The Expression Of FasB/C/A Proteins Of Group A Streptococci In E.coli And Preliminary Function Study

Streptococcus pyogenes (group A streptococci, GAS) is an important human pathogen capable of causing a wide variety of infections in a human host. Such infections comprise comparatively mild superficial diseases of the throat and skin and, less often, infections of deep-seated connective tissues and, occasionally, septicaemia.In order for pathogenic bacteria to colonize and cause an infection in a host, adaptive regulation of virulence factors/traits is required to compete with host defence mechanisms, growth-limiting conditions and other environmental conditions at the site of infection. Several pathogenic species achieve this goal through either population density-dependent regulation or growth phase-dependent regulation of their virulence factors.The fas operon was found to control growth phase-associated expression of certain GAS virulence genes. This operon is unusual as it contains two potential sensor kinases and one response regulator. It is differentially expressed across bacterial growth phases, and the types of virulence factors controlled by this novel two-component regulatory system exhibit features that demonstrate similarities to the agr system in S. aureus. Downstream of the fas operon is a gene encoding a short RNA molecule. Its expression was dependent on a functional fasA regulator gene. Using deletion mutants and complemented strains, it has been demonstrated that the small fasX RNA is the main effector molecule of this operon controlling virulence factor expression by as yet unknown mechanisms.FasB, FasC and FasA, the three key proteins in the fas operon were chosen in our research. First, recombinant FasA and NusA-FasA fusion protein were expressed by using two different expression vectors and the anti-FasA sera were prepared. Then, the endocellular parts of FasB and FasC were expressed as the forms of NusA-FasB and NusA-FasC fusions. Different methods were used to test whether there is an existing binding between FasA/FasB or FasA/FasC. It has been shown that there is no direct binding existed between FasA /FasB or FasA /FasC. Meanwhile, according to the reported fasX's potential promoter region, we designed specific primers to amplify the promoter region of fasX. By using electrophoretic mobility shift assay, which is an effective method to identify the interaction between proteins and nucleic acids, we tested whether FasA could bind fasX's potential promoter region. Results showed that there is no direct interaction between FasA and fasX's potential promoter region. Our results suggested that a related molecular-mediated phosphorelay might exist between FasB/C and FasA.So far, the study on fas operon is still at the initial stage. Our work is only preliminary study but it will be of great help in understanding function of fas operon in GAS infections.