Browsing by Subject "Vibrio parahaemolyticus"
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Item Characterization of the Type III Effector VOPA from Vibrio Parahaemolyticus(2007-12-18) Trosky, Jennifer E.; Orth, KimVibrio parahaemolyticus is a marine bacterium and causative agent of gastroenteritis associated with the consumption of contaminated seafood. It is endemic to Southeast Asia and is the leading cause of gastroenteritis in Japan. Sequencing of the Vibrio parahaemolyticus genome revealed the presence of a type III secretion system (TTSS) encoded within a pathogenicity island. Within this pathogenicity island a homologue of the Yersinia type III effector YopJ was found and is referred to as VopA (Vibrio outer protein A). The founding member of the family, YopJ from Yersinia spp., inhibits the MAPK and the NFκB signaling cascades within the host cell, thereby inhibiting the host's innate immune response. Recently our lab elucidated the mechanism of YopJ's inhibition by demonstrating that YopJ acetylates MKKs and inhibits kinase activation by blocking phosphorylation (1). The molecular characterization of VopA has focused on its effect on signaling pathways. In contrast to YopJ, VopA only inhibits MAPK signaling and shows no effect on the NFκB pathway in mammalian cells. In addition, VopA, like YopJ, utilizes an evolutionary conserved mechanism for inhibition of signaling which is demonstrated by VopA's ability to inhibit MAPK signaling in Saccharo ces cerevisiae. I have shown that VopA is an acetyltransferase targets the MKK within the MAPK cascade revealing an activity similar to YopJ's. Through mass spectrometric analysis, I found that VopA modifies MKK on four different residues. Three of the residues, S207, K210, and T211, that are located in the activation loop, are the same residues modified by YopJ. The fourth residue, K172, is only modified by VopA and is a conserved lysine in the catalytic loop of MKKs that is required for ATP binding. I have shown that VopA's modification of this residue disrupts ATP binding and allows for the inhibition of an activated kinase.Item Characterization of VIBRIO Parahaemolyticus-induced Intestinal inflammation in the mouse(2009-09-04) Lee, Olivia; Orth, KimThe Gram-negative marine bacterium Vibrio parahaemolyticus is a leading cause of gastroenteritis from the consumption of contaminated seafood. Some recent outbreaks are attributed to a rise in ocean temperatures, a trend that is likely to continue as a result of global warming and emphasizes the need to study the virulence mechanisms of the pathogen. V. parahaemolyticus utilizes a type III secretion system to inject effectors that disrupt signal transduction in eukaryotic hosts during infection. Herein, we describe an in vivo model of infection by V. parahaemolyticus using germfree mice. V. parahaemolyticus is recovered from the cecum and colon of mice infected by oral gavage. Mice infected with a pathogenic, wild type strain of V. parahaemolyticus exhibit intestinal inflammation characterized by epithelial damage, submucosal edema, crypt abscess and hyperplasia, and infiltration of the lamina propria by neutrophils. Using mutant strains of V. parahaemolyticus, we have determined that thermostable direct hemolysin, the most well characterized virulence factor produced by V. parahaemolyticus, and T3SS2, one of the two type III secretion systems present in this bacterium, both contribute to enteropathogenesis. Analysis of the expression of inflammatory cytokines IL-1? and IFN-gamma and chemokines KC, MIP-2 alpha, and CXCL-9 reveals that the duration of the inflammatory response is extended in the presence of TDH. In addition to delineating the roles of the virulence factors TDH, type III secretion system 1, and type III secretion system 2, in the infection process, we have developed an experimental system that will enable further characterization of type III effectors of V. parahaemolyticus.Item Characterization of Vibrio VopS, an AMPylator of Rho GTPases(2009-06-19) Yarbrough, Melanie Leann; Orth, KimVibrio parahaemolyticus is a gram-negative marine bacterium that causes gastroenteritis associated with the consumption of contaminated shellfish. The emergence of pandemic strains of V. parahaemolyticus has increased the need for characterization of the virulence factors of this pathogen. Sequencing of the genome of a clinical isolate revealed the presence of two type III secretion systems (T3SSs), one on each chromosome. The T3SS on chromosome one (T3SS1) has been shown to be responsible for cytotoxicity in HeLa cells, and it shares a high degree of homology to the T3SS of the Yersinia spp. Our studies have shown that infection of HeLa cells with a strain of V. parahaemolyticus capable of secreting only from T3SS1 indicated that T3SS1 mediates several events during infection including the rapid induction of autophagy, cell rounding, and finally lysis of the cell. Defining the T3SS1-mediated events of infection gives insight into virulence mechanisms of V. parahaemolyticus that have not been well characterized and provide a basis for the elucidation of the functions associated with T3SS1 effectors. One of the T3SS effectors, VopS, contains a Filamentation induced by cAMP (Fic) domain that we have shown is critical for the function of this effector. Our studies have found that VopS inhibits Rho GTPase signaling during infection by directly modifying Rho, Rac, and Cdc42, preventing their interaction with downstream effectors. These observations reveal a unique activity for VopS, which targets a pathway that is critical in the cellular response to V. parahaemolyticus infection. In addition, they provide insight into a novel post-translational modification that may expand our knowledge of eukaryotic cell signaling. Fic domains are found in proteins from several bacterial and eukaryotic species and are recognized by their conserved motif, HPFX(D/E)GNGR. The presence of Fic domains in higher eukaryotes suggested that this modification could be utilized in cell signaling. Our preliminary studies indicated that AMPylation is utilized by eukaryotes. We have shown that a Fic protein from humans, HYPE, possesses auto-AMPylation activity, confirming our hypothesis that these domains are involved in AMPylation. Ongoing and future studies seek to identify the substrates of HYPE activity and identify other components involved in this new layer of eukaryotic cell signaling.Item Characterization of VOPQ, A Type III Secreted Effector Protein from Vibrio Parahaemolyticus(2009-06-15) Burdette, Dara Lesley; Orth, KimVibrio parahaemolyticus is a Gram-negative bacterium responsible for gastroenteritis associated with the consumption of raw or undercooked shellfish. Its most well-characterized virulence factors are hemolysins that cause b-hemolysis on a special blood agar. Mutants lacking these hemolysins are still virulent in animal and tissue culture models of infection. These phenomena can be attributed in part to one of two type III secretion systems; one on chromosome 1 and the other on chromosome 2. We demonstrate that Vibrio parahaemolyticus utilizes the type III secretion system on chromosome 1 to induce a temporally regulated series of events that initiates with the induction of autophagy, followed by cellular rounding and finally cellular lysis and death. To the best of our knowledge, no other Gram-negative extracellular bacterium has been shown to induce autophagy during infection. To understand the mechanism of Vibrio parahaemolyticus induced cell death, we focused our analysis on VopQ, a type III secreted effector encoded by the type III locus on chromosome 1. We demonstrate that VopQ contributes to cytotoxicity as DvopQ strains induce cell lysis less efficiently. In addition, VopQ is necessary and sufficient for the induction of autophagy during infection. VopQ-mediated autophagy occurs independently of phosphatidylinositol 3-kinases and prevents phagocytosis. Additional experiments using Saccharomyces cerevisiae demonstrate VopQ induces autophagy and cell death through an evolutionarily conserved mechanism. Results presented herein delineate a novel virulence mechanism used by Vibrio parahaemolyticus to cause disease. This study also highlights the effector VopQ as a novel inducer of autophagy and a key mediator of cytotoxicity during infection.Item Characterization of VPA0450, A Type III Secreted Effector Protein from Vibrio Parahaemolyticus(2011-08-26T17:34:00Z) Broberg, Christopher Allen; Orth, KimVibrio parahaemolyticus is a Gram-negative, halophilic bacterium first isolated over 60 years ago after a major outbreak of food poisoning in Japan. It is now recognized as a significant cause of gastroenteritis associated with the consumption of raw or undercooked seafood. The recent emergence of pandemic strains has made the study of V. parahaemolyticus a priority in the field of bacterial pathogenesis. Virulence caused by V. parahaemolyticus has traditionally been attributed to the presence of one or more thermostable direct hemolysins. Genome sequencing of V. parahaemolyticus identified two distinct Type III Secretion Systems (T3SS). T3SS1, on chromosome 1, was shown to translocate four effectors, VopQ, VopR, VopS, and VPA0450, resulting in cytotoxicity of cultured host cells. VopQ has been shown to rapidly induce autophagy upon translocation into a host cell. VopS AMPylates Rho-family guanosine triphosphatases leading to the collapse of the actin cytoskeleton and host cell rounding prior to lysis. Herein we show that VPA0450 is a phosphatidylinositol phosphatase with homology to the inositol polyphosphate 5-phosphatase catalytic domain of the eukaryotic enzyme synaptojanin. VPA0450 was sufficient to induce membrane blebbing and the delocalization actin-binding proteins from the plasma membrane. VPA0450 contributes to cytotoxicity as strains deleted for vpa0450 induced cell lysis less efficiently than wild-type strains. VPA0450 compromised membrane integrity by hydrolyzing the D5 phosphate from phosphotidylinositide (4,5) bisphosphate, thereby disrupting adaptor protein binding sites required for proper membrane and cytoskeleton dynamics, likely contributing to cell death by facilitating lysis. Preliminary studies have shown the C-terminus of VPA0450 is necessary for localization of this effector to the plasma membrane, possibly by binding membranes and phosphoinositides. An improved system was developed for making chromosomal gene deletions in V. parahemaolyticus. New parent strains were created in which the positive regulators of each T3SS were deleted. Additional strains demonstrated that the cytotoxicity seen during infection with T3SS1 positive strains is attributed solely to T3SS1 effectors. Infection with a strain deleted for vopQ, vopS and vpa0450 uncovered the phenotype for VopR. Bioinformatic analysis of VopR identified effector homologs in other pathogens, homologous eukaryotic enzymes, and a catalytic triad.Item Structural and Kinetic Characterization of Protein Ampylation by VopS Fic Domain(2012-07-20) Luong, Phi Hoang; Orth, KimThe bacterial pathogen Vibrio parahaemolyticus manipulates host signaling pathways by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylating a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. We describe features of the VopS crystal structure, including a hairpin element that is responsible for protein–protein interaction and residues involved in ATP binding. Steady-state analyses of VopS point mutants provide kinetic understanding on the functions of conserved residues for the AMPylation activity. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. The structure of VopS and its ternary reaction mechanism provide critical groundwork for future studies on AMPylators, a novel family of enzymes that modify hydroxyl-containing residues with AMP. We also developed molecular tools to facilitate the study of protein AMPylation in collaboration with Howard Hang at The Rockefeller University. An ATP analogue, N6pATP, was developed that utilizes click chemistry to allow for the detection of AMPylated proteins by fluorescent or biotin tags. N6pATP can be utilized in in vitro AMPylation reactions catalyzed by known AMPylators including Fic domain and adenylyltransferase domain proteins. Further, we showed that N6pATP can be used for the detection and purification of endogenous AMPylated proteins. Preliminary studies were performed on another effector protein of unknown function, VopQ from Vibrio parahaemolyticus. The protein sequence of VopQ does not resemble any known protein domains. Various constructs were made for VopQ, and here I describe the purification and crystallization of VopQ.Item The Association of Virulent Vibrio Spp. Bacteria on Gafftopsail and Hardhead Catfish in Galveston Bay(2011-10-21) Gilbert, Leslie DeanneVibrio vulnificus (Vv) and V. parahaemolyticus (Vp) are gram negative, halophilic bacteria that occur naturally in estuarine waters of Galveston Bay. Both bacteria have the potential to cause infections in humans either via consumption or direct contact. Finfish are a potential vector for these bacteria. Previous work by Brinkmeyer determined that these bacteria are present on the benthic dwelling catfish, Ariopsis felis and Bagre marinus, using a conventional microbial method. The present work focused on using Quantitative Polymerase Chain Reaction (QPCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) to not only determine presence of these bacteria, but also to quantify them and look at community structure. QPCR was able to detect bacteria presence in 34 percent, 31.6 percent, and 0 percent for V.vulnificus, V.parahaemolyticus. thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes, respectively. Statistical analysis of the QPCR results found that there was no significant difference between the length of fish, location of catch or species of fish in relation to the abundance of bacteria. T-RFLP was able to detect the presence of bacteria in approximately 70 percent of the samples surveyed. Bands produced from T-RFLP were able to be grouped into five different ranges. The most frequently occurring band fell in the range of 213-219 base pairs, and the most common number of bands per sample was 1 band. This study found that both QPCR and T-RFLP were better assays than conventional microbial methods for detecting the presence of V. vulnificus and V. parahaemolyticus on catfish fins. QPCR proved to be the most rapid detection method. Based on this study, it was determined that these Vibrio spp. bacteria have some type of relationship with A. felis and B. marinus. This information may be useful to the medical community for determining when there is a greater risk of infection via catfish puncture wounds.