Molecular And Cellular Mechanisms Of The Cytotoxicity Induced By Vibrio Parahaemolyticus Isolates And Their Virulence Related Factors

Vibrio parahaemolyticus (V. parahaemolyticus, Vp), a member of the genus Vibrio, is Gram-negative, halophilic, heterophilic bacteria. Among pathogenic vibrios, Vp is one of the emerging food-borne pathogens, that are commercially associated with various shellfish species throughout the world. It is strictly environmental and many strains are pathogenic to human being, but its mechanisms of pathogenicity for human still not yet understood. It’s widely distributed, seasonally abundant, belong to warm climates estuarine, marine, and coastal environments all over the world as a natural inhabitant. It is commonly found free swimming or attached to underwater surfaces or commensally associated with different shellfish species. The virulent strains cause distinct diseases, including wound infections, septicemia, and most commonly, acute self-limiting gastroenteritis in human characterized by diarrhea, headache, vomiting, nausea, and abdominal cramps, acquired of this bacterium to human through direct transmission via consumption of raw or undercooked seafood, especially shellfish and indirect transmission via cross-contamination of Vp to other food during preparation, which considered as a major cause of gastroenteritis worldwide. In the immunocompromised host with significant systemic disease, it can lead to death. This pathogen is a common cause of food-borne illnesses in many Asian countries, including Japan and China, it was also observed in the United States.Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Vp has two type three secretion systems (T3SSs), which are a needle-like apparatus enable bacteria to deliver their effectors proteins into host cytoplasm triggering its cytotoxicity and enterotoxicity via their effectors. Over than 80 serotypes have been described, based on the somatic (O) and capsular (K) antigenic properties. O3:K6 is the most predominant of Vp pandemic strains that produces a thermostable direct hemolysin (TDH) and/or a TDH-related hemolysin (TRH). Recent advances in Vp genomics identified pathogenicity islands (Vpals) which either located on chromosome in both epidemic and pandemic strains or comprising additional colonization factors.Vp can cause intestinal fluid accumulation and watery diarrhea in mammalian models such as rabbit and rat, red leg disease of shrimp, and off-board of abalone. Although the mechanisms underlying the diarrheal action are not fully understood, the intestinal disease is linked to some virulence properties including adhesiveness and other virulence factors such as exoenzymes and toxins (TDH or TRH). These virulence factors have been confirmed to be cytotoxic and enterotoxic in several cell models and animal models, nevertheless, little is known if these cytotoxic and enterotoxic effects are host specific. In addition, the original host of Vp pandemic strains is still a mystery issues to human.One of the snags facing researchers who try to study this bacterium is the uncompleted understanding of pathogenicity as the previous feedback concluded that the linkage between strains isolated from the environment, seafood, and human clinical isolates are poorly understood in contrast to the other foodborne infections. In addition to that the caused infection by Vp has no relation with socioeconomic status, meteorological changes, the quality of the water supply and general sanitation. Many studies have been done on the virulence factors of this bacterium. Previous studies compared the pandemic and non-pandemic strains to understand the evolution of the pandemic strain, but no definitive explanation for its emergence has been discovered.The main research objectives and goals of present research was undertaken to:investigate the cytotoxic effects caused by Vp pandemic and non-pandemic strains on human non-phagocytic cells for better understand of its virulence mechanisms in vitro; to develop and characterize the Vp mutant strains of some virulence-related genes, and compare their effects on cytotoxicity and enterotoxicity comparing to their parent strain RIMD2210633; finally, explore the innate immune responses against Vp in vitro.To better understand the pathogenesis of Vp, we established cellular infection model in vitro using a non-phagocytic cell line Caco-2 as the preliminary part of our research, then, according to our findings in the second part of the investigation was designed as an exploratory study to clarify Vp pathogenicity in vitro and in vivo, with a particular focus in some genes at the genome sequences of Vp. The main results were as follow:A. All V. parahaemolyticus (pandemic and non-pandemic) clinical virulent isolates adhered to Caco-2 monolayers and could invade non-phagocytic epithelial cells. They also survived and persisted in non-phagocytic cells by modulating reactive oxygen species (ROS), allowing its replication, and resulting in complete cellular destruction.B. Additionally, we investigated the host cell response to some genes (VPA1656, VP2118, and VPA1321) through the construction of gene deletion mutant in RIMD2210633 which hypothesized to be required for organism virulence during cellular and molecular mechanisms of pathogenicity. Overall, the findings of this part included:VPA1656 is insignificant to V. parahaemolyticus virulence under normal circumstance. A VPA1656 gene deletion mutant (RIMDΔVPA17656) showed no significant difference in cytotoxicity and enterotoxicity effect in comparing to the parent (wild-type, WT) strain. Where the results revealed that mutant strain could adhere and also invade the monolayer cells, plus to the modulation by decreasing of ROS more than uninfected cells as well as its other related parameters measured (including NO and pH) using FACS in contrast to increase of both Ca2+ and MMP. LDH and cell death effect caused by this mutant strain was nearly the same to those by WT strain. Also, there was no significant difference in the infant rabbit infection model, as the results of colonization and survival of the mutant were equivalent to that from WT strain. Furthermore the fluid accumulation ratio significantly not differed, compared with the parent strain, the histopathological images showed complete necrosis in the most parts of the intestinal tract of infected infant rabbits in response to both of mutant and WT strains.Vp2118 plays a great role in V. parahaemolyticus survival and persistence. Reactive oxygen species (ROS), including superoxide anion radical, which induce a chronic risk of oxidative damage to many cellular macromolecules resulting in cellular damage. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vp can survive ROS generated by intestinal cells in vitro. However, there is little information concerning SODs in Vp. This part of study aims to clarify the role of Vp SODs against ROS activity. Vp SOD gene is named as FeSOD (VP2118). VP21J8 mutant strain of Vp (RIMD) was constructed and its SOD activity and resistance to oxidative stresses generated by Caco-2 cells in vitro and also that activity and effects on infant rabbits were measured. In a VP2118 gene deletion mutant (RIMDΔVP2118), SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. A significant decrease in adhesion and invasion indices in Caco-2 cells and lowered resistance to ROS were observed, which could be recovered by VP2118 gene complementation, in comparing with WT strain. The results revealed that the mutant strain had a less cytotoxic effect on non-phagocytic cells as there was a markedly decrease in LDH, cell apoptosis, and death percentage. Moreover, the bacterial load and survive was unequal and relatively decreased compared to the parent strain. The fluid accumulation ratio was low, furthermore, the histopathology images of the most intestinal tracts parts presented mild focal necrosis legions, in comparing to complete necrosis of intestinal tracts in infant rabbits infected with WT. The production of pro-inflammatory and anti-inflammatory cytokines of infected human non-phagocytic cells were measured and compared, the levels of them in infected cells by the mutant were significantly declined compared to those for WT strain. There was a positive correlation between the cell toxic parameters in vitro and the entertotoxicity parameters (bacterial colonies and survive plus to fluid accumulation ratio) in vivo. It could conclude that VP2118 is the primary Vp SOD gene and has a vital role for anti-oxidative stress responses. The Vp FeSOD VP2118 may enhance ROS resistance generated by non-phagocytic epithelium cell in vitro and could promote its survival in the intestinal tract to facilitate host tissue infection in infant rabbits as animal model in vivo.VopC unshares in V. parahaemolyticus pathogenicity. A VPA1321 gene deletion mutant constructed in RIMD had a highly significant difference from WT as it was unable to invade cell except less than 1% of mutant strain still could invade the cells, furthermore this mutant strain still had the ability to adhere to monolayer cell. Otherwise, the virulence not less than RIMD in vitro as the modulation of the measured parameters (ROS, NO, pH, Ca2+, and MMP) and the same observation detected when evaluate the LDH from cells treated with both of mutant and WT strains, moreover completely cell destruction had been seen.In conclusion, we have characterized aspects of non-phagocytic cells in response to infection with Vp (RIMD, pandemic, and non-pandemic strains) virulent clinical strains using flow cytometry, as well as adaptations acquired by this gastrointestinal pathogen to evade the host innate responses that may potentially contribute to pathogenesis by preventing rapid elimination from infected host tissues, where the pathogenicity of Vp is based on its capacities for adhesion and invasion, surprisingly, enhanced of ROS resistance period could promote the survival of Vp inside the intestinal tract, facilitating tissue infection by repressing the host’s oxidative stress response, as infections modulated ROS production in infected cells. And in response to ROS production, Vp was found to induce bacterial antioxidant genes encoding anti-oxidant enzymes, such as FeSOD (VP2118) to facilitate pathogen survival. That agrees with the hypothesis of superoxide dismutases (SODs) genes which is vital for anti-oxidative stress responses which can enhance ROS resistance and could promote Vp survival inside the intestinal tract to facilitate tissue infection, the results that assured in vivo using an infant rabbit model.