Investigation and Characterization of Antibacterial Proteins from the Eastern Subterranean Termites Reticulitermes flavipes in Response to Multidrug Resistant Bacteria
Type of DegreePhD Dissertation
Entomology and Plant Pathology
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Immune system of insects has been of great interest for discovering novel compounds against microbes. Subterranean termites (Blattodea: Isoptera: Rhinotermitidae), especially the Reticulitermes species, have a wide distribution in the U.S. These termites have developed disease resistance mechanisms that facilitated their survival and propagation as they nest and forage in soil. However, an improved understanding of the mechanisms governing antimicrobial production and the spectrum of antibiotic properties are necessary and would be helpful to develop novel strategies for discovering new antimicrobials against bacterial pathogens including multidrug resistant bacteria (MDR) as well as exploring new approaches to control termites. To assess the presence of antibacterial proteins in R. flavipes, termite colonies were collected on the Auburn University, and maintained in Urban Entomology Laboratory. First, the presence of antibacterial activities of the cell free whole body crude extract as well as five size- fractionated solutions of unsterilized R. flavipes workers were investigated against a common soil bacterium Bacillus subtilis using the inhibition zone assay. The activity against B. subtilis was observed in both crude extract and all size fractions. Next, the spectrum of antibacterial activity of the extract and the origin of antimicrobials were investigated against a panel of bacteria including three MDR and four non-MDR human pathogens. The crude extract of naïve (control) termites showed a broad activity against the non-MDR bacteria but it was ineffective against the three MDR pathogens Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and Acinetobacter baumannii. Interestingly, feeding termites with either heat-killed P. aeruginosa or MRSA dramatically induced activities against MDR, and maintain or slightly increased activities against most of the non-MDRs. Further investigation demonstrated that hemolymph, not the hind-gut, was the primary source of antibiotic activities. In the effort to discover new therapeutic approaches against two common multidrug resistant opportunistic bacterial pathogens, P. aeruginosa and MRSA, the alterations in hemolymph protein profiles of P. aeruginosa and MRSA induced termites were investigated, aiming to identify proteins with antimicrobial activities. The protein profiles were determined through two proteomic approaches via two-dimensional gel electrophoretic analyses and liquid- chromatography-MS/MS analysis. Two-dimensional gel electrophoretic analyses indicated that 38 and 65 proteins of the 493 hemolymph protein spots were differentially expressed at least 2.5- fold in P. aeruginosa and MRSA-fed termites, respectively. Mass spectrometry (MS) analysis indicated a total of 578 proteins, and 80 and 36 proteins were differentially expressed at least 2.5-fold in response to P. aeruginosa and MRSA-challenge, respectively. Many of these differentially expressed hemolymph proteins (actins, tublins, transferrin, dehydrogenases, peroxiredoxin, catalase and etc.) were known to be involved in immune-related processes including iron metabolism, antioxidant-related response, general stress response, and immune effectors. This research provided the first evidence of constitutive and inducible activities expressed by R. flavipes against human bacterial pathogens, and alternations of termite hemolymph proteins in response to bacterial challenges. These findings suggest an exploration of humoral as well as cellular immunity in R. flavipes upon being fed with multidrug-resistant bacteria.