Vibrio cholerae Type Three Secretion System Regulators and Effectors: Identification and Role in Pathogenesis

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2010.AM-19226 is a pathogenic, O39 serogroup Vibrio cholerae strain that lacks the toxin co-regulated pilus (TCP) and cholera toxin (CT), which are virulence factors for colonization and toxin production encoded by epidemic O1 and O139 serogroup V. cholerae strains. Instead, strain AM-19226 carries a 48.5 kb pathogenicity island that encodes a Type III Secretion System (T3SS). A T3SS is a conserved virulence mechanism carried by many gram-negative pathogens, and is a multi protein complex that translocates virulence (effector) proteins from the bacterial cytoplasm into eukaryotic host cells. Annotation of the AM-19226 T3SS island DNA sequence identified more than 50 ORFs (open reading frames): ten encode proteins of the secretion apparatus; two (A33_1664 and A33_1675) are predicted to encode transcriptional regulators that are similar to the ToxR protein (a transmembrane protein that regulates TCP and CT expression in epidemic strains); and more than 35 ORFs encode hypothetical proteins having no known homology. We hypothesized that the T3SS encoded ToxR-like proteins are important for T3SS gene regulation, and predicted that many of the hypothetical ORFs encode unique T3SS effector proteins that mediate the TCP/CT independent pathogenesis of strain AM-19226. Two approaches were used to determine the role of the T3SS encoded transcriptional regulators in pathogenesis. First, strains carrying deletions in ORFs A33_1664 and A33_1675 were shown to be severely attenuated for colonization in vivo using the infant mouse model, suggesting that they encode proteins essential for pathogenesis. Second, lacZ transcriptional reporter fusions were constructed to T3SS gene promoter regions to evaluate the role of A33_1664 and A33_1675 gene products in T3SS gene expression. Growth of reporter strains under different in vitro conditions identified bile and deoxycholate as strongly inducing T3SS structural gene expression. This effect was mediated by the transcriptional regulators encoded by A33_1664 and A33_1675, named vttRA and vttRB respectively. Saccharomyces cerevisiae has been used as a model system to study effector protein activity, since many signaling pathways and cellular mechanisms are conserved among yeast and higher eukaryotes. In different species, effectors are typically encoded by genes located in the T3SS pathogenicity island. Therefore 20 of the V. cholerae ORFs encoding hypothetical proteins were expressed in yeast and observed for growth defects. The results identified a total of 12 ORFs that inhibited yeast growth. A FRET based TEM1-reporter fusion assay determined that 3 of the 12 ORFs encode bona fide effector proteins that were translocated into eukaryotic cells in vitro in a T3SS dependent manner. One protein, encoded by locus A33_1663, was chosen for further study, since BLAST analysis suggested that it is a novel effector protein with conserved sequences found only in T3SS positive V. cholerae and V. parahaemolyticus strains. The role of A33_1663 in pathogenesis was explored using several approaches. A strain deleted for this locus showed a ~five-fold reduction in its colonization ability in vivo. The results of A33_1663-lacZ transcriptional reporter fusion activity showed that A33_1663 expression can be induced by growth in bile, and is co-regulated with the structural genes by VttRA and VttRB. Suppression of the yeast growth defect induced by A33_1663 was used to identify yeast proteins that interact with the A33_1663 product. Growth was assayed in yeast strains deleted for the components of different MAPK signaling pathways. Deletion of the BCK1 and SLT2 genes, components of the protein kinase C-mediated cell wall integrity (CWI) MAPK pathway, did not suppress the yeast growth inhibition by A33_1663. However, deletion of the RLM1 gene, encoding a transcriptional factor in the CWI MAPK pathway, partially suppressed the yeast growth defect induced by A33_1663 expression. The data suggest that Rlm1p may be a target of A33_1663 activity, however, this activity may not require the activation of Rlm1p by its MAPK Slt2p. Tandem affinity purification analysis, using A33_1663 as the bait protein in yeast, did not identify Rlm1p, suggesting an indirect interaction between the two proteins. Collectively, the data presented within describe the identification and initial characterization of essential components of V. cholerae T3SS mediated pathogenesis. Importantly, the studies identified two T3SS encoded transcriptional regulators that control T3SS gene expression and are essential for pathogenesis. In addition, multiple effector proteins were identified using yeast as a model system, and genetic studies suggest that the effector protein encoded by A33_1663 may contribute to pathogenesis by interacting with eukaryotic transcription factors or their downstream targets involved in cell signaling pathways