Function Study About Receptor Synthesis Related Gene Cluster Of Yersinia Enterocolitica O:3 Specific Phage PhiYe-F10

Phages are the most abundant and diverse microbial entities in the biosphere.The first step of phage infection is the recognition of the receptor.Bacteria can escape from phage lysis through the loss or change of the phage receptor.Therefore,the research on phage receptor has always been the most concerned part of the research on the interaction between phage and host bacteria.Yersinia enterocolitica is one of the three pathogenic yersinia and global zoonotic pathogen.The majority of patients infected with the bacteria by the fecal-oral route and resulted in Yersiniosis.Yersiniosis can cause abdominal pain and diarrhea,which are easily misdiagnosed as appendicitis or even removed by surgery due to the similar clinical manifestations with appendicitis.In addition,many patients suffered from chronic complications of Yersiniosis,such as reactive arthritis and endocarditis,which seriously affect the quality of patients’ life as a result of loss effective treatment due to misdiagnosis and other reasons.The infection of yersinia enterocolitica has a worldwide distribution.Hundreds of countries had infection.Currently,yersinia enterocolitica is divided into more than 60 serotypes according to the differences of lipopolysaccharide(LPS)structure.In recent years,O:3 serotype strains gradually increased and became the dominant serotype.PhiYe-F 10 is a lytic phage which is specific to yersinia enterocolitica O:3.This phage isolated from the porcine anal swabs collected from the slaughterhouse of Henan province during the monitoring of yersinia enterocolitica in China by our research group through plaque formation experiment.We also isolated a pathogenic yersinia enterocolitica 3/O:3 HNF10 carrying toxic plasmid pYV.Our research group has confirmed that o-antigen is the receptor of phage phiye-F10,and gene rfbC that was in rfb gene cluster is the synthesis regulator gene of the receptor of phage phiye-f10 rfb gene cluster includes 10 open reading frames,among which rfbABC and rfbDEFGH are related to o-antigen synthesis.We successfully constructed single mutant strains HNF 10-△rfbA,HNF 10-△rfbB,HNF 10-△rfbF,HNF10-△rfbG and complemented strains HNF10-△rfbA/CrfbA,HNF 10-△rfbBICrfbB,HNF10-△rfbF/CrfbF and HNF10-△rfbG/CrfbG by using homologous recombination.Phage sensitivity test showed that the deletion of rfbA gene reduced the sensitivity of mutant strain to phage.The deletion of gene rfbB,rfbF,rfbG all caused mutant strains resistant to phage phiYe-F10.In order to explore the reasons for the changes in the sensitivity of each deletion strain,we first conducted the polyvalent serum agglutination experiment of yersinia enterocolitica O:3.The results showed that the mutant strain HNF10-△rfbA still retained agglution with O:3 antiserum,while serum agglutination results of the mutant strain HNF 10-△rfbB,HNF 10-△rfbF,HNF10-△rfbG were negative.And the corresponding gene complemented strains restored the ability of agglutination with O:3 serum.Thus,we supposed that the deletion of genes rfbB,rfbF and rfbG leads to the structural changes and even deficiency of o-antigen,making the phage phiYe-F 10 unable to infect bacteria due to the deficiency of receptor.To reveal the reason why HNF 10-△rfbA reduced the sensitivity to phiYe-F10,we monitored the growth curve of HNF10-△rfbA for 14 hours.The OD600 values of wild type strain began to decrease after two hours adding phage phiYe-F10.While the growth curve of HNF10-△rfbA began to decrease after 5 hours adding phage phiYe-F10(approximately 2×10~5PFU of phiYe-F10 in 1ml).The growth curve of HNF10-△rfbA/CrfbA and wild type strain were basically identical.The results showed that the deletion of gene rfbA significantly increased the adsorption or nucleic acid injection time of phage phiYe-F10.To confirm whether gene rfbA affect phage adsorption,we conducted a phage adsorption experiment.The experimental results showed that compared with wild strain HNF 10,adsorption rate of HNF 10-△rfbA decreased obviously,while HNF 10-△rfbA/CrfbA had no significant difference.By comparing the phenotype of mutant strains,complemented strains and wild strain that are sensitive to phage HNF10 and the product function of each gene in rfb gene cluster involved in O-antigen synthesis,we confirmed that gene rfbA was a synthesis related gene of phage phiYe-F10 receptor and its coding product probably involed in the synthesis regulation of the phiYe-F10 receptor domain recquired in the reversible binding or irreversible attachment between the phage phiYe-F10 tail filament protein and host bacteria.The genes rfbB,rfbF and rfbG were indispensable genes required for the synthesis of the phiYe-F10 receptor,and the deletion of these genes resulted in the failure of synthesis of phage phiYe-F10 receptor.The genes rfbD,rfbE and rfbH knockout strains were not obtained by attempting many times.We supposed genes rfbD,rjbE and rfbH may be essential for bacterial survival under our experimental conditions and their functions need to be further studied.The comprehensive analysis of function of individual genes in rfb gene cluster that were involved in the phage receptor synthesis will provide a theoretical basis for not only the further development of utilizing phages for the prevention and control of infection and spread but also the control of yersinia enterocolitica O:3 spread in global.In addition,the study of phage receptor synthesis related genes has important guiding significance for the development of phage therapy which provides a more scientific alternative method to solve the increasingly serious problem of bacterial drug resistance.