Functional characterization of rhomboid proteases in pyogenic gram positive cocci

Abstract

Background: Rhomboid proteins are widely distributed in bacteria including the Gram positive pyogenic cocci. Rhomboid proteases are novel proteins that are increasingly being implicated in the pathogenecity of these organisms, though the precise medically significance of their biological roles is unclear. Result: Bioinformatics analysis reveals that staphylococcus and streptococcus rhomboids are true intramembrane serine proteases and are catalytically true serine proteases. Rhomboid proteases are also observed to display diversity across the Gram positive cocci and the pattern of evolution of staphylococcus rhomboids mirror the evolution of the staphylococcus species. Also using Cre-lox strategy we have deleted the rhomboid-encoding gene (sa1641) in staphylococci aureus with the aid of selection markers. Bypass of the strict enzyme modification systems of staphylococci aureus using a novel heat-treatment protocol and preparation of electro competent cells with a modified washing solution was crucial in this transformation. Targeting sa1641 gene, competent staphylococcus aureus cells were electroporated with shuttle plasmid pBT2 and gene deletion achieved with use of permissive and non permissive temperatures. Relevance: We anticipated that this study will be used to generate valuable insight into the role of rhomboid proteases in pyogenic cocci and could further unravel novel targets that can possibly be exploited in the development of new medical interventions against infections due to these organisms. Conclusion: Rhomboid proteins are diverse and could be used as one of the evolutionary markers. It is possible to achieve rhomboid gene knockout in pyogenic Gram positive cocci.