The role of calcium in the regulation of Xylella fastidiosa virulence

Abstract

The Gram-negative plant pathogenic bacterium Xylella fastidiosa inhabits the xylem vessels of the host, where it forms biofilms thought to be responsible for disease symptoms. Even though knowledge regarding the mechanism of pathogenicity is still inconclusive, symptoms depend on the ability of the bacterium to spread, colonize, and form biofilms. I hypothesize that sap mineral components have a role in the bacterial pathogenicity and virulence by promoting adhesion, aggregation, and twitching motility. The general objective of this research was to establish the role of the specific mineral element calcium (Ca) in the virulence traits of X. fastidiosa. A positive correlation between the increase in the concentration of Ca and the formation of biofilm and twitching motility was observed. The addition of Ca significantly increased the adhesion force to surfaces and autoaggregation and promoted the up-regulation of genes involved in the production of exopolysaccharide in biofilm cells. Further experiments were conducted to understand the molecular mechanisms of Ca-regulated twitching motility. Expression analyses of the type IV pili genes pilA, pilT, pilB, and the three pilY1 homologs (PD0023, PD0052, PD1611) were performed on cells incubated in microfluidic chambers under replete and depleted Ca conditions. Up-regulation was found for pilT, encoding the retraction ATPase in charge of pili depolymerization, and one of the pilY1homologs (PD1611), encoding a protein located at the tip of the pili. Sequence analysis identified a Ca-binding domain only in the in the up-regulated pilY1-PD1611 homolog. The deletion mutagenesis of pilY1-PD1611 results in reduced motility and a Ca-blinded phenotype, whereas twitching motility of a pilY1 PD0023

mutant was not affected by Ca supplementation. Results indicate that the mechanism of Ca-dependent twitching motility is associated with Ca-binding by only one of the pilY1 homologues found in X. fastidiosa. In planta experiments using the system model Nicotina tabaccum were carried out to determine the effect of Ca on X. fastidiosa infection. Results indicated that Ca supplementation increases bacterial populations and intensifies disease symptoms. In turn, bacterial infection elicits an increase in the accumulation of Ca in the plant above the levels caused by the fertilization with Ca. Additionally, a general change in the mineral composition of the infected plants was observed compared with the non-infected plants. The results of this research suggest that Ca acts as a key regulator of X. fastidiosa virulence.