Structural And Functional Insight Into The Hcp Of T6SS Related Protein Of Pathogenic Bacteria

The type VI secretion system(T6SS)is amacro-molecular machinery,distributing widely in Gram-negative bacteria.It plays an important role in many processes of bacterial life cycles,such as interspecies competition,biofilmformation and virulence-related processes.Opportunistic pathogens use the T6SS as a weapon for intruding prokaryotic or eukaryotic cells by inject toxin into them.The T6SS is structurally and functionally similar to the contractile phage tails,containing at least 13 essential subunits.Amongst them,Valine-glycine repeat protein(VgrG)and haemolysin co-regulated protein(Hcp)are secreted and significant for the function of the T6SS,and present the trait of T6SS.The Hcp can function as a transporter and chaperone of diverse effectors from bacterial T6SS,and as a vital component of T6SS.Hcp 1 protein,forming a hexameric ring and belonging to the Hcp family,is encoded by the secretion island I(HSI-I).Besides Hcp protein,VgrG and PAAR,two of critical components of T6SS,can also transport toxin effectors.The crystal structure of PaHcpl and Tse2 of Pseudomonas aeruginosawere worked out in 2006 and 2016,respectively.It was reported that Hcpl interacted directly with Tse2 and discovered that Tse2 bound the inner surface of PaHcpl through transmission electron.We try to obtain the crystal structure of PaHcpl-Tse2 protein complex by X-ray crystallography and illuminate the binding model of them in terms of structural biology,then prvide a fundamental insight into the transport mechanism of Hcp protein.Finally,we get the crystal of PaHcpl-Tse2 protein complex but failed to work out the crystal structure of them.The StHcpl from Salmonella typhimurium was recently found to plays an essential role in T6SS antibacterial activity by the specific interaction with T6SS toxin effector Tae4 to establish infection within the host gut.Several Hcp homologs structures have been reported till now,including Hcpl from Acinetobacter baumannii,EvpC from Edwardsiella tarda,Hcpl mutant from enteroaggregative Escherichia coli(EAEC)and Hcpl from Burkholderia pseudomallei.Significantly,all of them are composed of a β-barrel domain forming hexameric rings oligomers with an outer diameter of～80 A and an inner diameter of～40 A.The internal pore can only accommodate small folded proteins(<20 kDa)or unfolded proteins.However,the crystal structure of StHcpl has not yet been reported.In this study,we have worked out the crystal structure of StHcpl and StHcp2 from Salmonella typhimurium.It was showed they were composed of a(3-barrel domain with extended loops and can form hexameric rings as observed in known Hcp homologs.Wild-type StHcp2 exists as a hexamer as well as partial tetramer in solution.Mutation of the extended loop was found to partly destabilize the hexametric conformation into monomers or cause the production of inclusion bodies,suggesting its important role in hexameric ring formation.The structure-function studies of StHcpl and StHcp2 will provide novel insights into mechanism and function of T6SS in S.typhimurium.