Stenotrophomonas maltophilia virulence: Characterization of its Type IV Secretion System and Siderophore Production

Nas, Megan Yasemin

doi:https://doi.org/10.21985/n2-4y8k-bt70

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Stenotrophomonas maltophilia virulence: Characterization of its Type IV Secretion System and Siderophore Production

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The Gram-negative Stenotrophomonas maltophilia is naturally found in water, soil and the plant rhizosphere, and is increasingly recognized as an important opportunistic and nosocomial infection. Despite its rising prevalence and drug resistance, there is little known about S. maltophilia pathogenesis. In this dissertation, I explored two aspects of S. maltophilia virulence. First, I confirmed that the S. maltophilia strain K279a encodes a type IV protein secretion system (T4SS). I demonstrated that a mutation in a component of the S. maltophilia T4SS core complex (virB10) results in enhanced lung epithelial cell detachment and death indicating that the S. maltophilia T4SS inhibits cell death. I showed that the S. maltophilia T4SS inhibits staurosporine induced cell death as well as caspase activation and thus determined the S. maltophilia T4SS has an anti-apoptotic effect on human epithelial cells. I was able to confirm this phenotype upon S. maltophilia infection of primary human bronchial/tracheal epithelial cells. I also tested the effect of the S. maltophilia T4SS on a human macrophage cell line (U937) and explanted macrophages obtained from A/J mouse femurs and determined that the S. maltophilia T4SS elaborates a pro-apoptotic effect on macrophages. Both phenotypes necessitated bacterial contact with the host cell. I also determined that neither apoptosis phenotype was attributed to a difference in parental strain vs virB10 mutant S. maltophilia attachment to the host cells. Moreover, I showed by intranasally infecting A/J mice with parental and virB10 mutant strains of K279a that the T4SS enhanced the growth of S. maltophilia in mouse lungs. I also determined that the S. maltophilia T4SS confers a growth advantage when co-cultured with E. coli and P. aeruginosa during a 24 h co-culture. Additionally, I discovered during a shorter co-culture with heterologous bacteria, including cystic fibrosis (CF) lung P. aeruginosa isolates, parental K279a can kill more competitors than the T4SS mutant. I developed a list of putative antibacterial T4SS substrates and by demonstrated that mutations in two candidates resulted in less interbacterial killing than the parental K279a. Thus, I attributed the S. maltophilia T4SS antibacterial effect to at least two bona fide antibacterial T4SS effectors, the lipase TfcA and the peptidoglycan hydrolase TfcB. I confirmed that both effectors are T4SS substrates utilizing a Cre recombinase mediated interbacterial translocation assay and determined that TfcA was sufficient to cause E. coli death upon expression in the target’s periplasm. I also embarked upon studying S. maltophilia siderophore production. I report that S. maltophilia harbors a complete system for siderophore with a locus containing enterobactin biosynthesis homologs. An S. maltophilia mutant strain with an entC deletion lost its ability to produce siderophore. Colorimetric assays specific for determining siderophore structure revealed that the S. maltophilia siderophore is, like enterobactin, a catecholate structure, but its organic solvent extraction and thin layer chromatography patterns were distinct from enterobactin. Together, both in silico and experimental work lead me to hypothesize S. maltophilia is produces a novel enterobactin derivative. Moreover, I showed that the siderophore is important for S. maltophilia growth in mouse lungs. Overall, this dissertation is the first report of the S. maltophilia T4SS dependent modulation of apoptosis and antibacterial effect as well as its production of a novel siderophore.

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