Abstract
Many infectious Gram-negative bacteria, including Salmonella typhimurium, require a Type Three Secretion System (T3SS) to translocate virulence factors into host cells. The T3SS consists of a membrane protein complex and an extracellular needle together that form a continuous channel. Regulated secretion of virulence factors requires the presence of SipD at the T3SS needle tip in S. typhimurium. Here we report three-dimensional structures of individual SipD, SipD in fusion with the needle subunit PrgI, and of SipD:PrgI in complex with the bile salt, deoxycholate. Assembly of the complex involves major conformational changes in both SipD and PrgI. This rearrangement is mediated via a π bulge in the central SipD helix and is stabilized by conserved amino acids that may allow for specificity in the assembly and composition of the tip proteins. Five copies each of the needle subunit PrgI and SipD form the T3SS needle tip complex. Using surface plasmon resonance spectroscopy and crystal structure analysis we found that the T3SS needle tip complex binds deoxycholate with micromolar affinity via a cleft formed at the SipD:PrgI interface. In the structure-based three-dimensional model of the T3SS needle tip, the bound deoxycholate faces the host membrane. Recently, binding of SipD with bile salts present in the gut was shown to impede bacterial infection. Binding of bile salts to the SipD:PrgI interface in this particular arrangement may thus inhibit the T3SS function. The structures presented in this study provide insight into the open state of the T3SS needle tip. Our findings present the atomic details of the T3SS arrangement occurring at the pathogen-host interface.
Highlights
Bacterial infections including Salmonellosis and Shigellosis affect millions of people every year
SipD from S. typhimurium, IpaD from Shigella flexneri and BipD from Burkholderia mallei are tip proteins that are thought to interact with their corresponding needle subunits PrgI, MxiH and BsaL, respectively, to make the needle tip complex [5]
Since the rise of pathogenic bacterial strains resistant to antibiotics, the need to develop potent anti-infective drugs is continually increasing. This necessitates a detailed knowledge of the bacterial host invasion process
Summary
Bacterial infections including Salmonellosis and Shigellosis affect millions of people every year. These bacteria use a T3SS to secrete virulence factors to manipulate host cells. The T3SS is a multi-component system that forms a continuous protein transport channel through the two bacterial membranes and the periplasmatic space that extends into the surrounding medium by a needle structure [1,2,3]. Tip proteins, which bind to the distal end of the T3SS needle, are thought to play an important role in this process [4,5,6]. The mechanism is unclear, tip proteins were shown to influence secretion and invasion of bacteria [7,8,9]
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