Biophysical studies of the Burkholderia minor translocon protein BipC by NMR and CD spectroscopy

Abstract The type III secretion system (T3SS) provides many Gram-negative pathogens a tool to initiate, maintain and proliferate infection in the host. The T3SS is a syringe-like apparatus composed of a base that transverses the bacterial membranes, an extracellular needle, a tip complex, and a translocon. The T3SS consists of over 20 different protein components that assemble to form a pore into the host membrane. The T3SS creates a pathway for the transport of bacterial effector molecules into the host cytoplasm. The effector molecules hijack and manipulate the host cytoskeleton and cell signaling pathways to promote invasion by the bacteria, circumvention of the host immune system, and maintenance of infection. Several Gram-negative pathogens utilize the T3SS for infection including Burkholderia pseudomallei (melioidosis), Salmonella typhimurium (infectious diarrhea/typhoid), Shigella flexneri (shigellosis), Pseudomonas aeruginosa (nosocomial infection), Yersinia pestis (bubonic plague), Chlamydia trachomatis (sexually transmitted disease), and enterohemorrahagic Escherichia coli (bloody diarrhea/urinary tract infection). These pathogens pose a serious threat to public health, when combined they cause millions of cases of illness and deaths and constitute a huge economical burden to the US and the rest of the world. Since its discovery and visualization about two decades ago, the bacterial T3SS has enticed intense scientific research focused on deciphering the assembly of the injectisome, its virulence mechanism and discovery or design of novel vaccines and anti-infectives. The needle-tip-translocon proteins of the T3SS are potential therapeutic targets because of their hydrophilicity and exposure to the extracellular space. The needle and the tip proteins in Salmonella and Shigella have been characterized but the assembly of the translocon proteins and tip-translocon interaction has not been extensively studied. This thesis explores the Burkholderia tip-translocon protein-protein interaction and the interaction of the translocon with a membrane. I utilized biophysical techniques namely solution nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to investigate the protein binding surfaces involved in tip-translocon interaction as well as the structural transition of the translocon protein on exposure to membrane mimic detergent micelles. I have herein shown that the Burkholderia minor translocon protein BipC might be binding at the mixed α-β region of the tip protein BipD extrapolated from the interaction study between BipC and BipD as well as its homologs Salmonella SipD and Shigella IpaD. The results help understand the interaction of the Burkholderia minor translocon protein with the tip protein and detergent micelles and thus add to the knowledge of T3SS assembly