Browsing by Subject "Vibrio parahaemolyticus"
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Item Analysis of Vibrio parahaemolyticus Virulence Systems(2014-08-14) Calder, Thomas James; Sperandio, Vanessa; Orth, Kim; Fontoura, Beatriz; Gardner, Kevin H.Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis from the consumption of raw or undercooked seafood. The pathogenicity of V. parahaemolyticus is attributed to several virulence factors, including two hemolysins and two type III secretion systems (T3SS1 and T3SS2). Herein, we compare the virulence of V. parahaemolyticus POR strains, which harbor a mutation in the T3SS needle apparatus, to the V. parahaemolyticus CAB strains, which contain mutations in transcriptional regulators for the T3SSs. Additionally, we characterize a novel T3SS2 effector termed VPA1380. From this study, we demonstrate that each structural or regulatory mutant of T3SS1 or T3SS2 alters the pathogenicity of the bacterium in a different manner. POR and CAB strains exhibited differences in biofilm growth, but shared similar levels of swarming motility and effector production/secretion. Additionally, while the cytotoxicity of these strains was similar, the CAB2 (T3SS1 regulatory mutant) strain was strikingly more invasive than the comparable POR2 (T3SS1 structural mutant) strain. In summary, by creating structural or regulatory mutations in either T3SS1 or T3SS2, differential downstream effects on other virulence systems were observed. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domain of several large bacterial toxins. Mutations of conserved catalytic residues and of residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in yeast cells deficient for the production of IP6. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator. Additionally, VPA1380 appeared to disrupt trafficking of dextran and transferrin, which may be due to VPA1380’s potential interaction with important retrograde factors. Elucidating the host targets and cellular effects of VPA1380 is important for understanding the pathogenic nature of V. parahaemolyticus for diagnostic and treatment and purposes.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(2011-08-10) 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 Fic-Mediated AMPylation in Bacterial Infection and Endoplasmic Reticulum Stress(2015-04-14) Woolery, Andrew Ryan; Liu, Qinghua; Orth, Kim; Cobb, Melanie H.; Sternweis, Paul C.The post-translational modification AMPylation is emerging as a significant regulatory mechanism in both prokaryotic and eukaryotic biology. This process involves the covalent addition of an adenosine monophosphate to a protein resulting in a modified protein with altered activity. Proteins capable of catalyzing AMPylation, termed AMPylators, are comparable to kinases in that they both hydrolyze ATP and reversibly transfer a part of this primary metabolite to a hydroxyl side chain of the protein substrate. To date, all AMPylators discovered contain one of two domains: the Fic domain or the adenylyl transferase domain. All currently characterized AMPylators are bacterial in origin and are primarily Type III or Type IV secreted effector proteins, which are injected into a host cell to manipulate host signaling to the microbe's advantage. Examples of these are VopS (Vibrio parahaemolyticus), IbpA (Histophilus somni) and DrrA (Legionella pneumophila). The discovery of SidD, a deAMPylator also from L. pneumophila, shows that this modification is dynamic and could likely have a regulatory role in eukaryotic biology. Supporting this idea is the presence of a single copy of the Fic domain in most metazoans, including humans. The substrates, localization, and function of Fic proteins and other AMPylators in eukaryotic biology are perhaps the largest open questions in this rapidly expanding field. The goal of my dissertation work was to expand the understanding of the effects of AMPylation in eukaryotic signaling. I approached this goal in three ways: by examining the effects of an AMPylator (VopS) with known targets (Rho GTPases) on different aspects of cell signaling, developing screening tools for AMPylation and attempting to elucidate some of the functions of the human AMPylator, FicD, in which the targets are unclear. I found that VopS, in addition to collapsing the host actin cytoskeleton, also inhibits many aspects of host defense signaling including NFB, MAP kinases and the phagocytic NADPH oxidase system. I explored the possibility of other potential substrates of VopS by collaborating on an extensive protein microarray screen for AMPylation, determining that the entire Rho GTPase family is AMPylated. I also discovered that the human AMPylator FicD is induced during the unfolded protein response, is localized to the endoplasmic reticulum and is capable of AMPylating the ER chaperone BiP/GRP78. The progress made in these studies will contribute to understanding the role of this enigmatic modification in mammalian cell signaling.Item Physical Studies of Actin Nucleation and Conformational Dynamics(2017-09-06) Zahm, Jacob Aaron; Tomchick, Diana R.; Rosen, Michael K.; Rice, Luke M.; Yu, HongtaoActin is a 42 kilodalton ATPase that exists ubiquitously in eukaryotic cells. Unlike other ATPases, however, actin, under suitable conditions, can polymerize, forming helical filaments. Cells, in orchestrating their myriad cellular processes, utilize actin's intrinsic capacity to polymerize, but do so in a tightly controlled fashion, such that new filaments only appear when and where the cell needs them to suit specific purposes. Such control exists at two different levels. Firstly, the stability of actin filaments is subject to "intrinsic" control arising from the state of bound nucleotide. ATP binding favors incorporation of actin monomers into filaments. This incorporation augments actin's ATP hydrolysis activity, and the conversion of ATP to ADP in the nucleotide binding cleft considerably destabilizes filaments, facilitating the return of filament subunits to free monomers. The structural mechanism through which nucleotide conveys information throughout the actin monomer to influence polymerization behavior remains poorly understood and represents a persistent fundamental biological question. In this work I, for the first time, apply modern muti-resonance NMR methods to begin to answer these questions. In addition to the aforementioned intrinsic control, cellular actin is subject to "extrinsic" control via the action of nucleation factors. In order to form a growing filament, actin must proceed through a nucleation step in which monomers must assemble into a thermodynamically and kinetically disfavored nucleus, which ultimately proceeds to a growing filament. Nucleation factors accelerate the rate of filament formation by binding to actin monomers and arranging them into the prerequisite nucleus. In this work, I reveal the crystal structure of actin monomers in complex with the bacterially derived nucleation factor, VopL. The structure represents the first high resolution snapshot of a filament-like nucleation intermediate, and reveals general principles underlying the action of nucleation factors.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 Tale of Two Hosts: Vibrio parahaemolyticus Toxicity Against Human and Shrimp(2016-11-17) Li, Peng; Liu, Qinghua; Orth, Kim; Fontoura, Beatriz; Michael, AnthonyBile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 (T3SS2) in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment. Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has generated severe damage to the global shrimp industry. AHPND is caused by toxic strains of Vibrio paraehaemolyticus that have acquired a "selfish plasmid" encoding the deadly binary toxins PirAvp/PirBvp. To better understand the genetic features of AHPND causing Vibrio paraehaemolyticus, we conducted a comparative analysis using the genome sequences of the clinical isolate RIMD2210633, environmental non-AHPND and toxic AHPND isolates of Vibrio paraehaemolyticus. Our studies discovered a distinguishing feature that a virulent Type VI Secretions System (T6SS1), like that in RIMD2210633, is present in all of the AHPND but none of the non-AHPND strains. This T6SS1 is potentially conserved in these virulent strains because of its anti-bacterial activities to compete against other commensal bacteria during host infection.Item [UT Southwestern Medical Center News](2008-08-18) McKenzie, AlineItem [UT Southwestern Medical Center News](2013-07-29) Wormser, DeborahItem [UT Southwestern Medical Center News](2010-08-19) Siegfried, AmandaItem Vibrio Effector Protein, VopQ Targets the Host Lysosome to Manipulate Autophagy(2014-07-23) Sreelatha, Anju; Tu, Benjamin; Orth, Kim; Goodman, Joel M.; Shiloh, MichaelVibrio parahaemolyticus is a gram-negative marine bacterium that is the major cause of gastroenteritis due to the consumption of contaminated raw or undercooked seafood. Vibrio parahaemolyticus harbors two Type III secretion systems. The first, T3SS1, orchestrates a temporally regulated cell death mediated by autophagy, membrane blebbing, followed by cell rounding and eventual lysis of the host cell. One T3SS1 effector protein, VopQ is both necessary and sufficient to induce rapid autophagy during the first hour of infection. Herein, I characterize the biochemical activity of the virulence factor VopQ, a novel Vibrio parahaemolyticus protein with no homology to any proteins outside of the Vibrio species. VopQ binds to the conserved Vo domain of the V-ATPase that is enriched on the lysosomal membrane and causes deacidification of the lysosomes within minutes of entry into the host cell. VopQ forms an ~18 angstrom gated channel that facilitates outward-rectified flux of ions across lipid bilayers. These studies show how a bacterial pathogen uses a novel, targeted pore forming effector to alter autophagic flux by manipulating the partitioning of small molecules and ions. Additionally, we demonstrate that VopQ is also a potent inhibitor of vesicular membrane fusion using in vitro membrane fusion. The inhibition of membrane fusion appears to be independent of VopQ’s pore-forming activity. VopQ inhibits the final step of membrane fusion by inhibiting trans-SNARE complex formation. In order to delineate the two inhibitory functions of VopQ, deacidification and membrane fusion, I use mutational, biochemical and crystallographic studies. Elucidating the molecular mechanism of VopQ not only provides a better understanding of Vibrio parahaemolyticus pathology but also offers new insight into the host cell mechanisms of autophagy and vesicle fusion.