Research On What Vibrio Cholerae Pathogenisity Related Genes Contribute To Colonization Ability In Reseroirs Host Intestines By Using Zebrafish Model

Vibrio cholerae is a gram-negative, facultative anaerobic, and aquatic dialister bacterium, and the pathogen that causes human diarrheal disease, Cholera. So far, although, there have been plenty of researches on the Vibrio cholerae genes regulations under conditions of laboratory and pathogensity, there are less studies on the survival of Vibrio cholerae in the natural waters and related genes expression and regulation, which is mainly due to the lack of suitable models. Studies have shown that fish may be the natural reservoirs of Vibrio cholerae. Thus, the zebrafish (Danio rerio) model has become a relatively simple and convenient animal model for studying the in vivo development or pathogenesis of microorganisms associated with animals in their hosts.In order to establish the zebrafish model for research the survival and colonization of Vibrio cholerae under natural conditions, we put zebrafish in liters filled with sterile tap water with about105CFU/mL Vibrio cholerae inside, which leads to the colonization in less than24h.Firstly, according to the competitive colonization experiments between key virulence genes in-frame deletion mutants and the wild-type V. cholerae, we found that the competitive indexes of△luxO and△aphA were separately the least0.122and the most7.655, which might infer that these two genes played most important roles in colonization. However, in the infant mouse model, the colonization ability of△aphA mutant inhibited was less than wild type strain, thus, the deletions of pathogenicity related genes had a relatively small influence on the colonizaiton of Vibrio cholerae in zebrafish model.Secondly, when inoculated alone, the number of△toxT mutant almost equalled with the wild type, presuming that the toxin co-regulated pilus (TCP) does not work during the colonization in zebrafish, which is the greatest difference while Vibrio cholerae colonizes in infant mouse. We supposed that the main aim of Vibrio cholerae colonized in zebrafish was to survive and to proliferate. Comparing the colonization numbers of in-frame deletion mutants and wild type Vibrio cholerae under two different inoculations,△hapR mutant may significantly increase11-fold the number of wild type strain, while wild type strain can make the amount of△aphB mutant decrease54-fold, when each two were inoculated together.In the last but not least important, we chose some in-frame deletion mutants from the LuxR family, LysR family and protease family to study further. Using bioassay method to examine the zebrafish gut extracts, we found there was tiny AHL, which made LuxR family gene vc1277(its competitive index is4.548) play a certain role. And researches implied that the GI tract of zebrafish could generate AHLs signal molecules, which are the inducers of LuxR proteins and combine with LuxR proteins to have an influence on the expressions of its downstream genes. While in LysR family, when inoculated alone, AoxyR mutant was about16-fold less in number than wild-type Vibrio cholerae; However, when inoculated together, the number of AoxyR mutant was nearly the same with wild-type Vibrio cholerae, while there were no differences in the same conditions of infant mouse model. Hence, the expressions of aerobic metabolism related genes regulated by OxyR played a role in anti-ROS for Vibrio cholerae in zebrafish GI tracts. Finally, among the selected protease gene in-frame deletion mutants, the number of AprtV mutant separated from waters was about83-fold less. This may suggest that prtV not only plays a role in the zebrafish gut colonization, but also in the survival of the natural aquatic environments.In summary, this study had shown that different genes influenced on the colonization abilities between infant mouse model and zebrafish model, and these genes have no direct relationships with pathogenicity. Therefore, this model may play a significant role in studying the survival mechanism of Vibrio cholerae in nature, as well as to.colonization in reservoir hosts. And even more, this model may provide a research platform for screening the key genes of Vibrio cholerae during the natural survival.