Screening And Biological Traits Of Salmonella Typhimurium Factor H Binding Protein

Salmonella enterica serovar Typhimurium are gram-negative pathogens that causes serious diseases such as gastroenteritis,septicemia and typhoid fever.To establish a successful infection,Salmonella must possess effective mechanisms for overcoming host innate immune defenses.The complement system is an important component of natural immunity,and it is the host's first line of defense against pathogens.Factor H is a negative regulator of the complement system that inhibits the alternative complement pathway,its primary function is to protect the host cells from unwanted complement damage.Microbial pathogens have evolved the ability to hijack host factor H by factor H-binding proteins(FHBPs),leads to reduced complement activation and bactericidal effect.At present,there are relatively few studies on factor H-binding proteins in Salmonella,and only Rck protein has been reported to have factor H-binding activity.Therefore,in this paper,we aim to find the FHBPs of Salmonella and reveal their binding ability to factor H.We identify novel S.Typhimurium FHBPs and characterize their biological function.We explore factor H-binding protein potential application in subunit vaccine.We aim to provide a new strategy for the prevention and treatment of Salmonella.In this study,we proved that S.Typhimurium can interact with Factor H to evade complement-mediated lysis.Then,we identify PK,EF-G,and OmpC interacted specifically with factor H.The mouse model was used to investigate whether these proteins could be potential candidate antigen.Finally,we analysis biological function of potential FHBPs of S.Typhimurium strain x3761.1.Identification of anti-complement killing of S.TyphimuriumTo test whether S.Typhimurium can escape the complement killing.In this study,we incubated strain ?3761 with NHS,demonstrated that the survival of S.Typhimurium in human serum was decreased in a time and concentration dependent manner.It is verify that the complement system involved in killing Salmonella.Then we incubated different strains with normal human serum.We found that the different virulence strains had different ability to escape from the serum-mediated killing,suggesting that some different genes in S.Typhimurium helped the pathogen escape from the serum-mediated killing.Pre-incubated with factor H attenuated the sensitivity of S.Typhimurium strain ?3761 to complement-mediated serum killing,suggesting factor H contributes to complement resistance.Therefore we assumed that factor H could interact with FHBPs and be involved in protection against complement killing.2.Screening and binding traits of S.Typhimurium factor H binding proteinTo explore factor H-binding proteins potential biological function.We identified six potential FHBPs by two-dimensional(2D)-Far-Western blot,and five of them were expressed as recombinant proteins,then we confirmed EF-G,OmpC and PK binding ability to FH by western blot and dot blot.To explore factor H-binding protein potential application in subunit vaccine,we evaluated the humoral immune level of the factor H-binding proteins in a mice model,through the animal protection test to evaluate the recombinant protein EF-G,OmpC and PK,two weeks after second immunization mice were challenged by intraperitoneal route of x3761,results show that the recombinant protein OmpC,EF-G and PK couldn't provide 100%protection against ?3761,3.Construction and biological function analysis of fhbps deletion strains of S.TyphimuriumTo examine the functions of potential fhbps of S.Typhimurium.We constructed mutants of ompC,sdhA and mdh.We incubated strain ?3761 or three mutants with NHS,and found that AompC strain in NHS was significantly lower than ?3761 strain.There is no significant difference in the survival rates of ?sdhA strain and Amdh strain compared with ?3761 strains in serum,confirming that factor H-binding proteins OmpC can protect Salmonella against serum killing by binding factor H.The in vitro assays indicated that the ompC,sdhA and mdh mutation strain does not affect ?3761 strain adhesion to HeLa cells.Furthermore,to evaluate the role of inactivation of ompC,sdhA and mdh on the virulence of ?3761 strain,BALB/c mice were inoculated by oral challenges with the ?3761 strain and three mutants,the in histopathological assays showed that deletion of ompC,sdhA and mdh had no significant protection on the lesions or viability in BALB/c mice of ?3761 strain infection.To investigate the effects of deletion of ompC in the pathogenicity of ?3761 strain,six groups BALB/c mice received intraperitoneal challenges with 2×10 CFU,2×102 CFU or 2×103 CFU of the wild-type strain ?3761 strain or the ?ompC strain.There was no significant difference between ?3761 strain and ?ompC strain in the virulence of S.Typhimurium.Taken together,mouse infection model suggested that OmpC is not a critical virulence factor of S.Typhimurium.In summary,OmpC protein can improve the survival rate of S.Typhimurium in the blood,but it is not a key factor critical virulence factor in the pathogenicity of S.Typhimurium.