Characterization of Type II Protein Secretion, Secreted Proteases, and Type IV Pili of Stenotrophomonas maltophilia and Roles in Pathogenesis

The Gram-negative bacterium Stenotrophomonas maltophilia is increasingly identified as a multidrug-resistant opportunistic pathogen, being associated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis of S. maltophilia virulence is quite minimally defined. In this thesis, I investigated two research aims. First, I report that S. maltophilia strain K279a encodes a functional type II protein secretion (T2S) system. Mutants of K279a that contain a mutation in the xps T2S locus exhibit a loss of at least seven secreted proteins and three proteolytic activities. Unlike culture supernatants from the parental K279a, supernatants from the xps mutants also failed to induce the rounding, detachment, and death of A549 cells, a human lung epithelial cell line. Supernatants of the xps mutants were also unable to trigger a massive rearrangement in the host cell's actin cytoskeleton that was associated with secreted products of K279a. The activities that were defined as being Xps dependent in K279a were evident among other respiratory isolates of S. maltophilia. Utilizing a similar type of genetic analysis, I found that a second T2S system (Gsp) encoded by the K279a genome is cryptic under all of the conditions tested. To begin to identify secreted effectors that may be responsible for the Xps-dependent phenotypes, I identified that K279a encodes two extracellular proteases, StmPr1 and StmPr2. Together these two proteins account for all of the T2S-dependent serine protease activity in K279a supernatants. StmPr1 and StmPr2 also contribute to the rounding and detachment of A549 cells, and StmPr1 was entirely responsible for the T2S-dependent effect on A549 cell viability. In the second aim, I examined type IV pili (Tfp) in S. maltophilia. I found that K279a possesses a complete set of Tfp genes, and that the loss of Tfp correlates with increased adherence to host cells and increased survival in the lungs of mice. Overall, this thesis represents the first examination of T2S and Tfp in S. maltophilia, and the data obtained indicate these two systems significantly influence S. maltophilia pathogenesis.