MOLECULAR DISSECTION OF ESAB COMPONENT OF TYPE VII SECRETORY SYSTEM OF VANCOMYCIN-RESISTANT S. AUREUS MU50

Abstract

Intro: Proteins secreted from type VII secretory system present in many bacteria are vital for the bacterial survival and for its pathogenicity. EsaB is one of the protein that regulates T7 system substrate production genetically but is essential for the secretory activity. EsaB is present in very low level in cell and its absence leads to iron acquisition genes upregulation. In Staphylococcus aureus, EsaB protein structurally related to ubiquitin. During Staphylococcus aureus infection bacteria survives due to T7 mediated mechanisms but it remains unclear. It has been reported that S. aureus becomes more prone to host defenses in the absence of T7 secretory system components. Methods: During this study, gene of type VII secretion protein esaB from Vancomycin-resistant S. aureus (Mu50) were cloned in pSpeedET vector and transformed into E. coli DH5α cells. Recombinant DNA plasmids with 100% correct sequence from positive clones were transformed in expression cell line E. coli BL21(DE3) cells, under IPTG induction. The affinity chromatography and size exclusion chromatography were used for purification. Findings: Our study successfully developed an indigenous method of cloning, expression, and purification of type VII Secretion Protein EsaB. The EsaB protein produced using this protocol could be used to develop functional assays. Discussion: Majority of bacteria possesses type VII secretion systems that aid the secretion of proteins in bacterial competition and virulence. At genetic level, EsaB doesnot regulate the production of T7 substrates or components but its absence results in dysregulation of particular class of genes. However, it is integral for the secretion activity of T7SS. This work is presenting the invitro cloning of genes of esaB and the subsequent production of its protein. Conclusion: This study has focused on an important bacterial system that accounts for virulence and bacterial competition. the factor cloned, expressed and purified during this study is an essential part of secretion via T7SS.