Typing Phage VP5 For Vibrio Cholerae O1 Biotype El Tor Strains And Its Receptor

National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention.State Key Laboratory for Infectious Disease Prevention and Control (China CDC).Cholerae is an acute lethal diarrhea caused by Vibrio cholerae. It has been found more than 200 kinds of different O antigen serogroups Vibrio cholerae, of which only-O1 and O139 serogroups can cause cholera epidemics. Seven cholera pandemics have occurred in history. It is still in the seventh pandemic caused by Vibrio cholerae El Tor biotype. Vibrio cholerae phagetyping is commonly used typing method. In china,O1 El Tor Vibrio cholerae is divided into 32 phagetypes and 12 biotypes by Vibrio cholerae phagetyping combined with the biological classification. These can be used as tools for trace infection sources, transmission and analysis of popular forms of epidemiological.To understand the mechanism of phage-biotype guide to cholera prevention and control, to understand the O1 El Tor Vibrio cholerae genetic diversity and differentiation, it is important to explore the morphology, genome features, the mechanism of Vibrio cholerae phage infection and the differences between the phage-sensitive strains and resistant strains.In all phage-biotypes those can cause cholera epidemics, only 6b is not sensitive to the phage VP5. Between 1998 to 2001, cholera epidemic caused by 6b phage-biotype Vibrio cholerae in Sichuan Province, while dominant type of epidemic strains in other provinces in the same period and that in Sichuan Province before 1998 and after 2001 are all 1b strains. We try starting from this phenomenon, to understand prevalent mechanism of 6b which insensitive to VP5 phage, to understand the process of Vibrio cholerae VP5 lysis. All prevailing strains in Sichuan Province from 1998 to 2001 determined as 1b and 6b by phage-biotyping were toxigenic. A total of 24 patterns were obtained in PFGE analysis, in which one predominant pattern consisted of 13 strains. Parts of 1b and 6b strains from Sichuan and parts of the lb strains from other provinces showed the same PFGE pattern. Mutation 11bp in ompW gene was found in 6b strains. Vibrio cholerae 01 6b strains in Sichuan Province from 1998 to 2001 have special genetic markers, and may evolutionary correlated with contemporaneous 1b strains.Phage VP5 observed with electron microscopy have hexagonal head and short-tailed. Therefore, we speculated that VP5 is icosahedron. We sequenced the whole genome of VP5 and get a sequence of length 39789bps, predicted 36 ORF of which 16 are in positive chain and 20 are in negtive chain. The average length of VP5 ORF is 941bp, the longest gene is 2349bp. In this genome rRNA and tRNA gene was not found. We confirmed OmpW protein in N16961 is the receptor of phage VP5, for the ompW deletion mutant in N16961 get resistant to VP5 and the complemeted mutant strain is sensitive to VP5. Expressed OmpW protein can neutralize Vibrio cholerae N16961 VP5 lysis, that supports the view.Initially, we knew morphology, genome and receptor of 01 El Tor Vibrio cholerae typingphage VP5, and we found the tracking markers of 6b strains. There is a continuing need for the development of simple animal models for the study of host-pathogen interactions. In the study of pathogenic bacteria, the appropriate model organism is very important for the experimental reliability, ease of use and analysis of experimental results. In order to understand the pathogenesis of bacterial pathogens or environmental survival related information, we need to simulate the case of infection or symbiosis during the process of interaction between pathogenic bacteria and model organisms. Currently, rabbits, mice, rats, etc. are used for Vibrio cholerae research. But those animal models have a variety of defects such as operate complexity, relatively expensive, and implementation difficulties to experiments e.g. large scale screening of strains. And because of the complexity of mammals, there are so many factors that could influence the results, which bring limitations to analysis of experimental results.Caenorhabditis elegans feed on bacteria, so it is easy to feed in the laboratory. Caenorhabditis elegans became a new model organism as a facile and inexpensive host for human bacterial pathogens. Since 1999 it has been used in Pseudomonas aeruginosa, Enterococcus faecium, Staphylococcus aureus, Shigella flexneri, Pseudomonas aeruginosa, Yersinia, Listeria, Salmonella enterica, Burkholderia, Streptococcus pyogenes, Salmonella typhimurium, Bacillus thuringiensis, Serratia marcescens, Cryptococcus, Microbacterium nematophilum et al, to identify virulence factors, pathogenetic mechanism, host-pathogen interactions, biofilms, drug-resistance. As well as facilitating the identification and study of virulence mechanisms, simple model systems may also permit direct genetic approaches for the study of host defenses.In this study, we first determine Caenorhabditis elegans can feed on Vibrio cholerae. Compared with those on E. coli OP50, Caenorhabditis elegans growth and development process did not change significantly on the Vibrio cholerae N16961. We have chosen 24 strains Vibrio cholerae 01 and 0139 serogroup, with CT positive or negative. These strains were verified by PCR the existence of prtV gene, which is considered needed for killing of Caenorhabditis elegans. E. coli OP50 as the control, we detect the ability of Vibrio cholerae 24 strains to kill Caenorhabditis elegans by making survival curves. The results showed that Vibrio cholerae used in experiments can make Caenorhabditis elegans survival time significantly shorter. Vibrio cholerae 01 serogroup were able to kill Caenorhabditis elegans worms as efficiently as Vibrio cholerae 0139 serogroup. But We found the CT-positive group killed worms more quickly than CT-negtive group.This study is a initial attempt to use Caenorhabditis elegans on the study of Vibrio cholerae. The many experimental advantages associated with the use of the nematode Caenorhabditis elegans led us to investigate its interaction with Vibrio cholerae. Finally, we provide evidence that Caenorhabditis elegans may be a useful model host for Vibrio cholerae.