Wu, Z. Saenphimmachak, C. Hoffman, L. D?Argenio, D. Miller, S. Ramsey, and B. Speert. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 103: 8487?8492. Sokol, P. A., and D. E. Woods. 1988. Effect of pyochelin on Pseudomonas cepacia respiratory infection. Microb. Pathog. 5:197?205. Son, M., W. Matthews, Y. Kang, D. Nguyen, and T. Hoang. 2007. In Vivo Evidence of Pseudomonas aeruginosa Nutrient Acquisition and Pathogenesis in the Lungs of Cystic Fibrosis Patients. Infection and Immunity, 75: 5313?5324. Spoering, A. M. Y. L., and K. I. M. Lewis. 2001. Biofilms and Planktonic Cells of Pseudomonas aeruginosa have Similar Resistance to Killing by Antimicrobials. Journal of Bacteriology. 183:6746-6751. Stapper, a. P. 2004. Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation. Journal of Medical Microbiology. 53:679-690. Stover, C. K., X. Q. Pham, a L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, a Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959-964. Struelens, M., J.Schwam, V. Deplano, and A. Baran. 1993. Genome macrorestriction analysis of diversity and variability of Pseudomonas aeruginosa strains infecting cystic fibrosis patients. J Clin Microbiol. 31: 2320?2326. Twining, S. S., Kirschner, S. E., Mahnke, L. A. & Frank, D. W. 1993. Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins. Invest Ophthalmol Vis Sci 34: 2699?2712. Van Delden, C., and B. H. Iglewski. 1998. Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerging Infectious Diseases. 4:551-560. Wall, D., P. Duffy, C. DuPont, J. Prescott, and W. Meijer. 2005. Isocitrate Lyase Activity Is Required for Virulence of the Intracellular Pathogen Rhodococcus equi. Infection and Immunity, 73: 6736?6741. 21 Wang, Q., Y. Zhang, C. Yang, H. Xiong, Y. Lin, J. Yao, H. Li, and G. Zhao. 2010. Acetlyation of Metabolic Enzymes Coordinates Carbon Source Utilization and Metabolic Flux. Science. 327: 1004-1007. Welsh, M. 1996. Cystic fibrosis. In: Molecular Biology of Membrane Transport Disorders. (Schultz SG, ed). New York: Plenum Press. 605?623 Welsh, M., F. Accurso, G. Cutting. 2001. Cystic fibrosis. In: The Metabolic and Molecular Basis of Inherited Diseases (Scriver CR, Beaudet AL, Sly WS, Valle D, eds). New York: McGraw-Hill, 5121?5188. Willcox, M. D. P., H. Zhu, T. C. R. Conibear, E. B. H. Hume, M. Givskov, S. Kjelleberg, and S. Rice. 2008. Role of quorum sensing by Pseudomonas aeruginosa in microbial keratitis and cystic fibrosis. Microbiology (Reading, England) 154:2184-94. Xie, Z. D., Hershberger, C. D., Shankar, S., Ye, R. W. & Chakrabarty, A. M. 1996. Sigma factor-antisigma factor interaction in alginate synthesis: inhibition of AlgT by MucA. J Bacteriol. 178: 4990?4996. Zeng, G., S. Ye, and T. J. Larson. 1996. Repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K-12: primary structure and identi?cation of the DNA-binding domain. J. Bacteriol. 178: 7080?7089. 22 Chapter 2 The Role of GlpR in the Regulation of the Glyoxylate Pathway in Pseudomonas aeruginosa Abstract Pseudomonas aeruginosa infections are the leading cause of morbidity and mortality for cystic fibrosis (CF) patients. P. aeruginosa establishes life-long infection in the CF lung by utilizing various adaptation strategies to cause a chronic infection. One of these strategies includes the upregulation of the genes encoding for the glyoxylate pathway enzymes, aceA and glcB which encode for isocitrate lyase (ICL) and malate synthase (MS), respectively. The glyoxylate pathway allows certain bacteria to grow on acetate or fatty acids as a sole carbon source to replenish intermediates of the tricarboxylic acid cycle, a pathway that is required by many microorganisms for pathogenesis. We determined previously that the glyoxylate pathway becomes deregulated in some isolates of P. aeruginosa adapted to the CF lung, including FRD1. The occurrence of these isolates suggests deregulation of the glyoxylate pathway may benefit P. aeruginosa growing within the CF lung. However, the mechanism(s) responsible for regulation of the glyoxylate pathway have yet to be elucidated. GlpR is a transcriptional repressor that regulates the genes responsible for glycerol metabolism in P. aeruginosa. We determined that GlpR also plays a role in regulating the glyoxylate pathway. Disruption of glpR in PAO1, an acute isolate of P. aeruginosa, resulted in high ICL and MS activity. This activity was correlated with increased expression of aceA and glcB, which encode for ICL and MS respectively. GlpR?s 23 role in the regulation of the glyoxylate pathway provides a novel perspective into the interplay between fatty acid and glycerol metabolism in P. aeruginosa. Introduction Pseudomonas aeruginosa is the major etiologic agent of chronic pulmonary infections in cystic fibrosis (CF) patients (Bragonzi et. al. 2009). P. aeruginosa is acquired early in life by the patient and persists within the lung for decades (Bragonzi et. al. 2009; Mahenthiralingam et. al. 1996). During infection of the CF lung P. aeruginosa acquires several mutations that facilitate its survival in the CF lung environment (Chambers et. al. 2005; Hoffman et. al. 2009; Smith et. al. 2006). Some of the alterations that promote the survival of P. aeruginosa include the overproduction of alginate, loss of flagella, and differential expression of genes responsible for virulence and catabolism (Chambers et. al. 2005; Smith et. al. 2006; Son et. al. 2007). These alterations facilitate P. aeruginosa to evade clearance by the host immune system and are important for the acquisition and metabolism of nutrients obtained from CF sputum. Sputum is the most likely source of nutrition for P. aerugionsa living within the CF lung (Son et. al. 2007). Sputum contains a complex mixture of host and bacterial derived products including amino acids and lipids (Palmer et. al. 2005). In addition, carbon sources such as glycerol and fatty acids are liberated by the hydrolysis of membrane lipids by secreted bacterial phospholipases (Lyczak et. al. 2002; Terry et. al. 1992; Williams et. al. 1994). Given the relative abundance of these particular carbon sources in the CF lung, it would be advantageous for P. aeruginosa persisting in that environment to adopt strategies for more efficient utilization of these nutrients including altering regulation of the required metabolic pathways. 24 In previous studies, we showed that aceA and glcB are constitutively upregulated in the chronic CF isolate of P. aeruginosa, FRD1 (Lindsey et. al. 2008, Hagins et. al. 2010). We also demonstrated that aceA is essential for infection in the alfalfa seedling and rat lung models of infection, and is required for optimal production of alginate and hydrogen cyanide (Lindsey et. al. 2008, Hagins et. al. 2009 and 2010). aceA and glcB, which encode for isocitrate lyase and malate synthase, respectively, are specific to the glyoxylate pathway (Dunn et. al. 2009), which is required by certain microorganisms for growth on acetate or fatty acids as the sole carbon source (Dunn et. al. 2009). The mechanism of deregulation of aceA and glcB in FRD1 is unknown. We previously showed that aceA and glcB are negatively regulated by RpoN in PAO1. However, RpoN is not responsible for the deregulation of aceA and glcB in FRD1 (Hagins et. al. 2010). Due to the availability of fatty acids and glycerol in the CF lung, catabolism of both carbon sources may require coordination of several metabolic pathways, including the glyoxylate pathway. In this study, we focused on the contribution of GlpR to regulation of the glyoxylate pathway in both chronic (FRD1) and wound (PAO1) isolates of P. aeruginosa. GlpR controls glycerol metabolism in P. aeruginosa by negative regulation of the glp regulon (Schweizer et. al. 1996). The glp regulon encodes for a membrane-associated glycerol diffusion facilitator (GlpF), a glycerol kinase (GlpK), a membrane protein involved in alginate biosynthesis (GlpM), and glycerol-3-phosphate dehydrogenase (GlpD) (Schweizer et. al. 1996). Glycerol is transported into the cell via the GlpT transporter system, which is separate from the glp operon and is not regulated by GlpR. The glp regulon is induced by the presence of glycerol-3-phosphate (G3P) or glycerol in the growth medium (Schweizer et. al. 1996). We reasoned that fatty acid and glycerol catabolism might be coordinately regulated since both 25 carbon sources are liberated from host membranes. Therefore, we tested the effect of a glpR null mutation on the glyoxylate pathway in P. aeruginosa. Materials and Methods Bacterial strains, plasmids, and media. Bacterial strains and plasmids used in this study are listed in Table 2.1. Unless otherwise indicated, bacteria were cultured in L-broth (LB) or on L-agar at 37?C. No-carbon-E minimal medium (NCE) supplemented with 0.1% (w/v) casamino acids (CAA) was used to assay for growth on minimal media (Davis et al., 1980). Glycerol was used at a concentration of 20 mM. UV-Vis absorption spectra were recorded on a Shimadzu UV-1601 Spectrophotometer using 1 cm path length cells. A 1:1 mixture of L-agar and Pseudomonas Isolation Agar (PIA) was used to select for P. aeruginosa transconjugants and to counter select for E. coli following triparental mating. Media were solidified with 1.5% (w/v) Bacto Agar (Difco). Antibiotics were purchased from Sigma-Aldrich (St. Louis, MO) and used at the following concentrations in this study: 100 ?g ampicillin (Amp) ml-1 for E. coli; 100 ?g carbenicillin (Cb) ml-1 for P. aeruginosa; 20 ?g gentamicin (Gm) ml-1 for E. coli and 200 ?g for P. aeruginosa; 20 ?g tetracycline (Tet) ml-1 for E. coli; 100 ?g ml-1 for P. aeruginosa, and 50 ?g kanamycin ml-1 for E. coli. To examine growth over a 24 hour period, cultures were grown in 24 well microtiter plate and monitored at A600 with a BioTek Synergy HT plate reader (BioTek, Winooski, VT). DNA manipulations, transformations, and conjugations. E. coli strain DH10B was routinely used as a host strain for cloning. DNA was introduced into E. coli by electroporation and into P. aeruginosa by conjugation as previously described. Plasmids were purified with QIAprep Spin Miniprep columns (Qiagen, Valencia, CA). DNA fragments were excised from agarose gels and purified using the Qiaex II DNA gel 26 extraction kit (Qiagen) according to the manufacturer?s instructions. Restriction enzymes and DNA modification enzymes were purchased from New England Biolabs (Beverly, MA). Either Pfu from Stratagene (La Jolla, CA) or Taq from New England Biolabs were used for PCR amplification of DNA. Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Construction of P. aeruginosa glpR mutants. To generate glpR mutants of P. aeruginosa, the suicide plasmid pLS1554 was constructed: a DNA sequence containing approximately 400 bp upstream and 430 bp downstream of the glpR coding sequence was PCR amplified from PAO1 cells with Pfu and cloned into the SmaI site of pBluescript K(+). The resulting plasmid was digested with SphI and the internal 1.3 kb fragment of the glpR coding sequence was removed and replaced with the aacC1 gene encoding gentamicin resistance as a SmaI fragment (Schweizer, 1993). This was followed by introduction of an origin of transfer (moriT) of RP4 on a 230 bp HindIII fragment (Suh et al., 2004). pLS1554 was introduced into P. aeruginosa strains FRD1 and PAO1 by triparental mating, and potential glpR mutants were isolated as gentamicin-resistant, carbenicillin-sensitive colonies, indicating a double crossover event. Replacement of the wild- type glpR gene with the glpR101::aacC1 allele was verified by PCR analysis. Construction of the glpR complemented strains. To complement the glpR mutation, the wild-type gene was PCR amplified from PAO1 using Pfu. The resulting fragment was cloned into the SmaI site of a plasmid which contains a regulatable promoter upstream of the multiple cloning site (Silo-Suh et al., 2005) to produce pLS1950. The resulting plasmid (pLS1950) was digested with HindIII and the moriT was 27 inserted to allow for mobilization of the plasmid into P. aeruginosa. The plasmid was mobilized into P. aeruginosa by triparental mating and potential complemented strains were isolated as carbenicillin resistant colonies. In cis complementation was verified by PCR analysis and the complemented PAO1 glpR isolates were designated PAO1 glpR + (JS146). Construction of glpD and glpR transcriptional fusions and biochemical assays. The glpD::lacZ and glpR::lacZ transcriptional fusions were constructed using the glpD and glpR gene fragments isolated from PAO1 via PCR using Pfu. The fragments, which included 500 bp upstream from the coding sequence, were cloned into the SmaI site of pSS223 (Suh et al., 2004). The plasmids (pLS1954 and pJS149), containing the 5? coding sequence for glpD and glpR, respectively, in the proper orientation, were verified by PCR and restriction digest. The plasmids containing the fusions were conjugated into FRD1 and PAO1 via triparental mating and plasmid integration events were selected by carbenicillin resistance. Biochemical assays. ?-galactosidase assays were preformed as described by Miller (Miller, 1972). Isocitrate lyase activity was measured according to the Sigma Aldrich protocol (EC 4.1.3.1), with minor modification. P. aeruginosa cells were harvested from stationary cultures and washed with saline. The cells were resuspended in Tris-EDTA (TE) Buffer pH 6.9 and broken open via ultrasonification with Fisher Scientific, Model 100 Sonic dismembrator using a microtip. Following centrifugation, the total protein in the cell free extracts was quantified using the Bradford method (Bio-Rad, Hercules, CA). The cell free extract was added to a mixture of imidazole buffer, magnesium chloride, isocitrate, and phenylhydrazine per the Sigma protocol. The increase in absorbance at A324 was monitored for 5 minutes at room temperature and activity 28 was expressed as ?A324 min-1 (mg protein)-1 in which the rate of ?A324 was determined using only the linear part of the reaction. Malate synthase activity was determined according to the Sigma Aldrich protocol (EC 4.1.3.2), with slight modifications: P. aeruginosa cells were harvested from stationary phase cultures and washed with saline. The cells were resuspended in TE Buffer pH 8.0 and sonicated. Following centrifugation, the total protein in the cell free extracts was quantified using the Bradford method (Bio-Rad). The cell free extract was added to a mixture of imidazole buffer, magnesium chloride, acetyl-CoA, glyoxylic acid, and dithionitrobenzoic acid per the Sigma protocol. The increase in absorbance at A412 was monitored for 5 minutes and activity was expressed as ?A412 min-1 (mg protein)-1 in which the rate of the ?A412 min-1 was determined using the linear rate of the reaction. Overexpression and purification of his-tagged GlpR from P. aeruginosa. A PCR product containing the glpR coding region was cloned between the NcoI/EagI site of the expression vector pET28-b (Novagen) and electroporated into DH10B, creating a His-tag at the C-terminus. Following confirmation of a positive clone the plasmid was subsequently electroporated into BL21 (DE3). The E. coli BL21 (DE3) strain harboring the His-tagged GlpR plasmid was induced with 1mM IPTG (Isopropyl ?-D-1 thiogalactopyranoside) in L-broth and induced at 28? C for 4 hours. The cells were harvested, washed, re-suspended and sonicated (Sonic Dismembrator 100, Fisher Scientific) in a lysis buffer containing 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole. The recombinant His-tagged GlpR protein was puri?ed on Ni-NTA resin according to the Qiaexpressionist protocol. 29 Gel mobility shift assay. DNA promoter fragments (60 bp) of aceA and glcB were synthesized with a 5? biotin end label (Integrated DNA Technologies). Binding reactions were performed according to the LightShift? Chemiluminescent EMSA Kit (Thermo Scientific) instructions. Briefly, promoter fragments were incubated with various concentrations of purified GlpR in a mixture of 1X Binding Buffer, 2.5% glycerol, 5mM MgCl2, 50 ng/uL Poly dI?dC, and 0.05% NP-40 % in a final volume of 20 ?L (Thermo Scientific). The reaction was incubated for 20 min at room temperature and separated on a native 5 % acrylamide gel. After migration the gel was transferred to a nylon membrane and developed using the LightShift? Chemiluminescent EMSA Kit (Thermo Scientific). The gel shift was visualized using the ImageQuant 4000 (GE Healthcare). 30 Strain or Plasmid Genotype, relevant characteristics Source Strains FRD1 CF isolate, mucoid Ohman et. al. (1981) PAO1 Wound isolate, nonmucoid Holloway et al. (1979) FRD1glpR (JS134) PAO1 glpR (JS97) FRD1 glpR+ (JS148) PAO1 glpR+ (JS145) FRD1glpR101::aacCI PAO1glpR101::aacCI FRD1 complemented for glpR PAO1 complemented for glpR This study This study This study This study PAO1glpR aceA::lacZ PAO1glpR glcB::lacZ PAO1glpR carrying aceA::lacZ fusion PAO1glpR carrying glcB::lacZ fusion This study This study PAO1 glpR::lacZ FRD1 glpR::lacZ PAO1 glpD::lacZ FRD1 glpD::lacZ PAO1 aceA::lacZ FRD1 glcB::lacZ BL21(DE3) Plasmids pLS1954 pLS1968 pJS149 pLS1950 pET28-b+glpR PAO1 carrying glpR::lacZ fusion FRD1 carrying glpR::lacZ fusion PAO1 carrying glpD::lacZ fusion FRD1 carrying glpD::lacZ fusion PAO1 carrying aceA::lacZ fusion FRD1 carrying aceA::lacZ fusion glpR His-tag expression strain glpR101 in pBluescript K+ glpD::lacZ transcriptional fusion in pSS223 glpR::lacZ transcriptional fusion in pSS223 glpR complementing plasmid for PAO1 and FRD1 His-Tag Plasmid This study This study Dr. Laura Silo-Suh Dr. Laura Silo-Suh Lindsey et. al. 2009 Hagins et. al. 2011 This study Dr. Laura Silo-Suh Dr. Laura Silo-Suh This study Dr. Laura Silo-Suh Dr. Laura Silo-Suh Table 2.1 Bacterial strains and plasmids. Abbreviations used for genetic markers are described by Holloway et al. (1979). Alternate strain names are shown in parentheses. 31 Results GlpR controls ICL and MS activity To identify possible regulators of aceA and glcB in P. aeruginosa we measured ICL and MS activity from parental strains (FRD1 and PAO1) and their glpR mutant derivatives. As shown in Figures 2.1 and 2.2, disruption of glpR resulted in high ICL and MS activity in PAO1 which suggests that glpR plays a role in regulation of the glyoxylate pathway in the acute wound isolate. However, a mutation in glpR had no effect on ICL and MS activity in the chronic CF isolate FRD1, both of which had abnormally high activity. PAO1 strains complemented for glpR with a wild type copy of the gene in cis restored normal ICL and MS activity. High ICL and MS activity correlate with increased aceA and glcB expression in the PAO1 glpR mutant To determine if altered aceA and glcB expression were the cause of high ICL and MS activity in PAO1, aceA::lacZ and glcB::lacZ fusions were utilized to measure promoter activity. aceA::lacZ expression was induced in stationary phase cultures in the PAO1 glpR mutant (Figure 2.3a). In contrast, glcB::lacZ expression was induced in the PAO1 glpR mutant throughout the entire growth period with a slight increase during late stationary phase (Figure 2.3b). The results indicate that upregulation of ICL and MS activity in the PAO1 glpR mutant correlates with increased transcription of the genes encoding for the enzymes. 32 Figure 2.1 Effect of glpR mutation on ICL activity in PAO1. ICL activity was assayed from overnight cultures of P. aeruginosa grown in L-broth. Complemented strains are designated as ?+? for those containing a wild-type copy of glpR from PAO1. Values represent the average of 2 experiments with standard error bars. 0 0.5 1 1.5 2 2.5 ICL Act ivit y [?A 32 4 min -1 (mg pro tein -1 )] FRD1 FRD1 glpR FRD1 glpR+ PAO1 PAO1 glpR PAO1 glpR+ 33 Figure 2.2 Effect of glpR mutation on MS activity in PAO1. MS activity was assayed from overnight cultures of P. aeruginosa grown in L-broth. Complemented strains are designated as ?+? for those containing a wild-type copy of glpR from PAO1. Values represent the average of 2 experiments with standard error bars. 0 0.5 1 1.5 2 2.5 MS Act ivit y [?A 41 2 min -1 (mg pro tein -1 )] FRD1 FRD1 glpR FRD1 glpR+ PAO1 PAO1 glpR PAO1 glpR+ 34 Figure 2.3a Effect of glpR mutation on aceA expression in PAO1. ?- galactosidase activity is presented in Miller units. PAO1 ?; PAO1 glpR ?. The results are representative of 2 experiments with standard error bars. Figure 2.3b Effect of glpR mutation on glcB expression in PAO1. ?- galactosidase activity is presented in Miller units. PAO1 ?; PAO1 glpR ?. The results are representative of 2 experiments with standard error bars. 0 200 400 600 800 1000 1200 1400 1600 1800 0.1 1 10 ?-Galact osid ase Act ivit y (M iller Unit s) OD 600 0 200 400 600 800 1000 1200 1400 1600 0.1 1 10 ?-Galact osid ase Act ivt y (M iller Un its) OD 600 35 Expression of glpR is higher in FRD1 compared to PAO1 As previously shown, a mutation in glpR had no effect on ICL and MS activity in FRD1 but resulted in increased ICL and MS activity in PAO1. A simple explanation for this observation would be an altered glpR in FRD1 due to a mutation. However, sequence analysis of the FRD1 glpR gene revealed no changes that would indicate that it is defective. To verify that glpR is unaltered in FRD1, glpR::lacZ and glpD::lacZ transcriptional fusions were constructed to compare the expression of these genes between FRD1 and PAO1. In L-broth, glpR::lacZ expression was increased in FRD1 compared to PAO1 and glpD::lacZ expression was decreased in FRD1 compared to the acute isolate PAO1 (Figures 2.4 and 2.5). A defective glpR would no longer be able to repress the expression of glpD. Therefore, high ICL and MS activity in FRD1 is not caused by reduced expression of glpR. We also considered whether high internal glycerol- 3-phosphate (G3P) concentrations in FRD1 would relieve repression of the genes potentially regulated by GlpR, including aceA and glcB. However, this is not supported by the reduced expression of glpD in FRD1 compared to PAO1. A simple explanation for the lack of a GlpR effect on the glyoxylate pathway in FRD1 is the loss of a major regulator for this pathway in FRD1 that overshadows the small effects of GlpR. 36 Figure 2.4 Expression of glpR::lacZ in PAO1 and FRD1. PAO1 ?; FRD1 ?. The results are representative of 2 experiments with standard error bars. Figure 2.5 Expression of glpD::lacZ in PAO1 and FRD1. PAO1 ?; FRD1 ?. The results are representative of 3 experiments with standard error bars. 0 200 400 600 800 1000 1200 0.1 1 10 ?-Galact osid ase Act ivit y (M iller Unit s) OD600 0 2000 4000 6000 8000 10000 12000 0.1 1 10 ?-Galact osid ase Act ivit y (M iller Un its) OD600 37 Growth on glycerol induces expression of glcB in PAO1 Expression of glpR is not substantially altered in FRD1 and does not appear to be responsible for increased ICL and MS activity in FRD1. However, GlpR does regulate the glyoxylate pathway in PAO1. Schweizer et. al. in 1996 previously reported that the glp regulon is induced by glycerol or glycerol-3-phosphate (G3P). Induction of aceA or glcB by the presence of glycerol or G3P in the growth medium would provide additional evidence of GlpR?s role in the regulation of the glyoxylate pathway. We attempted to measure aceA expression in PAO1 grown on G3P, however, in contrast to Schweizer?s findings, PAO1 was unable to utilize G3P as a sole carbon source (Figure 2.6) and the addition of G3P to L-broth had no effect on aceA::lacZ expression (data not shown). PAO1 was able to utilize glycerol as a carbon source when supplemented with 0.1% casamino acids. Therefore, we compared the expression of aceA and glcB in PAO1 grown in L-broth versus minimal medium with glycerol as a carbon source. Growth on glycerol increased expression of glcB in PAO1 compared to L-Broth but there was no difference in aceA expression between the two carbon sources (Figure 2.7). Therefore, GlpR appears to regulate aceA and glcB differently and has a more dramatic effect on glcB expression and MS activity. 38 Figure 2.6 Growth of PAO1 on glycerol-3-phosphate. Cultures were Grown for 22 hours 20mM glycerol-3-phosphate. Figure 2.7 aceA and glcB expression in PAO1 in LB vs. glycerol. Cultures were grown overnight in L-Broth or 20mM glycerol supplemented with 0.1% casamino acids. 0 0.2 0.4 0.6 0.8 1 0 5 8 13 14 18 20 22 A 600 Time (h) 0 200 400 600 800 1000 1200 1400 ?-Galact osid ase Act ivit y (M iller Unit s) PAO1 aceA::lacZ PAO1 glcB::lacZ 39 GlpR binds to the glcB promoter E. coli GlpR was shown previously to bind a consensus sequence forming an inverted repeat (Weissenborn and Larson, 1992) (Figure 2.8a). Similar sequences to the E. coli GlpR binding site were identified upstream of GlpR regulated genes in P. aeruginosa (Schweizer et. al. 1996). However, these sequences were never verified to bind to P. aeruginosa GlpR. Using a putative consensus sequence we generated from known GlpR regulated genes in P. aeruginosa, we identified several potential GlpR binding sites upstream of the glcB promoter (Fig 2.8b). To determine whether P. aeruginosa GlpR binds the glcB promoter, we fused GlpR to a hexa histidine-tag at the C-terminal end and purified the protein from E. coli grown in L-broth (Figure 2.9 a-b). The purified protein was tested in a gel-mobility shift assay with a 60 nucleotide fragment of the glcB promoter containing a putative site (Figure 2.10a). As expected the Gel- shift assay showed that GlpR binds to the glcB promoter and that the complex can de dissociated in a competition reaction containing 100X unlabeled DNA containing the putative GlpR binding site (Figure 2.10b). In addition, no interaction was observed between the purified GlpR and the control DNA lacking the putative GlpR binding site (2.10c), which indicates that the binding between GlpR and the glcB promoter is specific. 40 TG[TTTTTAGATTTATCTGGAACAAAGTACAGTTTTTTTGCGAACATTGAGCC TGGCCAACG]TGACCGTGAAGCGTCATCCAGTCGTAACGCGACGCGTAACCA CTGATTTTTCCCGCGGCATCATGTAGTATGCCGCGGCTCGGACTACAAGGCC GTGCGGCCCGGGTCCAGAGCTGGTCTAGAGCAGAGTGAGGCAAACA ATG glcB 1 M glpR > Figure 2.8 Comparison of putative glpR binding sites. A. The E. coli consensus half site (E.c.) and putative half sites from GlpR regulated genes in P. aeruginosa (P.a.) are given. W=A or T. B. The upstream non-coding sequence of glcB is presented. Putative GlpR binding sites are indicated in bold. Sequence used for gel shift assay is in brackets. A B glcB 2 41 Figure 2.9a Overexpression of the 28 kDa protein GlpR in E. coli. GlpR from P. aeruginosa was overexpressed in E. coli using the T7 vector pET28-b (Merck, Rockland, MA) in two different clones. Cultures were grown in L-broth and induced with 1mM IPTG. Proteins were separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue. Figure 2.9b Purification of the 28 kDa protein GlpR in E. coli. GlpR from P. aeruginosa was overexpressed in E. coli using the T7 vector pET28-b. GlpR was induced for 4 hours. kDa 40- 25- Induction (hrs) M 0 2 4 6 0 2 4 6 28 kDa kDa 40- 25- 15- 28 kDa M Uninduced 4hr Purified Clone 1 Clone 2 42 Figure 2.10 Gel shift assays using the putative GlpR binding sites. a). Gel shift assays using a glcB fragment. b). Competition assay using 100X unlabeled probe. c). Negative control. The amounts of GlpR added to the reaction mixtures are indicated above each lane. 0 2 ug 4 ug 6 ug 8 ug A C B 43 Discussion Understanding the mechanisms pathogens use to maintain chronic infections is necessary in order to develop successful therapeutic approaches that target these mechanisms. ICL, the first enzyme unique to the glyoxylate pathway is required for P. aeruginosa virulence in rat lungs (Lindsey et. al. 2008). ICL is also required for the optimal production of alginate and hydrogen cyanide (Lindsey et. al. 2008 and Hagins et. al. 2009). To date, transcriptional regulators of the glyoxylate pathway in P. aeruginosa have not been identified with the exception of RpoN?s indirect role in the regulation of this pathway (Hagins et. al. 2010). In an effort to determine the mechanism of deregulation of ICL in FRD1, we focused on the contribution of GlpR in the regulation of the glyoxyate pathway because GlpR controls glycerol metabolism. Glycerol and fatty acids are liberated from membrane lipids by the action of phospholipases, and both compounds serve as important carbon sources for P. aeruginosa during infection. We predicted that efficient catabolism of both substrates may require coordination of some metabolic pathways including the glyoxylate pathway. As shown in this study, ICL and MS activity were induced in a PAO1 glpR mutant of P. aeruginosa. Increased enzymatic activity of ICL and MS in PAO1 correlated with increased expression of aceA and glcB, respectively. These results suggest that GlpR plays a role in negative regulation of the glyoxylate pathway similar to its effect on the glp regulon in the absence of glycerol or glycerol-3-phosphate. To determine if GlpR regulated glcB directly we conducted an electrophoretic mobility gel shift assay using a His-tagged GlpR and promoter sequences derived from glcB. As expected, GlpR binds to the glcB promoter that contains putative GlpR binding sites. In contrast, we were unable to identify a GlpR consensus site in the aceA promoter. Therefore, GlpR 44 regulates glcB directly and aceA via another mechanism. These results are reflected by expression of the glcB and aceA genes in a glpR mutant. While glcB::lacZ shows increased expression throughout a growth cycle in the glpR mutant compared to the parental strain, aceA::lacZ expression is only affected in late stationary phase. Soh et. al. (2001) also noted high levels of ICL activity in late cultures of Streptomyces clavuligerus during growth on 0.5% - glycerol, which may suggest that ICL plays an important physiological role when oxygen availability is limited (Soh et. al. 2001). In addition, Wayne et. al. (1981) reported a five-fold increase in ICL activity in anaerobic Mycobacterium tuberculosis cultures that were grown over a 28 day period. Results from our lab (Chapter 3) demonstrated that FRD1 shows a growth advantage on glycerol compared to PAO1. This growth advantage correlated with the overproduction of the exopolysaccharide alginate which provides an oxygen-limited growth environment for FRD1. The CF lung is comprised of various oxygen rich and poor niches. Therefore, P. aeruginosa within the CF lung would have to adapt to utilizing available nutrients under these conditions, including limited oxygen availability (Hoffman et. al. 2010). Taken together, these findings suggest that microanaerobic growth conditions might be necessary for the efficient catabolism of glycerol by P. aeruginosa. Thus, GlpR?s role in P. aeruginosa may include activating genes during anaerobic conditions including activation of aceA. In addition, some bacteria use an alternate set of genes for catabolizing glycerol or G3P during anaerobic conditions. For example, the glpA operon in E. coli encodes an anaerobic glycerol-3-phosphate dehydrogenase that is activated during anaerobic growth of G3P (Iuchi et. al. 1990). However, a glpA homolog in P. aeruginosa has yet to be identified. In addition, anaerobic catabolism of glycerol by P. aeruginosa has not been demonstrated or characterized to date. 45 In summary, we present evidence that GlpR, a transcriptional regulator involved in glycerol utilization in P. aeruginosa, also regulates malate synthase, an enzyme unique to the glyoxylate pathway. We suggest that coordinate regulation of glyoxylate and glycerol may be advantageous to P. aeruginosa during catabolism of lipids. However, further investigation is required to reveal the benefits of this process. 46 References Bragonzi, A., M. Paroni, A. Nonis, N. Cramer, S. Montanari, J. Rejman, C. Di Serio, G. D?ring, and B. T?mmler. 2009. Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence. American Journal of Respiratory and Critical Care Medicine 180:138-145. Chambers, D., F. Scott, R. Bangur, R. Davies, a Lim, S. Walters, G. Smith, T. Pitt, D. Stableforth, and D. Honeybourne. 2005. Factors associated with infection by Pseudomonas aeruginosa in adult cystic fibrosis. The European Respiratory Journal?: Official Journal of the European Society for Clinical Respiratory Physiology 26:651-656. Davis, R.W., Botstein, D., and Roth, J.R. 1980. Advanced Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Dunn, M. F., J. a Ram?rez-Trujillo, and I. Hern?ndez-Lucas. 2009. Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology (Reading, England) 155: 3166-3175. Hagins, J. M., R. Locy, and L. Silo-Suh. 2009. Isocitrate lyase supplies precursors for hydrogen cyanide production in a cystic fibrosis isolate of Pseudomonas aeruginosa. Journal of Bacteriology 191:6335-6339. Hagins, J. M., J. A. Scoffield, S.-J. Suh, and L. Silo-Suh. 2010. Influence of RpoN on isocitrate lyase activity in Pseudomonas aeruginosa. Microbiology 156:1201-1210. Hoffman, L. R., A. R. Richardson, L. S. Houston, H. D. Kulasekara, W. Martens-Habbena, M. Klausen, J. L. Burns, D. a Stahl, D. J. Hassett, F. C. Fang, and S. I. Miller. 2010. Nutrient availability as a mechanism for selection of antibiotic tolerant Pseudomonas aeruginosa within the CF airway. PLoS Pathogens 6:e1000712. Iuchi, S., S. T. Cole, and E. C. Lin. 1990. Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control. Journal of Bacteriology 172:179-184. Jiang, P., and J. E. Cronan. 1994. Inhibition of Fatty Acid Synthesis in Escherichia coli in the Absence of Phospholipid Synthesis and Release of Inhibition by Thioesterase Action. Journal of Bacteriology 176: 2814-2821. Lindsey, T. L., J. M. Hagins, P. a Sokol, and L. a Silo-Suh. 2008. Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase. Microbiology (Reading, England) 154:1616-1627. 47 Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2002. Lung Infections Associated with Cystic Fibrosis. Infection and Immunity. 15:194-222. Mahenthiralingam, E., Campbell, E. Foster, J. Lam, D. Speert. 1996. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol 34: 1129?1135. McCowen, S. M., P. V. Phibbs, and T. W. Feary. 1981. Glycerol catabolism in wild-type and mutant strains of Pseudomonas aeruginosa. Curr. Microbiol. 5:191?196. Miller, J.H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Miller, R.M., Tomaras, A.P., Barker, A.P., Voelker, D.R., Chan, E.D., Vasil, A.I., and Vasil, M.L. 2008. Pseudomonas aeruginosa twitching motility-mediated chemotaxis towards phospholipids and fatty acids: specificity and metabolic requirements. J. Bacteriol. 190: 4038? 4049. Palmer, K.L., Mashburn, L.M., Singh, P.K., and Whiteley, M. 2005. Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology. J. Bacteriol. 187: 5267?5277. Schobert, M., and D. Jahn. 2010. Anaerobic physiology of Pseudomonas aeruginosa in the cystic fibrosis lung. International Journal of Medical Microbiology. 300:549-556. Schweizer, H. and C. Po. 1996. Regulation of Glycerol Metabolism in Pseudomonas aeruginosa: Characterization of the glpR Repressor Gene. Journal of Bacteriology. 178: 5215? 5221. Silo-Suh, L., S.-J. Suh, P. a Sokol, and D. E. Ohman. 2002. A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma- 22) and RhlR contribute to pathogenesis. Proceedings of the National Academy of Sciences of the United States of America 99:15699-15704. Silo-Suh, L., Suh, S.J., Phibbs, P.V., and Ohman, D.E. 2005. Adaptations of Pseudomonas aeruginosa to the cystic fibrosis lung environment can include deregulation of zwf, encoding glucose- 6-phosphate dehydrogenase. J. Bacteriol. 187: 7561?7568. Smith, E., E.Buckley, D. Wu, Z. Saenphimmachak, C. Hoffman, L. D?Argenio, D. Miller, S. Ramsey, and B. Speert. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci. 103: 8487?8492. Soh, B., and P. Loke. 2001. Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585. Acta (BBA)-Gene Structure and Expression 1522:112-117. 48 Son, M., W. Matthews, Y. Kang, D. Nguyen, and T. Hoang. 2007. In vivo Evidence of Pseudomonas aeruginosa Nutrient Acquisition and Pathogenesis in the Lungs of Cystic Fibrosis Patients. Infection and Immunity. 75: 5313?5324. Struelens, M., J.Schwam, V. Deplano, and A. Baran. 1993. Genome macrorestriction analysis of diversity and variability of Pseudomonas aeruginosa strains infecting cystic fibrosis patients. J Clin Microbiol. 31: 2320?2326. Soh, B., and P. Loke. 2001. Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585. Acta (BBA)-Gene Structure and Expression 1522:112-117. Suh, S. J., L. Silo-Suh, D. E. Woods, D. J. Hassett, S. E. West, and D. E. Ohman. 1999. Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. Journal of Bacteriology. 181:3890-3897. Suh, S.J., Silo-Suh, L.A., and Ohman, D.E. 2004. Development of tools for the genetic manipulation of Pseudomonas aeruginosa. J. Microbiol. Methods. 58: 203?212. Terry, J. M., S. E. Pi?a, and S. J. Mattingly. 1992. Role of energy metabolism in conversion of nonmucoid Pseudomonas aeruginosa to the mucoid phenotype. Infection and Immunity 60:1329-1335. Wayne, L. G., and K. Y. Lin. 1982. Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions. Infection and Immunity 37:1042-1049. Weissenborn, D., N. Wittekindtn, and T. Larson. 1992. Structure and Regulation of the glpFK Operon Encoding Glycerol Diffusion Facilitator and Glycerol Kinase of Escherichia coli K-12. Journal of Bacteriology. 267: 6122-6131. Williams, S. G., J. a Greenwood, and C. W. Jones. 1994. The effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of Pseudomonas aeruginosa. Microbiology (Reading, England) 140:2961-2969. 49 Chapter 3 The Chronic Cystic Fibrosis Isolate, FRD1, is Enhanced for Growth on Glycerol Abstract Pseudomonas aeruginosa is the major etiologic agent of chronic pulmonary infections in cystic fibrosis (CF) patients. During establishment of chronic infections, the pathogen develops various adaptation strategies including redirecting metabolic pathways to utilize readily available nutrients present in the host environment. The airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa including phosphotidylcholine, a major component of host cell membranes. P. aeruginosa can degrade phosphotidylcholine to glycerol and fatty acids to increase the availability of glycerol in the CF lung. The goal of this study was to characterize and compare glycerol metabolism between an acute and a chronic isolate of P. aeruginosa. We show here that the chronic CF isolate, FRD1, displays a growth advantage on glycerol compared to the acute isolate PAO1. The enhanced ability of FRD1 to metabolize glycerol is correlated with alginate overproduction because FRD1 algT and algD mutants were unable to grow on glycerol. In addition, the alginate producing PAO1 mucA mutant showed increased growth on glycerol compared to the parent strain. Thus, alginate appears to be important for optimal glycerol utilization by P. aeruginosa. Finally, the addition of glycerol to L-broth enhanced alginate production by PAO1 suggesting that the CF lung may provide a 50 nutritional environment that promotes alginate production by P. aeruginosa even before the bacteria convert to the mucoid phenotype. Introduction Pseudomonas aeruginosa is the leading cause of lung dysfunction and death in cystic fibrosis (CF) patients (Bragonzi et. al. 2009). CF patients acquire P. aeruginosa from the environment at an early age and this bacterium establishes persistent lung infections during the patient?s lifetime (Bragonzi et. al. 2009; Mahenthiralingam et. al. 1996). P. aeruginosa produces several virulence factors that facilitate the survival of P. aeruginosa during infection, including phospholipases, lipases, proteases, and exotoxins. During chronic infection in the CF lung, P. aeruginosa undergoes several phenotypic and genetic adaptations to persist in the lung and evade clearance by the host immune system and antibiotic therapy. Some of these adaptations include loss-of-function mutations, deregulation of metabolic genes, loss of motility, antibiotic resistance, and overproduction of the exopolysaccharide alginate (Chambers et. al. 2005; Hagins et. al. 2011; Hoffman et. al. 2009; Lindsey et. al. 2008; Silo-Suh et. al. 2005; Smith et. al. 2006). These adaptations appear to not only be necessary for avoiding clearance in the lung, but also for the acquisition and catabolism of nutrients found within the CF lung (Son et. al. 2007). Current evidence suggests that P. aeruginosa and other bacteria that colonize the CF lung grow within the airway sputum (Palmer et. al. 2005; Son et. al. 2007). CF sputum contains a complex mixture of host secretions, inflammatory components, dead host and bacterial cells, nucleic acids, and bacterial products, and acts as a surface for biofilm development. Sputum also serves as a source of nutrition for colonizing bacteria (Palmer et. al. 2005). In the lung, nutrients 51 such as glycerol become available carbon sources due to the degradation of host cell membranes. Phosphotidylcholine, a major component of both membranes and lung surfactant, is degraded by P. aeruginosa to 1,2-diacylglycerol and phosphorylcholine using phospholipase C (Lyczak et. al. 2002; Terry et. al. 1992; Williams et. al. 1994). 1,2-diacylglycerol is then hydrolyzed to glycerol and fatty acids by lipases. The action of phospholipase C and lipases increases the availability of glycerol to P. aeruginosa as a potential carbon source (Lyczak et. al. 2002; Palmer et. al. 2005; Son et. al. 2007; Terry et. al. 1992; Williams et. al. 1994). Transcriptome studies reveal that amino acids and lipids are probable growth substrates for chronic P. aeruginosa isolates growing within CF sputum, while acute wound isolates primarily use amino acids during growth on CF sputum (Palmer et. al. 2005; Son et. al. 2007). Although amino acids and lipids appear to be a primary source of nutrition for P. aeruginosa in the lung, studies also indicate that genes responsible for glycerol metabolism are deregulated in some CF isolates of P. aeruginosa (Son et. al. 2007). Constitutive expression of genes involved in glycerol metabolism suggests that glycerol could be an important nutrient for P. aeruginosa during chronic infection (Son et. al. 2007). Unfortunately, glycerol utilization by P. aeruginosa is poorly understood in chronic CF infection. In this study, we examined glycerol metabolism in a CF and non-CF isolate of P. aeruginosa. We determined that a CF isolate of P. aeruginosa, FRD1, displays a growth advantage on glycerol compared to the wound isolate, PAO1. This growth advantage on glycerol correlates with the mucoid phenotype of FRD1, which results from the overproduction of alginate. This is supported by a growth analysis of CF P. aeruginosa isolates recovered from the lungs of a single patient. Many of the mucoid isolates from this collection show a growth 52 advantage on glycerol comparable to a wound isolate of P. aeruginosa. The mechanism by which alginate production facilitates growth on glycerol is presently unclear. Materials and Methods Bacterial strains, plasmids, and media. Bacterial strains used in this study are listed in Table 3.1. Unless otherwise indicated, bacteria were cultured in L-broth (LB) or on L-agar at 37?C. No-carbon-E minimal medium (NCE) supplemented with 0.1% (w/v) casamino acids (CAA) was used to assay for growth on minimal media (Davis et al., 1980). Glycerol was used at a concentration of 20 mM with or without CAA supplementation. UV-Vis absorption spectra were recorded on a Shimadzu UV- 1601 Spectrophotometer using 1 cm path length cells. Antibiotics were purchased from Sigma- Aldrich (St. Louis, MO) and used at a concentration of 200 ?g gentamicin (Gm) ml-1. To examine growth over a 24 hour period, cultures were grown in 24 or 96 well microtiter plate and monitored at A600 with a BioTek Synergy HT plate reader (BioTek, Winooski, VT). Biochemical assays. Alginate was isolated from P. aeruginosa culture supernatants that were dialyzed against distilled water as previously described (Suh et al., 1999), and the alginate level (i.e. uronic acid) was quantified by the carbazole method (Knutson & Jeanes, 1968) using Macrocystis pyrifera alginate (Sigma-Aldrich) as a standard. ?-galactosidase assays were preformed as described by Miller (Miller, 1972). 53 Strain or Plasmid Genotype, relevant characteristics Source FRD1 CF isolate, mucoid Ohman & Chakrabarty (1981) PAO1 Wound isolate, nonmucoid Holloway et al. (1979) FRD1 algT (LS586) FRD1 algT101::aacCI Silo-Suh et al. (2002) FRD1 algD (LS75) FRD1 algD101::aacCI Dr. Laura Silo-Suh PAO1 mucA (LS856) P3, P6, P13, P18-P19, P22, P24-P27 ENV2, ENV10, ENV46, ENV54 CF Isolates FRD1 algD::lacZ (SS934) PAO1 mucA101::aacCI Clinical Isolates Environmental Isolates Sequential Isolates algD transcriptional fusion (pSS223) Dr. Laura Silo-Suh Dr. Laura Silo-Suh Mahenthiralingam et al. (1994) Dr. Laura Silo-Suh Dr. Sang-Jin Suh PAO1 algD::lacZ (SS956) algD transcriptional fusion (pSS223) Dr. Sang-Jin Suh Table 3.1 Bacterial strains and plasmids. Abbreviations used for genetic markers are described by Holloway et al. (1979). Alternate strain names are shown in parentheses. Results The chronic CF isolate FRD1 displays a growth advantage on glycerol compared to the acute wound isolate PAO1. We tested the ability of FRD1 and PAO1 to use glycerol as a sole carbon source. FRD1 demonstrated an enhanced ability to grow on glycerol as a sole carbon source compared to PAO1 (Figure 3.1a). Interestingly, the addition of 0.1% casamino acids restored PAO1 growth on glycerol and boosted the growth of FRD1 (Figure 3.1b). Neither strain showed significant growth on 0.1% casamino acids alone (Figure 3.2). We considered that FRD1?s enhanced growth on glycerol might involve upregulation of the glp operon that encodes for various proteins involved in the transport and catabolism of glycerol. However, glpR expression (glycerol repressor) was upregulated and glpD expression (glycerol dehydrogenase) was downregulated in FRD1 compared to PAO1 (Chapter 2 results), which is inconsistent with that scenario. 54 Figure 3.1 FRD1 Displays a Growth Advantage on Glycerol (a) FRD1 and PAO1 in minimal medium with 20mM glycerol. (b) FRD1 in glycerol ; FRD1 in glycerol + 0.1% CAA PAO1 in glycerol ; PAO1 in glycerol + 0.1% CAA All cultures were grown for 24 hours. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 4 7 10 13 16 19 21 22 23 A 600 Time (hr) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 2 6 8 11 13 15 18 20 23 A 600 Time (hr) b a 55 Figures 3.2a and b. FRD1 and PAO1 grown on 0.1% casamino acids. (a) FRD1 (b) PAO1 Cultures were grown in minimal medium with 0.1% CAA for 22 hours. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 2 4 6 8 10 12 14 16 18 20 22 A 600 Time (hr) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 2 4 6 8 10 12 14 16 18 20 22 A 600 Time (hr) a b 56 Utilization of glycerol by clinical, environmental, and sequential CF isolates of P. aeruginosa To determine if enhanced growth on glycerol is common in other isolates of P. aeruginosa, we tested the ability of several clinical (non-CF acute clinical isolates), environmental, and sequential CF isolates of P. aeruginosa for the ability to use glycerol as a sole carbon source. The acute clinical and environmental isolates displayed growth similar to PAO1 on glycerol (Figure 3.3). All of the clinical and environmental isolates used in this study were non-mucoid. In contrast, some of the sequential isolates recovered from CF patients showed increased growth on glycerol at comparable levels to FRD1 (Figure 3.4) and some of these isolates were mucoid. The mucoid phenotype is caused by an overproduction of the exopolysaccharide alginate. These results suggest that the ability to efficiently utilize glycerol is an adapted phenotype that arises in chronic CF isolates of P. aeruginosa and may be influenced by the presence of alginate. 57 Figure 3.3 Survey of glycerol utilization in P. aeruginosa isolates. Cultures of P. aeruginosa isolates were grown in minimal medium with 20 mM glycerol for 24 hours in a 24-well microtiter plate. Values represent the average of 3 experiments. (? standard error). FRD1 ; PAO1 ; Clinical Isolates ; Environmental Isolates ; Lanes 1, P3; 2, P6; 3, P13; 4, P18; 5, P19; 6, P22; 7, P24; 8, P25; 9, P26; 10, P27; 11, ENV2; 12, ENV10; 13, ENV46;14, ENV54; 15, C1; 16, C2; 17, C3; 18. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 A 600 F P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 58 Figure 3.4 Survey of glycerol utilization in sequential P. aeruginosa CF isolates. Isolates were grown in minimal medium with 20 mM glycerol for 24 hours in a 24- well microtiter plate. Values represent the average of 3 experiments. (? standard error). FRD1 , PAO1 , * = mucoid. * * * * * * * * * * * * * * * * * * * * * 0 0.2 0.4 0.6 0.8 1 1.2 A 600 F P Patient 1 Patient 12 Patient 13 Patient 16 Patient 17 Patient 19 59 Alginate enhances P. aeruginosa growth on glycerol FRD1 algT and FRD1 algD mutants were tested for growth on glycerol to determine if AlgT or alginate plays a role in glycerol metabolism. algT encodes for an alternative sigma factor that has a global effect including activation of the alginate biosynthetic genes. The alginate biosynthetic genes are located in a single operon beginning with algD, which encodes for GDP-mannose dehydrogenase. Disruption of algT or algD results in the loss of alginate and the mucoid phenotype by FRD1. Alternatively, deletion of mucA, the anti-sigma factor of AlgT, produces a mucoid phenotype in PAO1. As shown in Figure 3.5, the FRD1 algT and algD mutants were defective for growth on glycerol compared to the parental strain. In contrast, activation of algT in the PAO1 mucA mutant enhanced its growth on glycerol compared to wild- type PAO1 (Figure 3.6). These results suggest that alginate influences P. aeruginosa growth on glycerol. A known environmental factor that affects glycerol metabolism in bacteria is oxygen availability. Some studies suggest that glycerol might be better metabolized during anaerobic or microaerophilic growth conditions compared to aerobic growth (Durin et. al. 2009). The presence of alginate in the growth medium may provide an oxygen-limited environment for FRD1 and enhance glycerol catabolism. It is tempting to speculate that overproduction of alginate in the CF lung helps P. aeruginosa catabolize glycerol in this environment. 60 Figure 3.5 algD and algT are required by FRD1 for optimal growth on glycerol. FRD1 ; FRD1 algD ; FRD1 algT . Cultures were grown in minimal medium with 20 mM glycerol for 24 hours. Figure 3.6 PAO1mucA mutants and wild-type PAO1. PAO1 ; PAO1 mucA . Cultures were grown in minimal medium with 20 mM glycerol for 24 hours. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 7 9 13 16 19 22 23 A 600 Time (hrs) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 4 6 8 11 13 15 17 21 23 A 600 Time (hrs) 61 Glycerol promotes alginate production by PAO1 To further study the connection between glycerol metabolism and alginate, we determined whether the addition of glycerol to L-broth would increase the production of alginate by PAO1. Comparatively, FRD1 produces copious amount of alginate when grown in L-broth (Chapter 4), whereas PAO1 produces negligible amounts. In this experiment, we grew FRD1 and PAO1 on L-broth with the addition of various amounts of glycerol. Increased alginate production correlated with the addition of glycerol for both isolates (Figures 3.7 and 3.9). These results show that glycerol promotes the production of the virulence determinant, alginate in FRD1 and PAOl. While growth on glycerol would likely produce precursors for alginate production, such as DHAP and fructose-6-phosphate (Figure 3.11), efficient utilization of F-6-P for alginate production would require activation of the alginate biosynthetic operon. Therefore, we compared the expression of an algD::lacZ fusion in FRD1 and PAO1 in the presence and absence of glycerol in L-broth. As shown in Figures 3.8 and 3.10, algD::lacZ expression increased with the addition of glycerol in FRD1 and PAO1. 62 Figure 3.7 Glycerol increases alginate production in FRD1. Cultures were grown overnight in L-broth with the addition of varying concentration of glycerol. Values represent the average of 3 experiments. (? standard error). Figure 3.8 Expression of algD in FRD1. Cultures were grown overnight in L- broth with the addition of varying concentrations of glycerol. ?- galactosidase activity is presented in Miller units. Values represent the average of 3 experiments. (? standard error). 0 400 800 1200 1600 2000 2400 0% 1% 2% 3% Algin ate mg mL -1 % Glycerol 0 4000 8000 12000 16000 0% 1% 2% 3% ?-Gal acto sid ase Activity (M iller Un its) % Glycerol 63 Figure 3.9 Glycerol increases alginate production in PAO1. Cultures were grown overnight in L-broth with the addition of varying concentrations of glycerol. Values represent the average of 3 experiments. (? standard error). Figure 3.10 Expression of algD in PAO1. Cultures were grown overnight in L- broth with the addition of varying concentrations of glycerol. ?- galactosidase activity is presented in Miller units. Values represent the average of 3 experiments. (? standard error). 0 50 100 150 200 250 0% 1% 2% 3% Alg inate m g m L -1 % Glycerol 65 70 75 80 85 90 95 0% 1% 2% 3% ?-Galact osid ase Act ivit y (M iller Unit s) % Glycerol 64 Figure 3.11 Pathway for alginate biosynthesis from glycerol. Discussion The ability to acquire nutrients from the host in vivo is essential for chronic P. aeruginosa isolates growing within the CF lung. CF sputum contains various nutrients that are potential carbon sources, including glycerol (Son et. al. 2007). In this study, we tested the ability of several P. aeruginosa isolates to utilize glycerol as a sole carbon source. The chronic CF isolate, FRD1, displayed a growth advantage on glycerol compared to the wound isolate, PAO1. The environmental and clinical isolates tested in this study were also deficient in their ability to use glycerol as a sole carbon source. However, several P. aeruginosa isolates recovered from CF patients displayed an enhanced ability to grow on glycerol, similar to that of FRD1. The ability to grow efficiently on glycerol correlated with the mucoid phenotype associated with chronic CF 65 isolates. Therefore, the overproduction of alginate may provide an advantage to chronic P. aeruginosa isolates for glycerol catabolism. In addition, glycerol catabolism can produce carbon precursors for alginate production by P. aeruginosa. The conversion of P. aeruginosa to the mucoid phenotype in the CF lung, which results from alginate overproduction, is associated with decreased lung function and an improved ability to resist antibiotics and phagocytosis (Bragonzi et. al. 2009; Mahenthiralingam et. al. 1996). We show here that alginate facilitates the acquisition of nutrients like glycerol, and in turn, glycerol fuels the production of alginate. The CF airway contains microenvironment pockets ranging from aerobic to anaerobic (Hassett et. al. 2009; Schobert et. al. 2010). P. aeruginosa, which normally prefers aerobic respiration, is able to persist in anaerobic environments because it can utilize NO3- or NO2- as terminal electron acceptors, and these nitrogen sources are abundant in the CF airway (Hoffman et. al. 2010; Schobert et. al. 2010). Under these conditions, P. aeruginosa is able to produce alginate, which also contributes to hypoxic conditions by restricting the influx of oxygen in the airway (Hasset, 1996). The efficient utilization of glycerol by P. aeruginosa appears to be dependent upon the presence of amino acids, as demonstrated by the presence of casamino acids in the growth medium in PAO1, or the production of alginate in FRD1. Although we did not measure oxygen concentrations, we speculate that PAO1 cultures grown with the addition of glycerol become microaerophilic due to the increased amount of alginate produced (Hasset, 1996). In addition to glycerol, other nutrients may be acquired anaerobically in the CF lung including arginine, and the arginine fermentation pathway is induced in this environment (Palmer et. al. 2005; Son et. al. 2007). 66 CF sputum contains an abundant source of fatty acids and catabolism of these nutrients would produce high concentrations of glycerol (Son et. al. 2007). P. aeruginosa derivatives that are capable of utilizing glycerol would have a growth advantage over other bacteria present in the CF lung. Interestingly, some of the genes responsible for glycerol metabolism are upregulated in P. aeruginosa isolates that have been recovered from the CF lung (Son et. al. 2007). In that study, GlpD (glycerol 3-phosphate dehydrogenase), was required for in vivo degradation of phosphatidylcholine (PC), which is the source of many host derived nutrients. Furthermore, glpD and other glp genes (glycerol uptake facilitator gene (glpF), regulator gene (glpR), kinase gene (glpK), and the glycerol-3-phosphate transporter gene (glpT)), were induced when PAO1 was grown on PC (Son et. al. 2007). Efficient utilization of host nutrients is essential for P. aeruginosa isolates growing within the CF lung. This study demonstrates that alginate helps facilitate the acquisition of certain host derived nutrients by P. aeruginosa and assigns another role for alginate in P. aeruginosa virulence. Further studies that analyze the metabolic capabilities of CF P. aerugionsa will provide insight as to how to target these adaptation strategies. 67 References Applebee, M. K., A. R. Joyce, T. M. Conrad, D. W. Pettigrew, and B. ?. Palsson. 2011. Functional and metabolic effects of adaptive glycerol kinase (GLPK) mutants in Escherichia coli. The Journal of Biological Chemistry 286: 23150-23159. Bragonzi, A., M. Paroni, A. Nonis, N. Cramer, S. Montanari, J. Rejman, C. Di Serio, G. D?ring, and B. T?mmler. 2009. Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence. American Journal of Respiratory and Critical Care Medicine. 180:138-145. Chambers, D., F. Scott, R. Bangur, R. Davies, a Lim, S. Walters, G. Smith, T. Pitt, D. Stableforth, and D. Honeybourne. 2005. Factors associated with infection by Pseudomonas aeruginosa in adult cystic fibrosis. The European Respiratory Journal?: Official Journal of the European Society for Clinical Respiratory Physiology. 26:651-656. Davis, R.W., Botstein, D., and Roth, J.R. 1980. Advanced Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Durnin, G., J. Clomburg, Z. Yeates, P. J. J. Alvarez, K. Zygourakis, P. Campbell, and R. Gonzalez. 2009. Understanding and Harnessing the Microaerobic Metabolism of Glycerol in Escherichia coli. Biotechnology. 103:148-161. Hagins, J., J. Scoffield, S.J. Suh, and L. Silo-Suh. 2011. Malate synthase expression is deregulated in the Pseudomonas aeruginosa cystic fibrosis isolate FRD1. Canadian Journal of Microbiology. 195:186-195. Hassett, D. J. 1996. Anaerobic production of alginate by Pseudomonas aeruginosa: alginate restricts diffusion of oxygen. Journal of Bacteriology. 178:7322-7325. Hoffman, L. R., A. R. Richardson, L. S. Houston, H. D. Kulasekara, W. Martens- Habbena, M. Klausen, J. L. Burns, D. a Stahl, D. J. Hassett, F. C. Fang, and S. I. Miller. 2010. Nutrient availability as a mechanism for selection of antibiotic tolerant Pseudomonas aeruginosa within the CF airway. PLoS Pathogens 6:e1000712. Lindsey, T. L., J. M. Hagins, P. Sokol, and L. a Silo-Suh. 2008. Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase. Microbiology. (Reading, England) 154: 1616-1627. Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2002. Lung Infections Associated with Cystic Fibrosis. Infection and Immunity. 15:194-222. Mahenthiralingam, E., Campbell, E. Foster, J. Lam, D. Speert. 1996. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J Clin Microbiol. 34: 1129?1135. 68 Palmer, K.L., Mashburn, L.M., Singh, P.K., and Whiteley, M. 2005. Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology. J. Bacteriol. 187: 5267?5277. Schobert, M., and D. Jahn. 2010. Anaerobic physiology of Pseudomonas aeruginosa in the cystic fibrosis lung. International Journal of Medical Microbiology. 300: 549-556. Silo-Suh, L., Suh, S.J., Phibbs, P.V., and Ohman, D.E. 2005. Adaptations of Pseudomonas aeruginosa to the cystic fibrosis lung environment can include deregulation of zwf, encoding glucose- 6-phosphate dehydrogenase. J. Bacteriol. 187: 7561?7568. Smith, E., E.Buckley, D. Wu, Z. Saenphimmachak, C. Hoffman, L. D?Argenio, D. Miller, S. Ramsey, and B. Speert. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 103: 8487?8492. Son, M., W. Matthews, Y. Kang, D. Nguyen, and T. Hoang. 2007. In Vivo Evidence of Pseudomonas aeruginosa Nutrient Acquisition and Pathogenesis in the Lungs of Cystic Fibrosis Patients. Infection and Immunity. 75: 5313?5324. Suh, S.J., Silo-Suh, L.A., and Ohman, D.E. 2004. Development of tools for the genetic manipulation of Pseudomonas aeruginosa. J. Microbiol. Methods, 58: 203?212. Terry, J. M., S. E. Pi?a, and S. J. Mattingly. 1992. Role of energy metabolism in conversion of nonmucoid Pseudomonas aeruginosa to the mucoid phenotype. Infection and Immunity. 60:1329-1335. Williams, S. G., J. a Greenwood, and C. W. Jones. 1994. The effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of Pseudomonas aeruginosa. Microbiology (Reading, England) 140:2961-2969. Wood, L. F., and D. E. Ohman. 2009. Use of cell wall stress to characterize sigma 22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa. Molecular Microbiology. 72:183-201. 69 Chapter 4 GlpR is Required for Virulence in a Chronic Isolate of Pseudomonas aeruginosa Abstract Pseudomonas aeruginosa is the major etiologic agent of chronic pulmonary infections in cystic fibrosis (CF) patients. During establishment of chronic infections, the pathogen develops various strategies, including altering the expression of virulence determinants, to adapt to the infection niche. Virulence factor production is controlled by a network of transcriptional regulators or alternative sigma factors. Factors that mediate the acquisition of certain carbon sources may also play a role in the expression of key virulence determinants that are involved in chronic CF infection. For this study we focused on characterizing the role of GlpR in virulence factor production by an acute (PAO1) and a chronic (FRD1) isolate of P. aeruginosa. GlpR is a transcriptional repressor that is required for glycerol metabolism in P. aeruginosa. The FRD1 glpR mutant was decreased for production of pyocyanin, pyoverdine and rhamnolipids compared to the parent strain. In the alfalfa seedling infection assay, the FRD1 glpR mutant was severely decreased in its ability to cause disease. Interestingly, the glpR mutation had no significant effect on PAO1 virulence. Finally, our data indicate that GlpR is involved in the emergence of persister cells. In summary, GlpR plays several important roles in the pathogenesis of CF P. aeruginosa. 70 Introduction Chronic infections caused by Pseudomonas aeruginosa are the major cause of lung dysfunction and mortality in cystic fibrosis (CF) patients. The ability of P. aeruginosa to cause or maintain an infection is dependent upon a large number of regulatory genes that control the expression of virulence determinants (Carterson et. al. 2003; Heurlier et. al. 2003; Juhas et. al. 2004; Suh et. al. 1999). P. aeruginosa produces several cell-associated and extracellular virulence factors (e.g., exotoxin A, exoenzyme S, cytotoxin, proteases, lipases, pyocyanin, rhamnolipids and phospholipases) that contribute to pathogenesis. However, the expression of these genes varies between acute and chronic infections (Bragonzi et. al. 2009; Govan, 1996; Nguyen et. al. 2006). Nutrient acquisition can also influence virulence factor production suggesting that some virulence factors may be coordinately regulated by nutritional sources. For example, Cryptococcus neoformans mutants that are unable to utilize glucose are severely reduced for virulence (Price et. al. 2011). Furthermore, genes responsible for arginine acquisition are required for the expression of several virulence genes in Streptococcus pneumonia (Kloosterman and Kuipers, 2011). As shown in Chapter 3, P. aeruginosa variants that efficiently catabolize glycerol arise and persist within the CF lung. Catabolism of glycerol enhances alginate production suggesting virulence of CF P. aeruginosa may depend upon the presence of this nutrient within the CF lung. In fact, glycerol metabolism can play a role in virulence factor production and virulence by bacteria. For example, the glpD gene, which encodes glycerol-3-phosphate oxidase, is required for toxicity and the production hydrogen peroxide by Mycoplasma pneumoniae (Hames et. al. 2008). In the same study, glpF and glpK, which encode for a glycerol facilitator and the glycerol kinase, respectively, were also necessary for pathogenicity (Hames et. al. 2008). GlpR is a 71 transcriptional regulator that negatively controls the genes required for glycerol metabolism in P. aeruginosa, which are located in the glp regulon (Schweizer et. al. 1996). The FRD1 glpR mutant displayed a white colony phenotype on agar plates in contrast to the blue-green color normally seen with the parental strain (data not shown). This suggested a loss of pyocyanin (blue fluorescence) and pyoverdine (green fluorescence) production. Therefore, we examined the role of GlpR in the production of various virulence determinants by P. aeruginosa. Materials and Methods Bacterial strains, plasmids, and media. Bacterial strains and plasmids used in this study are listed in Table 4.1. Unless otherwise indicated, bacteria were cultured in L-broth (LB) or on L-agar at 37?C. UV-Vis absorption spectra were recorded on a Shimadzu UV-1601 Spectrophotometer using 1 cm path length cells. Antibiotics were purchased from Sigma-Aldrich (St. Louis, MO) and used at the following concentrations in this study: Gentamicin at 200 ?g/ml and ofloxacin at 10 ?g/ml for P. aeruginosa. Alfalfa seedling infection assay. Seeds of alfalfa variety 57Q77, a wild-type strain not bred for pest resistance, were provided by Pioneer Hi-Bred International. The alfalfa seedling infection assay was conducted as previously described (Silo-Suh et al., 2002) with the following modifications: FRD1 and derivatives were inoculated onto wounded alfalfa seedlings using 105 colony forming units (CFU) per seedling while PAO1 and derivatives were inoculated using 104 CFU per seedling. Water agar plates containing inoculated seedlings were sealed with Parafilm and placed in a 72 30?C incubator without light. Disease symptoms were scored 6?7 days following inoculation by visual inspection. Seedlings with symptoms of infection were scored positive. FRD1, FRD1 glpR, PAO1 and PAO1 glpR were tested on 50 seedlings for each experiment. Data were expressed as the mean ? standard error and analyzed for significance using an ANOVA (InStat; Graph Pad Software). A value of P<0.05 was considered significant. Biochemical assays. Alginate was isolated from P. aeruginosa culture supernatants that were dialyzed against distilled water as previously described (Suh et al., 1999), and the alginate level (i.e. uronic acid) was quantified by the carbazole method (Knutson & Jeanes, 1968) using Macrocystis pyrifera alginate (Sigma-Aldrich) as a standard. Pyocyanin was purified and measured from 20 h cultures as described by Essar et al. (1990). Pyoverdine was measured as previously described by Suh et al., 1999 with several modifications. Briefly, P. aeruginosa was grown in Kings B medium for 16 to 17 h at 37?C with aeration. The culture supernatants were serially diluted in 10 mM Tris-HCl (pH 7.5) and excited at 400 nm, and the emission at 460 nm was recorded using Black 96 welled Costar plates read in a BioTek Synergy HT. Cyanide was assayed according to Carterson et al. (2004), as previously reported (Hagins et al. 2009), and normalized to CFU of bacteria recovered from each Pseudomonas Isolation Agar plate. Cyanide levels were quantified by comparison with KCN standards using the same protocol and presented as micromoles per 109 CFUs. Rhamnolipid was purified and measured as previously described (Du Plessis, 2005). 73 Persister cell assay. Persistence was measured by determining survival upon exposure to antibiotics in a time dependent manner as described by Mulchaly et. al. (2010). Briefly, 16 hour cultures of P. aeruginosa derivatives were washed, diluted, and inoculated into 3 mL?s of Mueller-Hinton Broth. Prior to exposure with oflaxicin, samples were washed and plated on Mueller-Hinton agar plates to calculate CFUs. Samples exposed to oflaxicin for 8 hours were treated in the same manner. Samples were allowed to grow on Mueller-Hinton plates for 48 hours before CFUs were calculated. Strain or Plasmid Genotype, relevant characteristics Source FRD1 CF isolate, mucoid Ohman et. al. (1981) PAO1 Wound isolate, nonmucoid Holloway et al. (1979) FRD1 glpR (JS134) PAO1 glpR (JS97) FRD1glpR101::aacCI PAO1glpR101::aacCI Chapter 2 Chapter 2 Table 4.1 Bacterial strains and plasmids. Abbreviations used for genetic markers are described by Holloway et al. (1979). Alternate strain names are shown in parentheses. Results GlpR is required for optimal pyoverdine and pyocyanin production by FRD1 Pyocyanin and pyoverdine are two quorum sensing-regulated virulence factors that are important for establishing infection (Govan et. al. 1996). We tested the effect of the glpR mutation on pyocyanin and pyoverdine production in PAO1 and FRD1. There was a three-fold 74 decrease in pyoverdine production and a two-fold decrease in pyocyanin production by the FRD1 glpR mutant compared to the parental strain (Figures 4.1 and 4.2). However, the PAO1 glpR mutant was slightly enhanced for the production of both products compared to its parental strain (Figures 4.1and 4.2). Therefore, both virulence factors are differentially regulated by GlpR in the chronic and acute isolates of P. aeruginosa. GlpR is required for optimal rhamnolipid production but not hydrogen cyanide production by FRD1 and PAO1 Rhamnolipids are quorum sensing regulated virulence factors that promote infiltration and adherence of P. aeruginosa in the CF airway (Zulianello et. al. 2006). Glycerol can provide intermediates for biosynthesis of rhamnolipids (Figure 4.5). However, in the absence of glycerol, upregulation of the biosynthetic genes for glycerol metabolism may divert intermediates away from rhamnolipid production. This scenario is consistent with our observation that the FRD1 glpR mutant did not produce detectable rhamnolipid under the conditions tested while the PAO1 glpR mutant was decreased two-fold for rhamnolipid production compared to the parental strain (Figure 4.3). Hydrogen cyanide (HCN) production is enhanced in some P. aeruginosa isolates recovered from the CF lung and HCN production is associated with decreased cellular functions (Ryall et. al. 2008; Hagins et. al. 2009). In contrast to the other virulence factors tested, there was a moderate increase in hydrogen cyanide production by the FRD1 glpR and PAO1 glpR mutants compared to their parental strains (Figure 4.4). 75 Figure 4.1 The effect of glpR on pyoverdine production in FRD1 and PAO1. Cultures were grown in L-broth overnight and assayed for pyoverdine production. Data represent the average of 3 experiments ? standard error. Figure 4.2 The effect of glpR on pyocyanin production in FRD1 and PAO1. Cultures were grown in L-broth overnight and assayed for pyocyanin. Data represent the average of 3 experiments ? standard error. 0 200 400 600 800 1000 Py ov erd ine (A 460 ) FRD1 FRD1 glpR PAO1 PAO1 glpR 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Py oc yan in (ug mL -1 ) FRD1 FRD1 glpR PAO1 PAO1 glpR 76 Figure 4.3 The effect of glpR on rhamnolipid production in FRD1 and PAO1. Cultures were grown in L-broth or L-broth + 1% glycerol overnight and assayed for rhamnolipid production. Data represent the average of 3 experiments ? standard error. Figure 4.4 The effect of glpR on hydrogen cyanide production in FRD1 and PAO1. Cultures were grown in L-broth overnight and assayed for hydrogen cyanide production. Data represent the average of 3 experiments ? standard error. 0 0.5 1 1.5 2 2.5 3 Rha mn olip id (A 625 ) FRD1 FRD1 glpR PAO1 PAO1 glpR 0 200 400 600 800 1000 1200 1400 1600 Hy drog en Cy an ide mM /10 9 c fu FRD1 FRD1 glpR PAO1 PAO1 glpR 77 Glycerol Fructose 6-Phosphate Acetyl-CoA Glucose 6-Phosphate Malonyl-CoA Glucose 1-Phosphate Fatty Acid Biosynthesis dTDP-D-Glucose ?-ketodecanoylester ?-hydroxydecanoyl-ACP dTDP-L-rhamnose ?-hydroxydecanoyl- ? hydroxydecanoyl-S-CoA L-rhamnosyl- ?-hydroxydecanoyl- ?-hydroxydecanote L-rhamnosyl-L-rhamnosyl- ?-hydroxydecanoyl- ?-hydroxydecanote Figure 4.5. Rhamnolipid biosynthesis from glycerol. (Template courtesy of Dr. Sang-Jin Suh) 78 The FRD1 glpR mutant overproduces alginate Alginate is an exopolysaccharride that is often produced by CF P. aeruginosa isolates, including FRD1. Alginate protects P. aeruginosa in a variety of ways during infection such as inhibiting the penetration of antibiotics and promoting the formation of biofilms in the CF lung (Govan et. al. 1996). It was previously reported that some genes in the glp regulon are required for alginate production. Schweizer et. al. (1995) reported that insertions in glpD had a polar effect on glpM, which abolished alginate production in P. aeruginosa when grown on certain carbon sources. In our study, a mutation in glpR resulted in an increase in alginate production by the FRD1 glpR mutant (Table 4.2). GlpR is required for FRD1 virulence on alfalfa We questioned whether reduced virulence factor production by the FRD1 glpR mutant affected its ability to cause disease. As shown in Table 4.2, glpR is required for FRD1, but not PAO1, virulence on alfalfa. Strain1 % Alfalfa Seedling Infection Alginate (mg.mL-1) FRD1 90?4 782?98 FRD1 glpR101::aacCI 29?4 1004?56 PAO1 93?3 NA PAO1 glpR::aacCI 83?1 NA Table 4.2 GlpR is required for optimal infection of CF isolates on alfalfa seedlings and alginate is increased in the FRD1 glpR mutant. 1. aacCI gentamicin resistant cassette. Values represent the number of seedlings showing maceration symptoms and is the average of 3 experiments with 50 seedlings each. ? Standard error. 79 Loss of GlpR increases persister cell formation by FRD1 and PAO1 Persister cells are near-dormant bacterial populations that are resistant to killing by antibiotics and contribute to the recalcitrance of many chronic infections (Lewis 2010). In this study, the FRD1 glpR and PAO1 glpR mutants showed increased resistance to killing by oflaxacin compared to the parent strains. These results are consistent with a previous study that showed overexpression of glpD, which encodes for a glycerol 3-phosphate dehydrogenase, resulted in increased tolerance to ampicillin and oflaxacin in E. coli (Spoering et. al. 2006). Loss of GlpR would relieve repression of the glp regulon and activate glpD expression in P. aeruginosa. Previous studies show that persister cells arise in the lung and are a common occurrence among late chronic isolates of P. aeruginosa (Mulcahly et. al. 2010). However, in contrast to published reports, the CF adapted isolate FRD1 did not produce more persister cells than PAO1 (Figure 4.6). Taken together, the results suggest that catabolism of glycerol in the CF lung may facilitate persister formation by P. aeruginosa. 80 1E-09 1E-08 0.0000001 0.000001 0.00001 0.0001 0.001 0.01 0.1 1 10 100 0 2 4 6 8 10 Per cen t Su rviv al Time (hr) Figure 4.6 The Effect of glpR on Persister Cell Formation by FRD1 and PAO1. Data represent the average of 4 experiments. FRD1 FRD1 glpR PAO1 PAO1 glpR 81 Discussion In this study, we examined the contribution of GlpR to virulence by both a chronic CF and an acute wound isolate of P. aeruginosa. Loss of GlpR affected the production of several virulence determinants with a more pronounced effect on FRD1 compared to PAO1. This was reflected in the significant loss of virulence by the FRD1 glpR mutant in the alfalfa seedling model of infection compared to the parent strain and to PAO1. Further analysis will have to determine whether virulence factors such as pyoverdine or pyocyanin are regulated at the level of gene expression or by the re-routing of carbon sources. The virulence factors regulated by GlpR are predicted to be affected by growth of P. aeruginosa on glycerol (Figure 4.7). Interestingly, the given profile mimics the known phenotype of CF adapted P. aeruginosa isolates in that some virulence factors are downregulated and others such as alginate, HCN production and persister cell formation are upregulated. Therefore, initiation of these phenotypes within the CF lung may begin in the early non-adapted bacteria by the presence of glycerol as a nutritional source in this environment. This begs the question of whether the CF adapted phenotype is merely the consequence of isolates adapted to use glycerol more efficiently as a carbon source. Glycerol Pyoverdine - Pyocyanin Rhamnolipid + Virulence HCN, Alginate, Persister Cell Formation Figure 4.7. Summary of virulence phenotypes regulated by GlpR and glycerol catabolism. 82 This is the first study to characterize the role of GlpR in virulence of P. aeruginosa. The results from this study show that GlpR appears to be required for the expression of several acute virulence determinants, particularly by FRD1. This was not an unexpected results based on the requirement for GlpR by other bacteria for virulence. Expression of glpR is upregulated almost three-fold during otitis media infection by Haemophilus influenza (Mason et. al. 2003). In Mycoplasma pneumoniae, GlpD (glycerol 3-phosphae dehydrogenase) and GlpQ (glycerophosphodiesterase) are required for cytotoxicity (Hames et. al. 2009; Schmidl et. al. 2011). Furthermore, glycerol metabolism results in the production of hydrogen peroxide, which is the major virulence factor in M. pneumoniae (Halbedel et. al. 2007). GlpR controls the expression of genes involved in glycerol catabolism and may regulate virulence factors such as alginate and rhamnolipids by affecting carbon intermediates. In this study, the addition of glycerol to the growth medium was able to alleviate the effect of a glpR mutation on rhamnolipid production by the acute isolate PAO1. Interestingly, the addition of glycerol to growth medium was able to diminish the effects of stress and compromised fitness in prfA mutants in Listeria monocytogenes (Bruno and Freitag, 2001). PrfA is global transcriptional regulator of virulence in L. monocytogenes (Bruno and Freitag, 2001). Similar to the FRD1 glpR mutant, prfA mutants are severely attenuated in virulence (Leimeister-Wachter et. al. 1990). The expression of virulence factors appears to be strongly modulated by carbon source availability, particularly in the host (Milenbachs et. al. 1997; Stoll et. al. 2008; Weir et. al. 2008). For example, sulfate and phosphates have an effect on alginate and exotoxin A production in P. aeruginosa (Weir et. al. 2008). In addition, increasing concentrations of iron 83 reduce the production of elastase, toxin A, pyocyanin, and other extracellular virulence determinants produced by P. aeruginosa (Sokol et. al. 1982). In the CF lung, glycerol, amino acids, and fatty acids are major nutritional sources for P. aeruginosa, as evident by the constitutive expression of genes involved in the metabolism of these carbon sources (Palmer et. al. 2005; Son et. al. 2007). During infection, the ability to metabolize certain host derived nutrients appears to signal specific bacterial responses that influence the differential expression of virulence genes or pathogenicity. For example, loss of dadA, a gene involved in alanine catabolism in P. aeruginosa, exhibits reduced competitive fitness in a rat lung model of infection (Boulette et. al. 2009). In addition, functional histidine catabolic genes are required for optimal infection of eukaryotic hosts and expression of Type III secretion genes in P. aeruginosa (Rietsch et. al. 2004). Moreover, the regulator involved in fatty acid metabolism, FadR, is required for Vibrio vulni?cus to cause disease in mammalian hosts (Brown and Gulig, 2008). The correlation that exists between carbon metabolism and virulence suggests that bacteria rely heavily on central catabolic genes to initiate or persist during an infection. The overlap in regulatory networks that govern the expression of virulence determinants and carbon metabolism appears to be beneficial for the adaptation of P. aeruginosa in various niches, particularly during chronic infection. 84 References Boulette, M. L., P. J. Baynham, P. Jorth, I. Kukavica-Ibrulj, A. Longoria, K. Barrera, R. C. Levesque, and M. Whiteley. 2009. Characterization of alanine catabolism in Pseudomonas aeruginosa and its importance for proliferation in vivo. Journal of Bacteriology 191:6329-6334. Bragonzi, A., M. Paroni, A. Nonis, N. Cramer, S. Montanari, J. Rejman, C. Di Serio, G. D?ring, and B. T?mmler. 2009. Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence. American Journal of Respiratory and Critical Care Medicine 180:138-145. Brown, R. N., and P. Gulig. 2008. Regulation of fatty acid metabolism by FadR is essential for Vibrio vulnificus to cause infection of mice. Journal of Bacteriology 190:7633-7644. Bruno, J. C., and N. E. Freitag. 2010. Constitutive activation of PrfA tilts the balance of Listeria monocytogenes fitness towards life within the host versus environmental survival. PloS one 5:e15138. Carterson, A. J., L. A. Morici, D. W. Jackson, A. Frisk, S. E. Lizewski, R. Jupiter, K. Simpson, D. A. Kunz, S. H. Davis, J. R. Schurr, D. J. Hassett, and M. J. Schurr. 2004. The Transcriptional Regulator AlgR Controls Cyanide Production in Pseudomonas aeruginosa. Journal of Bacteriology. 186: 6837-6844. Ciofu, O., L. F. Mandsberg, T. Bjarnsholt, T. Wassermann, and N. H?iby. 2010. Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants. Microbiology (Reading, England) 156:1108-1119. Du Plessis, D. J. F. 2005. Regulation of rhamnolipid biosynthesis in the Pseudomonas aeruginosa PAOI biofilm population, MSc dissertation, University of Pretoria, Pretoria, viewed 18 April 2011 < http://upetd.up.ac.za/thesis/available/etd-08182008-085625. Essar, D. W., Eberly, L., Hadero, A. & Crawford, I. P. 1990. Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. J Bacteriol 172: 884?900. Govan, J. R., and V. Deretic. 1996. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiological Reviews 60:539-574. Hagins, J. M., R. Locy, and L. Silo-Suh. 2009. Isocitrate lyase supplies precursors for hydrogen cyanide production in a cystic fibrosis isolate of Pseudomonas aeruginosa. Journal of Bacteriology 191:6335-6339. Halbedel, S., C. Hames, J. St?lke. 2007. Regulation of Carbon Metabolism in the Mollicutes and Its Relation to Virulence. J Mol Microbiol Biotechnol. 12:147-154. 85 Hames, C., S. Halbedel, M. Hoppert, J. Frey, and J. St?lke. 2009. Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae. Journal of Bacteriology 191:747-753. Heurlier, K., V. De, G. Pessi, C. Reimmann, and D. Haas. 2003. Negative Control of Quorum Sensing by RpoN (54) in Pseudomonas aeruginosa PAO1. Journal of Bacteriology 185: 2227- 2235. Hoffman, L. R., H. D. Kulasekara, J. Emerson, L. S. Houston, J. L. Burns, B. W. Ramsey, and S. I. Miller. 2009. Pseudomonas aeruginosa lasR mutants are associated with cystic fibrosis lung disease progression. Journal of cystic fibrosis?: Official Journal of the European Cystic Fibrosis Society. European Cystic Fibrosis Society. 8:66-70. Juhas, M. 2004. Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa. Microbiology 150:831-841. Keren, I., N. Kaldalu, A. Spoering, Y. Wang, and K. Lewis. 2004. Persister cells and tolerance to antimicrobials. FEMS Microbiol. Lett. 230:13?18. Kloosterman, T. G., and O. P. Kuipers. 2011. Regulation of arginine acquisition and virulence gene expression in the human pathogen Streptococcus pneumoniae by transcription regulators ArgR1 and AhrC. The Journal of biological chemistry 286:44594-44605. Knutson, C. A. & Jeanes, A. 1968. A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem 24: 470?481. Leimeister-Wachter M, Haffner C, Domann E, Goebel W, Chakraborty. 1990. Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of Listeria monocytogenes. PNAS. 87: 8336?8340. Lewis, K. 2010. Persister cells. Annual review of Microbiology. 64:357-372. Mason, K. M., R. S. M. Jr, O. Lauren, and L. O. Bakaletz. 2003. Nontypeable Haemophilus influenzae Gene Expression Induced In Vivo in a Chinchilla Model of Otitis Media. Infect. Immun. 71: 3454-3462 Milenbachs, a a, D. P. Brown, M. Moors, and P. Youngman. 1997. Carbon-source regulation of virulence gene expression in Listeria monocytogenes. Molecular microbiology. 23:1075-1085. Mulcahy, L. R., J. L. Burns, S. Lory, and K. Lewis. 2010. Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. Journal of Bacteriology 192: 6191-6199. Nguyen, D., and P. K. Singh. 2006. Evolving stealth: genetic adaptation of Pseudomonas aeruginosa during cystic fibrosis infections. Proceedings of the National Academy of Sciences of the United States of America 103: 8305-8326. 86 Palmer, K. L., L. M. Mashburn, P. K. Singh, and M. Whiteley. 2005. Cystic Fibrosis Sputum Supports Growth and Cues Key Aspects of Pseudomonas aeruginosa Physiology. Journal of Bacteriology 187: 5267-5277. Price, M. S., M. Betancourt-quiroz, and L. Jennifer. 2011. Cryptococcus neoformans Requires a Functional Glycolytic Pathway for Disease but Not Persistence in the Host. doi:10.1128/mBio.00103-11. Host. mBio 2(3). Rietsch, A., M. C. Wolfgang, J. J. Mekalanos, A. Rietsch, M. C. Wolfgang, and J. J. Mekalanos. 2004. Effect of Metabolic Imbalance on Expression of Type III Secretion Genes in Pseudomonas aeruginosa Infection and Immunity. 72: 1383?1390. Ryall, B., J. C. Davies, R. Wilson, a Shoemark, and H. D. Williams. 2008. Pseudomonas aeruginosa, cyanide accumulation and lung function in CF and non-CF bronchiectasis patients. The European Respiratory Journal?: Official Journal of the European Society for Clinical Respiratory Physiology. 32:740-747. Schweizer, H. P., and C. Po. 1996. Regulation of glycerol metabolism in Pseudomonas aeruginosa: characterization of the glpR repressor gene. Journal of Bacteriology 178:5215-5221. Schweizer, H. P., C. Po, and M. K. Bacic. 1995. Identification of Pseudomonas aeruginosa glpM, whose gene product is required for efficient alginate biosynthesis from various carbon sources. Journal of Bacteriology. 177:4801-4808. Schmidl, S. R., A. Otto, M. Lluch-Senar, J. Pi?ol, J. Busse, D. Becher, and J. St?lke. 2011. A trigger enzyme in Mycoplasma pneumoniae: impact of the glycerophosphodiesterase GlpQ on virulence and gene expression. PLoS Pathogens 7:e1002263. Silo-Suh, L., Suh, S. J., Sokol, P. A. & Ohman, D. E. 2002. A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma- 22) and RhlR contribute to pathogenesis. Proc Natl Acad Sci. 99: 15699? 15704. Sokol, P. A., C. D. Cox, and B. H. Iglewskil. 1982. Pseudomonas aeruginosa Mutants Altered in Their Sensitivity to the Effect of Iron on Toxin A or Elastase Yields. Journal of Bacteriology. 151:783-787. Son, M. S., W. J. Matthews, Y. Kang, D. T. Nguyen, and T. T. Hoang. 2007. In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients. Infection and Immunity. 75: 5313-5324. Spoering, A. L., M. Vulic, and K. Lewis. 2006. GlpD and PlsB participate in persister cell formation in Escherichia coli. Journal of Bacteriology. 188:5136-5144. Stoll, R., S. Mertins, B. Joseph, S. M?ller-Altrock, and W. Goebel. 2008. Modulation of PrfA activity in Listeria monocytogenes upon growth in different culture media. Microbiology (Reading, England) 154: 3856-3876. 87 Suh, S. J., L. Silo-Suh, D. E. Woods, D. J. Hassett, S. E. West, and D. E. Ohman. 1999. Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. Journal of Bacteriology. 181:3890-3897. Weir, T. L., V. J. Stull, D. Badri, L. a Trunck, H. P. Schweizer, and J. Vivanco. 2008. Global gene expression profiles suggest an important role for nutrient acquisition in early pathogenesis in a plant model of Pseudomonas aeruginosa infection. Applied and Environmental Microbiology 74: 5784-5791. Zulianello, L., C. Canard, T. K?hler, D. Caille, J.-S. Lacroix, and P. Meda. 2006. Rhamnolipids are virulence factors that promote early infiltration of primary human airway epithelia by Pseudomonas aeruginosa. Infection and Immunity. 74: 3134-47. 88 Chapter 5 This chapter contains published and unpublished miscellaneous results Introduction We determined previously that the glyoxylate pathway becomes deregulated in some isolates of P. aeruginosa that have adapted to the CF lung, including FRD1. The occurrence of these isolates suggests deregulation of the glyoxylate pathway may benefit P. aeruginosa growing within the CF lung. However, the mechanism(s) responsible for the deregulation of this pathway have yet to be elucidated. This chapter, outlined in three data sections, details some of the preliminary studies that lead to the search for the mechanism of deregulation of the glyoxylate pathway. 1. Expression of rpoN in FRD1 and PAO1 Published in: Hagins, J. M., J. A. Scoffield, S.-J. Suh, and L. Silo-Suh. 2010. Influence of RpoN on isocitrate lyase activity in Pseudomonas aeruginosa. Microbiology (Reading, England) 156:1201-10. Summary We previously reported that RpoN negatively regulated aceA expression and ICL activity in the acute isolate PAO1. Expression of rpoN was analyzed in FRD1 and PAO1 to support the regulation data. 89 2. Preliminary Characterization of Malate Synthase in P. aeruginosa Published in: Hagins, J., and J. Scoffield. 2011. Malate synthase expression is deregulated in the Pseudomonas aeruginosa cystic fibrosis isolate FRD1. Canadian Journal of Microbiology 195:186-195. Summary P. aeruginosa establishes life-long chronic infections in the CF lung by utilizing various strategies for adaptation. Some of these strategies include upregulation of glcB, which encodes for malate synthase (MS). However, regulation of glcB expression is poorly understood. The goal of this analysis was to better understand the regulation of glcB in order to provide clues to its role (s) in P. aeruginosa pathogenesis. 3. Unpublished Data Summary This section discusses the attempt to identify the mechanism of aceA deregulation in FRD1. We first measured aceA expression in P. aeruginosa derivatives that were disrupted for known regulators of ICL or carbon catabolism. Materials and Methods Bacterial strains and plasmids used in this study are listed in Table 5.1. Leucine was used at 1%, arabinose at 20mM, and Palmitic acid was dissolved in 10% (w/v) Brij 58 solution and used at 2.5 mM (pH with 1M KOH), Heptanoic acid was used at 5 mM. PA1015, PA3508, PA3604, and PA4341 are transposon insertion mutants that were purchased from the University of Washington Pseudomonas mutant stock center. 90 Strain or Plasmid Genotype, relevant characteristics Source FRD1 CF isolate, mucoid Ohman et. al. (1981) PAO1 Wound isolate, nonmucoid Holloway et al. (1979) FRD1 glcB (JH104) PAO1 glcB (JH105) FRD1 glcB+ (JH148) PAO1 glcB+ (JH151) FRD1 glcB::lacZ (JH133) PAO1 glcB::lacZ (JH135) FRD1 aceA glcB (LS1916) PAO1 aceA glcB (LS1917) PAO1 rpoN::lacZ FRD1 rpoN::lacZ PAO1 lrp PA1015 (iclR1) PA3508 (iclR2) PA3604 (erdR) PA4341 (iclR3) PA1015 (iclR1) aceA::lacZ PA3508 (iclR2) aceA::lacZ PA3604 (erdR) aceA::lacZ PA4341 (iclR3) aceA::lacZ FRD1 glcB101::aacCI PAO1 glcB101::aacCI FRD1 glcB complemented for glcB PAO1 glcB complemented for glcB FRD1 carrying glcB::lacZ fusion PAO1 carrying glcB::lacZ fusion FRD1 glcB101::aacCI aceA102::tetA PAO1 glcB101::aacCI aceA102::tetA PAO1 carrying rpoN::lacZ fusion FRD1 carrying rpoN::lacZ fusion PAO1 lrp101::aacCI Mutation in a probable IclR transcriptional regulator Mutation in a probable IclR transcriptional regulator Mutation in rsponse regulator erdR Mutation in a probable IclR transcriptional regulator (iclR3) PA1015 carrying aceA::lacZ fusion PA3508 carrying aceA::lacZ fusion PA3604 carrying aceA::lacZ fusion PA4341 carrying aceA::lacZ fusion Hagins et. al. 2011 Hagins et. al. 2011 Hagins et. al. 2011 Hagins et. al. 2011 Hagins et. al. 2011 Hagins et. al. 2011 Hagins et. al. 2011 Hagins et. al. 2011 Hagins et. al. 2011 Hagins et. al. 2011 Dr. Laura Silo-Suh Univ. of Washington Univ. of Washington Univ. of Washington Univ. of Washington This study This study This study This study Table 5.1 Bacterial strains and plasmids. Abbreviations used for genetic markers are described by Holloway et al. (1979). Alternate strain names are shown in parentheses. 91 Results 1. Published Results Expression of rpoN in PAO1 and FRD1 In Hagins et. al. 2010, we reported that RpoN negatively regulates aceA expression and ICL activity in PAO1. However, expression of aceA::lacZ was much higher in FRD1 compared with the PAO1 rpoN mutant (Hagins et. al. 2010). This suggests that deregulation of RpoN- mediated repression is not solely responsible for high expression of aceA in FRD1. To confirm that our data were not due to differential expression of rpoN between PAO1 and FRD1, we analyzed rpoN::lacZ expression in both strains. As shown in Figure 5.1, the rpoN::lacZ transcriptional fusion was expressed at a slightly lower level in FRD1 compared with PAO1 throughout a growth cycle, and therefore it is unlikely to account for the nine-fold difference in ICL activity between the two isolates. 92 Figure. 5.1. Expression of an rpoN::lacZ transcriptional fusion. ?-Galactosidase assays were conducted using cultures grown in L-broth. Activity is expressed in Miller units and is an average of 3 experiments conducted in duplicate. FRD1 PAO1 0 500 1000 1500 2000 2500 0.1 1 10 ?-Galact osid ase Act ivit y (M iller Unit s) OD 600 93 2. Published Results glcB expression is deregulated in FRD1 To initiate characterization of glcB expression in P. aeruginosa, a glcB::lacZ transcriptional fusion was constructed and introduced into the FRD1 and PAO1 genomes. As shown in Figure 5.2, expression of glcB::lacZ is significantly higher in FRD1 than in PAO1 over a growth cycle when grown in L-broth. Because a peptide-rich medium such as L-broth has not previously been shown to induce glcB expression in bacteria, this result suggested that regulation of glcB is altered in FRD1. Alternatively, upregulation of glcB in FRD1 may be a consequence of high glyoxylate concentrations provided by increased aceA activity in this isolate. However, the glcB::lacZ fusion retained high expression even in the FRD1 aceA mutant, suggesting that glcB is not induced by high glyoxylate concentrations in the parental background (Figure 5.2). The promoter of glcB from FRD1 is identical to the published PAO1 sequence for over 200 bp upstream of the open reading frame (data not shown). Therefore, the simplest explanation for altered glcB regulation in FRD1 compared with PAO1 is the loss of a negative regulator. We previously demonstrated that aceA, which encodes for the first enzyme of the glyoxylate pathway in P. aeruginosa, is also deregulated in FRD1. Because aceA and glcB are both single open reading frames and located distantly from each other in the P. aeruginosa genome, it is likely these genes share a common regulatory mechanism that became altered following adaptation of P. aeruginosa to the CF lung. 94 0 500 1000 1500 2000 2500 3000 3500 4000 4500 0.1 1 10 ?-Galact osid ase act ivit y (M iller Unit s) OD 600 Figure 5.2. glcB::lacZ expression in FRD1 and PAO1. ?- Galactosidase assays were conducted using cultures grown in L- broth. Activity is expressed in Miller units and is an average of 3 experiments conducted in duplicate. FRD1 FRD1 aceA PAO1 95 Effect of carbon sources on glcB expression MS encoded by aceB has been shown to be regulated by acetate, fatty acids, glyoxylate, and glycolate (Pellicer et. al. 1999; Lorca et. al. 2007), while MS encoded by glcB has been shown to be induced by arabinose, glycolate, glyoxylate, or acetate (Pellicer e.t al. 1999; Garc??a-de los Santos et. al. 2002). To more accurately determine the effect of carbon sources on glcB transcription in P. aeruginosa, expression studies were carried out on FRD1 and PAO1 carrying a glcB::lacZ transcriptional fusion and grown in the presence of different carbon compounds. Several compounds known to induce aceA also induced glcB expression in the PAO1 background, including leucine, heptanoic and palmitic acid (Figure 5.3). In contrast, glcB expression was highly expressed in FRD1 in most of the compounds tested, suggesting that expression of this gene is largely deregulated in FRD1 (Figure 5.3). P. aeruginosa failed to grow appreciably in minimal medium with either glyoxylate or glycolate as the sole carbon source, preventing evaluation of these compounds as sole inducers of glcB expression (data not shown). 96 0 2000 4000 6000 8000 10000 12000 Arabinose Leucine Heptanoic Acid Palmitic Acid ?-Galact osid ase Act ivit y (M iller Unit s) Figure 5.3. Effect of various carbon sources on glcB::lacZ expression in FRD1 and PAO1. ?-Galactosidase assays were conducted using cultures grown in L-broth. Activity is expressed in Miller units and is an average of 3 experiments conducted in duplicate. FRD1 PAO1 97 Malate synthase is required for hydrogen cyanide but not alginate production by Pseudomonas aeruginosa. We observed that disruption of glcB in P. aeruginosa led to reduced production of hydrogen cyanide but not alginate (Figures 5.4 and 5.5). Glyoxylate formed by ICL appears to be converted to glycine, which is the preferred substrate for hydrogen cyanide synthase (Castric 1977; Hagins et. al. 2009). While loss of MS activity would likely lead to increased glyoxylate, high glyoxylate concentrations inhibit HCN production, possibly by competing with glycine for binding to HCN synthase (Hagins et al. 2009). Therefore, the reduced HCN production by the P. aeruginosa glcB mutants compared with the parental strains likely results from increased cellular glyoxylate concentrations. Consistent with this hypothesis, the double aceA glcB mutant in the PAO1 background show higher HCN production than the single glcB mutant. This suggests that disruption of aceA alleviates the build-up of cellular glyoxylate concentrations formed in the absence of MS activity. Higher HCN production is not observed in the FRD1 double mutant background compared with FRD1. However, this was not an unexpected result because disruption of aceA reduces HCN in FRD1 but does not affect HCN production by PAO1 (Hagins et. al. 2009). Complementation of the FRD1 and PAO1 glcB mutants with a wild-type copy of the gene from FRD1 under the control of a regulatable promoter restored HCN activity in both backgrounds. 98 0 200 400 600 800 1000 1200 1400 1600 Hy dro gen Cy an ide ?M /10 9 CFU 's Figure 5.4. Malate synthase is required for hydrogen cyanide production by Pseudomonas aeruginosa. Cyanide concentrations were normalized to the colony-forming units (CFUs) of bacteria recovered from Pseudomonas Isolation Agar plates. Values represent the mean ? standard error of 2 independent experiments conducted in duplicate. 99 0 200 400 600 800 1000 1200 Algin ate (mg mL -1 ) FRD1 FRD1 glcB Figure 5.5. Malate synthase is not required for alginate production by FRD1. Alginate was assayed from cultures grown in L-broth and values represent the mean ? standard error of 3 experiments. 100 3. Unpublished Results Isocitrate Lyase Regulation Deregulation of the glyoxylate pathway in FRD1 suggests the loss of a negative regulator as FRD1 adapted to the CF lung. In an attempt to identify the mechanism responsible for the deregulation of the glyoxylate pathway in FRD1, we tested the effect of several mutations in transcriptional regulators on aceA expression and ICL activity in PAO1. In E. coli, IclR regulates the gloxylate pathway in conjunction with FadR (Gui et. al. 1996). Therefore, we tested the effect of mutations in three iclR homologs on aceA::lacZ expression in PAO1. Disruption of three iclR homologs resulted in a significant reduction in aceA::lacZ expression in PAO1, which suggests that these genes may play a role in activating the glyoxylate pathway (Figure 5.6). Similar results were seen in a mutation in the response regulator erdR (PA4341). ErdR regulates the acsA gene, which is required for acetate activation in P. aeruginosa (Kretzschmar et. al. 2010). Due to the induction of glcB expression on leucine, we also tested the effect of a mutation in lrp on ICL activity. Lrp (Leucine-responsive Regulatory Protein) proteins are global transcriptional regulator involved in cellular metabolism and respond to exogenous amino acids (Brinkman et. al. 2003). As shown in figure 5.7, disruption of lrp resulted in an increase in ICL activity in PAO1, which suggests a possible role in the negative regulation of the glyoxylate pathway. However, sequence analysis of the lrp gene from FRD1 revealed only 3 silent mutations, therefore, it is unlikely that this regulator is responsible for deregulation of the glyoxylate pathway in FRD1 (data not shown). 101 0 200 400 600 800 1000 1200 ?-Galact osid ase Act ivit y (M iller Unit s) Figure 5.6. Effect of various mutations on aceA::lacZ expression in P. aeruginosa. ?-Galactosidase activity was assayed from overnight cultures of P. aeruginosa grown in LB. Values represent the mean (?standard error) of two experiments conducted in duplicate. PAO1 PAO1 iclR1 PAO1 iclR2 PAO1 iclR3 PAO1 erdR 102 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 IC L Act ivit y [?A 32 4 min -1 (mg pro tein -1 )] Figure 5.7. Effect of a mutation in the lrp gene on ICL activity in PAO1. Malate synthase activity was assayed from overnight cultures of P. aeruginosa grown in LB. Values represent the mean (? standard error) of two experiments. PAO1 PAO1 lrp 103 Discussion Initial characterization of malate synthase revealed that it is deregulated in a chronic isolate of P. aeruginosa. In addition, glcB expression was induced on carbon sources that require the glyoxylate pathway. Although MS was required for the optimal production of hydrogen cyanide, it was not required for alginate production. In an attempt to identify the mechanism responsible for deregulation of the glyoxylate pathway, we tested the effect of several mutations in iclR homologs, erdR, and lrp on aceA expression or ICL activity. All of the iclR homologs increased aceA expression, which suggests they probably play a role in the activation of the glyoxylate pathway. Similar results were seen in the erdR mutant. However, a mutation in lrp, which encodes for the leucine-responsive regulatory protein, showed increase ICL activity, which suggests a role in negative regulation, however, LRP appeared to be unaltered in FRD1. The glyoxylate pathway has been shown to be important for pathogenesis of several microorganisms. We reported that ICL in P. aeruginosa is required for alginate production and for virulence in the rat lung and alfalfa models of infection (Lindsey et. al. 2008), in addition to optimal production of hydrogen cyanide (Hagins et. al. 2009). In two separate studies, Mycobacterium tuberculosis and Rhodococcus equi strains lacking ICL were demonstrated to be reduced for virulence (McKinney et. al. 2000; Wall et. al. 2005). In addition, ICL was required for persistence of M. tuberculosis. Similarly, ICL is required for persistence in Burkholderia pseudomallei during pulmonary melioidosis infection (Schaik et. al. 2009). Taken together, these studies demonstrate that the glyoxylate pathway is not only important for growth on certain carbon sources but also for virulence and persistence. 104 Several transcriptional regulators of the glyoxylate pathway have been identified in other bacteria. For example, in E. coli, the glyoxylate pathway is controlled by two regulators, FadR and IclR (Gui et. al. 1996). However, neither appears to regulate the glyoxylate pathway in P. aeruginosa (Hagins). In addition, RamB has been shown to regulate the glyoxylate pathway in M. tuberculosis and Corynebacterium glutamicum (Cramer et. al. 2007; Micklinghoff et. al. 2009). To date, putative regulators of the glyoxylate pathway have not been identified with the exception of RpoN, an alternative sigma factor (Hagins et. al. 2010) in P. aeruginosa. Identification of the mechanism(s) responsible for the deregulation of the glyxoylate pathway in the chronic P.aeruginosa isolate, FRD1, would give a clearer indication of how P. aeruginosa uses this pathway to persist in the lung and perhaps lead to the development of improved therapies. 105 References Barth, a L., and T. L. Pitt. 1995. Auxotrophic variants of Pseudomonas aeruginosa are selected from prototrophic wild-type strains in respiratory infections in patients with cystic fibrosis. Journal of Clinical Microbiology 33:37-40. Barth, a L., and T. L. Pitt. 1996. The high amino-acid content of sputum from cystic fibrosis patients promotes growth of auxotrophic Pseudomonas aeruginosa. Journal of Medical microbiology 45:110-119. Bragonzi, A., M. Paroni, A. Nonis, N. Cramer, S. Montanari, J. Rejman, C. Di Serio, G. D?ring, and B. T?mmler. 2009. Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence. American Journal of Respiratory and Critical Care Medicine 180:138-145. Brinkman, A. B., T. J. G. Ettema, W. M. D. Vos, and J. V. D. Oost. 2003. MicroReview The Lrp family of transcriptional regulators. Molecular Microbiology 48:287-294. Castric, P.A. 1977. Glycine metabolism by Pseudomonas aeruginosa: hydrogen cyanide biosynthesis. J. Bacteriol. 130: 826? 831. Cramer, A., M. Auchter, J. Frunzke, M. Bott, and B. J. Eikmanns. 2007. RamB, the transcriptional regulator of acetate metabolism in Corynebacterium glutamicum, is subject to regulation by RamA and RamB. J. Bacteriol. 189:1145?1149. Davis, R. W., Botstein, D. & Roth, J. R. 1980. Advanced Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Dunn, M. F., J. a Ram?rez-Trujillo, and I. Hern?ndez-Lucas. 2009. Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology (Reading, England) 155: 3166-3175. Figurski, D. H. & Helinski, D. R. 1979. Replication of an origin- containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76: 1648?1652. Govan, J. R. & Nelson, J. W. 1993. Microbiology of cystic fibrosis lung infections: themes and issues. J R Soc Med 86: 11?18. Gui, L., a Sunnarborg, and D. C. LaPorte. 1996. Regulated expression of a repressor protein: FadR activates iclR. Journal of Bacteriology 178: 4704-4709. Holloway, B.W., Krishnapillai, V., and Morgan, A.F. 1979. Chromosomal genetics of Pseudomonas. Microbiol. Rev. 43: 73? 102. 106 Hagins, J.M., Locy, R., and Silo-Suh, L. 2009. Isocitrate lyase supplies precursors for hydrogen cyanide production in a cystic fibrosis isolate of Pseudomonas aeruginosa. J. Bacteriol. 191: 6335?6339. Hagins, J.M., Scoffield, J.A., Suh, S.J., and Silo-Suh, L. 2010. Influence of RpoN on isocitrate lyase activity in Pseudomonas aeruginosa. Microbiology 156: 1201?1210. Kretzschmar, U., V. Khodaverdi, and L. Adrian. 2010. Transcriptional regulation of the acetyl-CoA synthetase gene acsA in Pseudomonas aeruginosa. Biological Reviews 685-690. Knutson, C.A., and Jeanes, A. 1968. A new modification of the carbazole analysis: application to heteropolysaccharides. Anal. Biochem. 24: 470?481. Lindsey, T. L., J. M. Hagins, P. a Sokol, and L. a Silo-Suh. 2008. Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase. Microbiology (Reading, England) 154: 1616-1627. Lorca, G.L., Ezersky, A., Lunin, V.V., Walker, J.R., Altamentova, S., Evdokimova. 2007. Glyoxylate and pyruvate are an-tagonistic effectors of the Escherichia coli IclR transcriptional regulator. J. Biol. Chem. 282: 476-491. Mahenthiralingam, E., Campbell, M. E. & Speert, D. P. 1994. Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis. Infect Immun 62: 596?605. McKinney, J. D., K. H?ner zu Bentrup, E. J. Mu?oz-El?as, a Miczak, B. Chen, W. T. Chan, D. Swenson, J. C. Sacchettini, W. R. Jacobs, and D. G. Russell. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406: 735-738. Micklinghoff, J. C., K. J. Breitinger, M. Schmidt, R. Geffers, B. J. Eikmanns, and F.-C. Bange. 2009. Role of the transcriptional regulator RamB (Rv0465c) in the control of the glyoxylate cycle in Mycobacterium tuberculosis. Journal of Bacteriology 191:7260-7269. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press Ohman, D. E., and M. Chakrabarty. 1982. Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin. Infection and immunity 37:662-669. Palmer, K. L., L. M. Mashburn, P. K. Singh, and M. Whiteley. 2005. Cystic Fibrosis Sputum Supports Growth and Cues Key Aspects of Pseudomonas aeruginosa Physiology. Infection and Immunity 187:5267-5277. 107 Pellicer, M.T., Fernandez, C., Bad??a, J., Aguilar, J., Lin, E.C., and Baldom, L. 1999. Cross- induction of glc and ace operons of Escherichia coli attributable to pathway intersection. Characterization of the glc promoter. J. Biol. Chem. 274: 1745?1752. Ryall, B., J. C. Davies, R. Wilson, a Shoemark, and H. D. Williams. 2008. Pseudomonas aeruginosa, cyanide accumulation and lung function in CF and non-CF bronchiectasis patients. The European Respiratory Journal?: Official Journal of the European Society for Clinical Respiratory Physiology 32:740-747. Smith, E. E., Buckley, D. G., Wu, Z., Saenphimmachak, C., Hoffman, L. R., D?Argenio, D. A., Miller, S. I., Ramsey, B. W., Speert, D. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 103: 8487?8492. Suh, S.J., Silo-Suh, L., Woods, D.E., Hassett, D.J., West, S.E., and Ohman, D.E. 1999. Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J. Bacteriol. 181: 3890?3897. Wall, D. M., P. S. Duffy, C. Dupont, J. F. Prescott, and W. G. Meijer. 2005. Isocitrate Lyase Activity Is Required for Virulence of the Intracellular Pathogen Rhodococcus equi. Infection and Immunity 73:6736-6741. 108 Chapter 6 Conclusion and Future Directions Conclusion The improved effectiveness and development of novel therapies used to treat chronic bacterial infections is dependent upon the ability to enhance our understanding of the intricate mechanisms used by bacteria to cause infection and persist in the host. P. aeruginosa establishes life-long chronic infections in the CF lung by utilizing various strategies for adaptation (Bragonzi et. al. 2009). One strategy used by some chronic isolates of P. aeruginosa, including FRD1, may include the upregulation of the genes encoding for the glyoxylate pathway enzymes, aceA and glcB, which encode for isocitrate lyase (ICL) and malate synthase (MS), respectively (Hagins et. al. 20010, 2011; Lindsey et. al. 2008). In addition, ICL has also been shown to be required for the optimal production of alginate and hydrogen cyanide, which are two virulence determinants commonly found within the CF lung (Hagins et. al. 2009; Lindsey et. al. 2008). Furthermore, ICL is required for infection in the rat lung and alfalfa models of infection (Lindsey et. al. 2008). Previous data suggest deregulation of the glyoxylate pathway may benefit P. aeruginosa growing within the CF lung. However, the mechanism(s) responsible for the deregulation of these genes have yet to be elucidated. The initial goal of my project was to determine the mechanism of regulation of the glyoxylate pathway in an acute isolate (PAO1) and CF isolate (FRD1) of P. aeruginosa. In an attempt to identify the mechanism of deregulation of the glyoxylate pathway I first tested IclR homologs that are known to regulate the glyoxylate 109 pathway in other bacteria. In E. coli, aceA is negatively regulated by FadR and IclR (Gui et. al. 1996). However, none of the IclR homologs were found to be negative regulators of the glyoxylate pathway in P. aeruginosa. Eventually, I determined that GlpR, a negative regulator of glycerol metabolism (Schweizer et. al. 1996), also negatively regulates the glyoxylate pathway in PAO1. However, GlpR was not responsible for the deregulation of the glyoxylate pathway in FRD1. My second objective was to characterize glycerol metabolism in several P. aeruginosa isolates. I discovered that some CF isolates, such as FRD1, are able to utilize glycerol more efficiently as a carbon source than non-CF isolates. Isolates that were able to grow proficiently on glycerol displayed the mucoid phenotype and it was determined that the overproduction of alginate was responsible for enhanced growth on glycerol. Alginate provides micraerophilic growth conditions and it is likely that P. aeruginosa metabolizes glycerol better when oxygen is limited. Genes involved in glycerol metabolism have been shown to be upregulated in the CF lung (Son et. al. 2007). In addition, glycerol is a likely nutritional source for P. aeruginosa growing within the CF lung. The enhanced ability to metabolize glycerol by CF isolates may provide some benefit to P. aeruginosa. The third goal of my project was to characterize the role of GlpR in P. aerugionsa virulence. I determined that GlpR is required for optimal virulence in the chronic CF isolate, FRD1, and that loss of glpR increases the formation of persister cells in both FRD1 and PAO1. Further analysis is needed to determine if GlpR regulates these virulence factors directly, or by re-routing carbon intermediates. This was the first study to demonstrate the potential connection between fatty acid and glycerol metabolism in P. aeruginosa. Moreover, this study showed the importance of glycerol metabolism on virulence in the chronic CF isolate, FRD1. In summary, 110 these data show that the glyoxylate pathway and glp regulon are important factors that may contribute to adaptation and persistence during chronic infection. Future Directions The major goal of my project was to characterize the regulation of the glyoxylate pathway in P. aeruingosa. Although GlpR appears to regulate the glyoxylate pathway in PAO1, the mechanism responsible for the deregulation of this pathway in FRD1 remains to be elucidated. More studies are needed to determine the cause of high ICL and MS activity in CF adapted isolates. In addition, this study focused largely on the contribution of GlpR to P. aeruginosa virulence. The contribution of GlpR regulated genes to P. aeruginosa should also be characterized. Identification of additional regulators of the glyoxylate pathway It is likely that the glyoxylate pathway is regulated by factors other than GlpR. Predictive approaches have failed to identify other transcriptional regulators and a non-predictive approach identified RpoN as an indirect regulator of the glyoxylate pathway (Hagins et. al. 2010). An alternative method for identifying regulators of this pathway is to perform a DNA pull down assay. Biotinylated aceA and glcB promoters can be complexed with proteins in a P. aeruginoa cell free extract and then isolated using a streptavidin column. The proteins can then be eluted from the DNA-protein complex and N-terminal amino acid sequencing conducted to identify the bound proteins. It is also possible that the glyoxylate pathway is regulated by factors involved in stress response. DNA alterations acquired by P. aeruginosa in the CF lung are likley caused by oxidative (Ciofu et. al. 2010), and possibly osmotic stress. Overexpression of ICL has been 111 demonstrated in P. fluorescens in response to aluminum stress (Hamel et. al. 2004). Other evidence suggests that in E. coli the glyoxylate pathway is additionally regulated by RpoS, the stress response regulator (Dong et. al. 2009). In fact, loss of RpoS increases isocitrate lyase and malate synthase activites under stress conditions (Maharjan et. al. 2005). Lastly, this study is one of the first to show a relationship between fatty acid and glycerol metabolism. It would be interesting to see if genes involved in fatty acid or acetate metabolism are required for growth on glycerol, and vice versa. Genes involved in glycerol metabolism have been shown to be upregulated during growth on acetate in Citrobacter sp. (Kim et. al. 2012). Anaerobic utilization of glycerol by P. aeruginosa. In this study, chronic mucoid CF isolates of P. aeruginosa were able to utilize glycerol more efficiently compared to the non-mucoid isolate, PAO1. I determined that this phenotype was due to the overproduction of alginate which may provide microaerophilic growth conditions. Further confirmation of these results would be to examine glycerol utilization by PAO1 under strict anaerobic conditions. Finally, it would be interesting to see if CF adapted isolates show a growth advantage on other carbon sources commonly found within the CF lung compared to PAO1 or other clinical isolates. In conclusion this study provides novel insight into the regulatory interchange involved in glycerol and fatty acid metabolism in P. aeruginosa, and will promote our understanding of how these two networks enable P. aeruginosa to establish and maintain chronic infections. Furthermore, it suggests a more important role for nutrient catabolism in P. aeruginosa adaptation and virulence in the CF lung. The absence of the glyoxylate enzymes in humans suggests the potential for controlling P. aeruginosa CF lung infections by targeting these 112 enzymes for therapy. Understanding how the glyoxylate pathway is regulated will provide a better understanding of the role it plays in pathogenesis. 113 References Bragonzi, A., M. Paroni, A. Nonis, N. Cramer, S. Montanari, J. Rejman, C. Di Serio, G. D?ring, and B. T?mmler. 2009. Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence. American Journal of Respiratory and Critical Care Medicine. 180:138-145. Ciofu, O., L. F. Mandsberg, T. Bjarnsholt, T. Wassermann, and N. H?iby. 2010. Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants. Microbiology (Reading, England) 156:1108-1119. Dong, T., and H. E. Schellhorn. 2009. Control of RpoS in global gene expression of Escherichia coli in minimal media. Molecular Genetics and Genomics. 281: 19-33. Gui, L., A. Sunnarborg, and D. LaPorte. 1996. Regulated Expression of a Repressor Protein: FadR Activates iclR. Journal of Bacteriology. 178: 4704?4709. Hagins, J., J. Scoffield, S. Suh and L. Silo-Suh. 2010. Influence of RpoN on isocitrate lyase activity in Pseudomonas aeruginosa. Microbiology. 156: 1201-1210. Hagins, J., J. Scoffield, S. Suh and L. Silo-Suh. 2011. Malate synthase expression is deregulated in the cystic fibrosis isolate FRD1. Canadian Journal of Microbiology. 195:186- 195. Hamel, R., V. D. Appanna, T. Viswanatha, and S. Puiseux-Dao. 2004. Overexpression of isocitrate lyase is an important strategy in the survival of Pseudomonas fluorescens exposed to aluminum. Biochemical and Biophysical Research Communications. 317:1189-1194. Kim, Y.-M., S.-E. Lee, B.-S. Park, M.-K. Son, Y.-M. Jung, S.-O. Yang, H.-K. Choi, S.-H. Hur, and J. H. Yum. 2012. Proteomic analysis on acetate metabolism in Citrobacter sp. BL-4. International Journal of Biological Sciences 8:66-78. Maharjan, R., P.-L. Yu, S. Seeto, and T. Ferenci. 2005. The role of isocitrate lyase and the glyoxylate cycle in Escherichia coli growing under glucose limitation. Research in Microbiology 156:178-183. Schweizer, H. and C. Po. 1996. Regulation of Glycerol Metabolism in Pseudomonas aeruginosa: Characterization of the glpR Repressor Gene. Journal of Bacteriology. 178:5215? 5221. Son, M., W. Matthews, Y. Kang, D. Nguyen, and T. Hoang. 2007. In Vivo Evidence of Pseudomonas aeruginosa Nutrient Acquisition and Pathogenesis in the Lungs of Cystic Fibrosis Patients. .Infection and Immunity. 75: 5313?5324.