Karla J. Satchell - US grants

[unreadable] DESCRIPTION (provided by applicant): Vibrio cholerae strains that do not produce cholera toxin induce a more inflammatory diarrhea than normal cholera disease, implicating other potent toxins in the pathogenesis of cholera. Several accessory toxins of V cholerae are purported to account for this reactogenic response. One of these factors is the newly discovered VcRtxA toxin of V cholerae. VcRtxA is a large protein toxin that is a unique member of the RTX family Production of this toxin has been evolutionarily conserved by Vibrio sp indicating that maintenance of this large toxin is essential for virulence or survival in the environment. Its cytotoxicity has been further shown to function by a novel mechanism This toxin causes depolymerization of actin stress fibers in both polarized and non-polarized cell lines by a unique pathway Concurrent with depolymerization, the actin molecules become covalently linked together into dimers, trimers, and higher order multimers This observation suggests that covalent crosslinking of actin by the toxin drives actin depolymerization This unusual reaction distinguishes VcRtxA from all other bacterial toxins that cause actin depolymerization. Research performed under this grant proposal will investigate further the novel biochemical properties of this important virulence factor The VcRtxA toxin is the largest single polypeptide protein toxin ever described, however, it is unclear whether the full 4545 amino acid protein is the toxic moiety The size of the toxic moiety and potential post-translational modifications will by identified by examining biochemical properties of the active form of purified toxin The catalytic activity will then be investigated and the role of a putative catalytic domain in this reaction will be assessed. It will then be established whether the toxin is active at the membrane or within the cytoplasm of the target cells, and the role of a putative actin-binding domain will be characterized. [unreadable] [unreadable]

Abstract The life-threatening diarrheal disease cholera is caused by toxigenic strains of the Gram-negative organism Vibrio cholerae. In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V. cholerae secretes a novel toxin that is the founding member of new family of Multifunctional, Autoprocossin RTX toxins (MARTX). The MARTX toxin of V. cholerae (MARTX-Vc) contributes to virulence in mice and may be an important factor contributing to persistent colonization both in cholera patients and non-symptomatic carriers.. MARTX-Vc is of intrinsic interest due to its novel biochemical properties and mode of action. At a predicted size of >480 kDa, the toxin is a multifunctional toxin. Thus far, we have described three activities associated with this toxin. We have demonstrated that this toxin depolymerizes actin stress fibers and covalently crosslinks actin into oligomers. We have demonstrated that this toxin causes rounding of cells due to inactivation of RhoGTPases by an unknown mechanism. Finally, this toxin has a cysteine protease domain with autoprocessing activity that may also have cellular targets resulting in cytotoxicity. Each of these activities has been mapped to a specific domain of the toxin. A fourth domain homologous to the alpha-beta hydrolase family of proteins is also a putative activity domain. In this grant proposal, we will pursue the multiple catalytic activities of the toxin by studying the mechanism of action of the independent activity domains. A structure [unreadable] function analysis of the actin crosslinking domain will be performed and the regions essential for catalysis and actin binding will be identified. The target of the RhoGTPase inactivating domain will be identified and characterized. The requirements for autocleavage site recognition and inositol hexakisphosphate binding will be investigated and other potential sites of cleavage identified. In all, this work will further our understanding of the mechanism of action of this toxin and provide insight into its role in disease and will also provide information about related toxins that share the novel functional domains carried by this unique multifunctional toxin. Lay summary The life-threatening diarrheal disease cholera is caused the Gram-negative organism Vibrio cholerae. In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V. cholerae secretes a novel toxin that is the founding member of new family of the RTX toxins, the Multifunctional-Autoprocessing RTX toxins (MARTX). The MARTX toxin of V. cholerae contributes to virulence in mice and may be an important factor contributing to persistent colonization both in cholera patients and non-symptomatic carriers. This proposal seeks to understand the cellular mechanism of action of this toxin that has three known enzymatic activities: actin crosslinking, Rho-inactivation, and autoprocessing. Completion of this research will impact not only our understanding of cholera pathogenesis, but also the function of other uncharacterized toxins produced by other human pathogens that share these unique enzymatic activities.

DESCRIPTION (provided by applicant): Vibrio cholerae O1 is the causative agent of pandemic cholera, a severe diarrheal disease still prevalent worldwide. The major etiologic agent of the Seventh Cholera Pandemic that began in 1961 and continues today is the cholera toxin producing El Tor O1 strains. These strains were first thought to have low virulence potential as they cause disease of less severity than "classical" strain responsible for earlier pandemics. However, the lowered virulence of El Tor strains is believed to have contributed to their pandemic spread since a majority of infected persons do not become ill but do become colonized and excrete the infectious organisms. Indeed, one factor thought to contribute to the emergence of El Tor strains is their ability to persist an average of 5 days in asymptomatic carriers compared to only 1.5 days for classical strains. Thus, El Tor strains have genetic factor(s) absent in classical strains that contribute to prolonged colonization. These genetic factors had not been previously identified. Using a new animal model of cholera amenable to studies of the host immune response, we have demonstrated that accessory toxins hemolysin and RTX are key factors in establishment of prolonged V. cholerae colonization of the crypts of the small intestine. This grant will continue examining the connection between hemolysin, RTX and innate immunity using coinfection studies to determine if colonization defects can be extracellulary complemented. This study will also investigate the cell types recruited to the gut associated lymphoid tissue during early V. cholerae infection. Finally, this project will specifically determine whether neutrophils and mast cells are key components of the innate immune response to V. cholerae and whether these cells are disabled by hemolysin and RTX toxin both in vivo. PUBLIC HEALTH RELEVANCE: Vibrio cholerae O1 is the causative agent of pandemic cholera, a severe diarrheal disease still prevalent worldwide. The epidemiology of modern cholera is distinct from the Classical disease as epidemics are caused by lower virulence strains that induce a high frequency of mild or asymptomatic infections. This prolonged period of vibrio excretion likely contributes to transmission of disease. Using a new animal model of cholera, accessory toxins hemolysin and RTX are shown to be key factors in prolonging intestinal colonization by V. cholerae. This project will investigate key components of the immune response to V. cholerae and identify is accessory toxins are associated with disabling innate immunity.

Project Summary Vibrio vulnificus is a natural inhabitant of coastal waters, including the US Gulf. The bacterium causes severe life threatening infections after consumption of contaminated seafood (especially raw oysters) and from wounds contaminated by seawater. Among food-borne pathogens, V. vulnificus is most notable for its high rates of hospitalization and death and its negative economic impact. Indeed, V. vulnificus accounts for 72% of deaths from Vibrio illnesses despite causing only 13% of infections. Although infections are rare, the number of serious infections has been increasing globaly due to climate change that has caused a rise in the number of days amenable to growth of V. vulnificus in coastal waters and the geographical area amenable to the pathogen. Attempts by various agency to warn and protect citezens by implementation of new policies or issuing warnings have met with resistance for it effect on the shellfish harvesting industry and the tourist economy. Thus, the study of V. vulnificus pathogenesis has become both a food safety and public policy priority. A significant virulence factor of V. vulnificus is the large Multifunctional-Autoprocessing RTX toxin (MARTXVv). This toxin is comprised of long repeat regions that are associated with cellular necrosis, but this activity is not sufficient for virulence. Rather, virulence is associated with ?effector domains? that are translocated across host plasma membrane by repeat regions, and then released to the cell cytosol by inositol hexakisphophate induced autoprocessing. Bioinformatics studies reveal that different clinical isolates of V. vulnificus express distinct forms of the toxin, with five different variants assembled from eight different MARTX effector domains. To date, the mechanism of action of five of these domains has been determined. In this project, we will investigate the mechanism of action of the remaining effector domains found in clinical isolates. Further, we will study the relative toxicity of different variants of MARTXVv toxin in pathogenesis by the food- borne route of infection.

DESCRIPTION (provided by applicant): Vibrio vulnificus is a natural inhabitant of coastal waters, including the US Gulf. The bacterium causes rapid septicemia after consumption of contaminated seafood (especially raw oysters), predominantly in persons with liver dysfunction. Among food-bourne pathogens, V. vulnificus is most notable for its high rates of hospitalization and death. Indeed, V. vulnificus accounts for 1% of deaths from food-bourne illness despite causing only 0.003% of illnesses. Attempts by the FDA to protect consumers by implementation of post- harvest processing mandates met with significant resistance from the shellfish harvesting industry forcing the FDA to review its policies. Thus, the study of V. vulnificus pathogenesis has become a food safety and public policy priority. Recent studies establish that cytotoxicity of V. vulnificus is predominantly associated with a large Multifunctional-Autoprocessing RTX toxin (MARTXVv). In this study, we demonstrate that this toxin is directly linked to pathogenesis by the intragastric route of infection. We propose to study the role of the MARTXVv toxin in pathogenesis using in vivo, cell culture, and biochemical systems. The focus of the proposal will be mechanisms directly impacting early infection after consumption of contaminated food and identification and characterization of specific regions of the toxin linked to tissue damage and immune system evasion.

Bacteria within the Vibrio genus have a huge impact on the biology of the earth, and especially on humans. A number of Vibrio spp. cause significant human disease via consumption of contaminated food and water, and many others cause disease in marine vertebrates and invertebrates, and even in corals, thus directly or indirectly affecting humans. November 12-15, 2017, we will be holding the conference ? ?Vibrio2017: The Biology of Vibrios? ? the seventh in a series of international conferences on the subject of the biology of Vibrios. This conference will build on the successes of the first six conferences held in Belgium, France, Brazil, Spain, Scotland, and France from 2005-2016, respectively. This conference will be held in the U.S. for the first time, geographically situated in Chicago for ease of travel for both domestic and international participants. The American Society for Microbiology is highly qualified and willing to organize this conference. The confirmed speaker list already includes a diverse set of scientists that is balanced for age, gender, and racial diversity, includes speakers from the US, Asia, and Europe, and covers topics across the spectrum of Vibrio physiology, ecology, and pathogenesis. We are seeking NIH support for this conference that most closely aligns with the National Institute for Allergy and Infectious Diseases. The requested funds from NIH will be utilized for three purposes: 1. To provide funds to increase the number of supported keynote speakers, in order to increase the scientific quality of the meeting, 2. To provide funds to defray the costs of audiovisual equipment rental for the conference, and 3. To offer additional travel awards for post-docs, graduate students, and undergraduate-level students whose abstracts are selected for presentation at the conference. This support from NIH will dramatically enhance the quality and accessibility of this conference to maximize attendance and ensure the participants gain useful knowledge that will ultimately assist to minimize the impact of Vibrios on the human population.

The goal of the Structural Genomics Centers for Infectious Diseases is to apply state-of-the-art, high-throughput structural biology technologies to experimentally characterize the three-dimensional atomic structure of targeted proteins on the NIAID Category A-C priority pathogens list and organisms causing emerging and re-emerging infectious diseases. This program also supports research projects that utilize the 3D structure information and experimental approaches to better characterize the molecular functional role of protein targets. The primary focus of the Center is pathogen proteins that are expected to have an important biological role and a potential impact on biomedical research.

ABSTRACT Vibrio vulnificus is a Gram-negative bacterial pathogen that causes severe life-threatening infections in humans after eating shellfish or swimming in warm seawater. In the US, there are about 200 cases annually with high rates of mortality and morbidity. While the number of cases is low compared to other food-borne pathogens, the high rates of death among patients exerts a significant burden on human life and the economy. This is expected to increase as incidence of V. vulnificus infections is rising due to the climate crisis. The most prominent virulence factor of V. vulnificus is a 5208 aa MARTX family toxin that delivers cytotoxic effectors to cells and these effectors are essential for virulence in a mouse model. While extensive information is known about the mechanisms of the effectors, very little is known about the remaining over 2800 aa of the protein toxin. These regions are known to be both necessary and sufficient for secretion of the toxin from the bacteria, interaction of the toxin with the eukaryotic cell surface, and translocation of the effectors across the plasma membrane. The project proposes to identify host cell receptors and host factors essential for intoxication using a genetic screen and selection for mutant cells that survive the cytolytic action of the toxin. We will also use a structure-function based approach to identify regions of the MARTX toxin essential for Type I secretion from the bacterium, binding of the toxin to a putative surface receptor, formation of a pore in the plasma membrane, and translocation of effectors to the eukaryotic cell cytosol. This study will advance our understanding of the function of the MARTX toxin repeat regions in detail and is expected to impact our understanding of pathophysiology during V. vulnificus infection.