Fluorescence polarization provides insights into the first steps of Salmonella type III secretion system tip maturation

Fluorescence polarization provides insights into the first steps of Salmonella type III secretion system tip maturation Salmonella spp. are Gram-negative pathogenic bacteria responsible for salmonellosis, one of the most common causes of acute gastroenteritis in the United States. Like many Gram-negative pathogens, Salmonella spp. utilize a type III secretion system (T3SS) as a primary virulence factor, allowing the direct exchange of effectors from the bacterial cytoplasm to the host cell. The T3SS is comprised of approximately 30 different proteins and shares many structural similarities with bacterial flagella. A basal body spans the inner and outer membranes, anchoring the T3SS to the bacterium and aiding in the control of the T3SS. The T3SS needle extends from the basal body and just beyond the bacterial surface. An oligomeric tip complex comprised of the Salmonella proteins SipD, SipB and SipC is required for invasion of host cells and subsequent infection. These proteins are recruited in an orchestrated manner to ultimately form a translocon pore in the host cell membrane, completing the conduit between the cells. The importance of this complex to virulence has made it a highly-anticipated target for anti-infective treatment, however, many of the necessary structural and mechanistic details of the complex formation remain unclear. Here, we investigate the mechanism of interaction between two of the tip proteins, SipD and SipB. To address the lack of stability in aqueous environments of SipB, we have developed a series of N-terminal deletion mutations, all lacking the putative hydrophobic regions near the C-terminus of SipB. These truncated forms of SipB can be expressed and purified in high quantities and maintain the stabilities necessary for subsequent in vitro analysis. Specifically, fluorescence polarization studies have provided valuable insight into the interactions between SipD and SipB that will act as a platform from which we will further dissect the mechanism of the Salmonella translocon formation and pursue the development of anti-infective treatments