Molecular analysis of switching in the bacterial type 3 protein secretion system
Type 3 secretion system (T3SS) is a complex and multi-subunit trans-membrane protein translocation system of gram negative bacteria having direct association with virulence of a number of animal and human pathogens, viz. Yersinia, Vibrio, Enteropathogenic E. coli (EPEC), Salmonella, Pseudomonas, etc. It works as an injection syringe to deliver bacterial cytoplasmic toxins to the host cell cytoplasm. Even though T3SS has attracted significant attention from the research community in the last 3 decades, many questions regarding the signals that target proteins to the channel, the order of events and the interactions that occur, and the energetics as well as the high resolution structure of the apparatus at work are unanswered. A major obstacle to further progress is that practically all of the studies till now have been analyzed in vivo which because of its inherent limitations has not been able to answer these questions. Considering these gaps in our understanding of T3SS, the present study has been designed to reconstitute for the first time in vitro the T3SS of Enteropathogenic Escherichia coli by applying the principles of molecular bacteriology, genetic engineering and biochemistry. This study on the model organism Enteropathogenic E. coli will be completed in three years. As inner membrane vesicles contain the T3SS, our first aim will be to isolate functional IMVs from EPEC, which then, will be used to study the membrane binding affinity of chaperone/effector complexes by density gradient ultracentrifugation. The minimal components of the T3SS will be identified with the help of a library of mutant strains of EPEC. These minimal components will be over-expressed alone or in combination using suitable operons. Then, IMVs will be prepared from the strain expressing minimal T3SS components. Hence, these IMVs will contain T3SS with minimal core components. Our next focus will be to evaluate these in vitro constructed T3SS embedded on the IMVs for protein translocation capability by using ATPase assay and fluorescence microscopy. So, in the proposed study, at the end of three years a functional in vitro T3SS model is expected to be developed, which will have the potential to substantially contribute towards the development of novel antibiotics, drug delivery systems, T3SS-based antibacterial vaccine and many more applications.