Search

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2014., Vibrio cholerae strain AM-19226, which causes sporadic cholera-like disease, encodes a horizontally-acquired type three secretion system (T3SS) as its major virulence mechanism. Disease mediated by strain AM-19226 in animal models requires a functional T3SS, which translocates bacterial effectors directly into the host cell cytoplasm. Putative open reading frames within the AM-19226 T3SS genomic island were screened for their ability to inhibit growth when expressed in Saccharomyces cerevisiae, and were further screened in yeast strains deleted for components of MAPK pathways to potentially identify interacting partners, and also tested for T3SS-dependent translocation into HeLa cells. Eleven translocated proteins with varying levels of translocation were identified. Previous studies showed that AM-19226 T3SS transcriptional regulators, VttRA and VttRB, are required for bile-dependent expression of T3SS structural genes in vitro. To better understand the scope of genes that are potentially regulated by VttRA and VttRB, deep RNA sequencing was performed on wild type AM-19226 and derivatives deleted for vttRA and vttRB grown in bile. Comparative transcriptome analysis revealed genes encoded outside the T3SS genomic island, such as those involved in biofilm formation, motility, and type six secretion as potentially regulated by VttRA/B. In addition, T3SS effectors also appeared to be positively regulated by VttRA/B. Infant mouse competition assays showed that strains individually deleted for effectors, Vops H, A, M, I, and W, exhibited a 100-1000 fold defect in colonization whereas others had ≈ 10-fold defect (VopG) or no defect (VopZ). Because colonization can alter host cell signaling pathways and AM-19226-mediated disease has an inflammatory component, we evaluated the chemokine profile of Caco2-BBE cells during AM-19226 infection. A standard ELISA detected increased levels of IL-8 during Caco2-BBE/AM-19226 co-culture that required a functional T3SS and bile. Collec

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2014., Vibrio cholerae is a Gram-negative bacterium that causes the diarrheal disease cholera. Pathogenic V. cholerae O1 and O139 serogroup strains cause epidemic disease using the toxin co-regulated pilus (TCP) and cholera toxin (CT), whereas non-O1/non- O139 serogroup strains cause sporadic disease, often using proteins and molecular mechanisms that are less well understood. One alternative mechanism is a type three secretion system (T3SS), found in many Gram-negative bacteria. T3SSs function to deliver bacterial effector proteins directly into the host cytosol where they alter host cell homeostasis and cause disease. AM-19226 is the prototype TCP-/CT-/T3SS+, non- O1/non-O139 serogroup strain that encodes the T3SS on a 50kb horizontally acquired genomic island. The AM-19226 T3SS island is predicted to encode ~50 proteins including components of the secretion apparatus, transcriptional regulators, one effector, a non-T3SS associated hemolysin (TRH), and ~25 hypothetical proteins of unknown function. Because T3SS function is essential for disease in animal models of infection, we hypothesized that the T3SS island-encoded effector proteins, together with other T3SS island proteins, collectively contribute to pathogenesis. We therefore performed experiments to examine the expression and function of T3SS island genes. We identified eleven additional effector proteins encoded within the island and focused on the role of a novel effector protein, VopK, during infection. While VopK is not required for colonization in the infant mouse model, it strongly inhibits growth when expressed in Saccharomyces cerevisiae, and we have identified specific VopK amino acid residues required for yeast growth inhibition. Furthermore, vopK is co-expressed with T3SS structural genes. trh expression is co-regulated with T3SS structural genes suggesting that TRH does play a role in virulence that is coordinated with T3SS activity. However, TRH is not required for colonization in vivo or mammalian ce

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 encode

Vibrio cholerae is a Gram-negative water borne human pathogen. During infection, this bacterium expresses a set of coordinately regulated virulence factors resulting in intestinal colonization, production of cholera toxin, and manifestation of the disease known as cholera. Regulated expression of virulence determinants in response to environmental signals is mediated by the ToxR and ToxS proteins. ToxR is an integral membrane protein, whose cytoplasmic N-terminus is homologous to the OmpR subfamily of transcriptional activators belonging to the two component family of regulatory proteins. The periplasmic C-terminal domain of ToxR is believed to interact with ToxS, an integral membrane protein residing mainly in the periplam. The observation that alkaline phosphatase sequences could functionally substitute for the ToxR periplasmic domain led to a model for ToxR activity based on dimerization. The hypothesis proposed that in response to environmental signals, the ToxR DNA binding domain assumed a transcriptionally active conformation as a result of dimerization initiated by the ToxR periplasmic domain. ToxS was postulated to play a role in dimer formation or stabilization. To test this hypothesis, we used the N-terminal DNA binding domain of phage $\lambda$ repressor as a reporter for heterologous sequences to propagate dimerization. $\lambda$-ToxR chimeric proteins were constructed, and dimerization evaluated by repression of an ${\rm O\sb{R}1}P\sb{R}$-lacZ reporter fusion. $\lambda$-ToxR proteins effectively dimerized in E. coli, although initial experiments in Vibrio cholerae did not produce similar results. Additional experiments suggested that ToxR sequences could act cooperatively. Reconstruction of the reporter fusion to include additional operator sites upstream of the $P\sb{R}$-lacZ fusion resulted in measurable oligomerization of $\lambda$-ToxR proteins. Conditions known to modulate ToxR activity did not regulate $\lambda$-ToxR oligomerization, suggesting th

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2016., Vibrio cholerae is a diverse species of pathogenic and non-pathogenic bacteria that naturally inhabits marine and coastal waters and can cause the severe diarrheal disease cholera. The pathogenic strains that cause epidemic outbreaks belong to the O1 and O139 serogroups, while sporadic disease is caused by non-O1/non-O139 serogroup strains. The non-O1/non-O139 serogroup strain, AM-19226, encodes a type three secretion system (T3SS) to mediate virulence. Regardless of the virulence factors they encode, all pathogenic strains of V. cholerae must rely on intricate regulatory networks to quickly and efficiently respond to changes in their surroundings in order to survive in their natural aquatic environment as well as the human host. The T3SS of V. cholerae strain AM-19226 is encoded on a horizontally acquired pathogenicity island. T3SS structural gene expression is controlled by two island-encoded transcriptional regulatory proteins named VttRB and VttRB. In addition to coordinating T3SS gene expression, VttRA and VttRB likely regulate the expression of ancestral, core genome encoded genes to control multiple phenotypes important for infection and coordinate the shift between the aquatic reservoir and the human host. We present genetic evidence demonstrating that ToxR, VttRA and VttRB work in a hierarchy to activate T3SS gene expression, which is required to cause cytotoxicity in a mammalian cell culture model of infection. We also evaluated a group of effector proteins conserved between T3SSpositive strains of Vibrio spp. that are encoded within the T3SS island structural operons controlled by VttRA and VttRB. We found that Vops H, A, and W were required for secretion of other effector proteins using a FRET-based translocation assay, and thus may have roles as T3SS apparatus proteins. Lastly, we identified an additional effector protein, named VopZZ, which is also under the transcriptional control of VttRA/B. We then turned our attention to regulatory loci outside of