Sujeet Kumar, student, VIT University (India)
Natividad Ruiz, faculty mentor
The rise in antibiotic resistance poses a serious threat to global health. Our research aims to understand the peptidoglycan (PG) biogenesis in Gram-negative bacteria. PG is an essential polymeric mesh-like structure that maintains cell shape and protects cells from osmotic lysis. Specifically, we study the function of MurJ, the lipid II flippase, that transports lipid II across the inner membrane. Since humans do not synthesize PG, MurJ serves as a vital target for antibiotic development.
MurJ is essential for the physiology of the bacterial cell. To elucidate MurJ conformational changes during Lipid II transport, we have developed a method that utilizes site-directed cysteine cross-linking in live bacterial cells coupled with proteolytic gel analysis to probe its specific conformations. Our method can be used in vivo to test and validate mechanisms resulting from structures and in silico predictions, as well as to investigate the effects of potential inhibitors of transport.
We demonstrated that MurJ, the bacterial Lipid II flippase, functions by an alternating access mechanism, and illustrated which step in the Lipid II transport cycle requires membrane potential. The knowledge gained from our research will aid in the development of novel anti-microbial remedies to control infections by Gram-negative pathogens.