Structural And Functional Analysis And Inhibitors Screening Of SctV From The Salmonella T3SS

Salmonella enterica is a common food-borne pathogen that infects both humans and animals.S.enterica infection is characterized by fever,abdominal pain,vomiting,and diarrhea.Importantly,it is urgent to devise novel antibiotics due to the development of multidrugresistant S.enterica.When S.enterica contacts with the intestinal epithelium,several bacterial effector proteins could be delivered into the host cells through the Type Ⅲ secretion systems(T3SSs).The results in the manipulation of many common celluar processes,including host immune responses,cytoskeletal dynamics,vesicle transport,and signal transduction pathways,to facilitate the infection in the host cells.T3SSs are protein transport nanomachines that are found in the most Gram-negative bacterial pathogens and symbionts.T3SS contains more than 20 proteins that spans three membranes,including the bacterial inner and outer membranes,as well as the host cell membrane.Many prominent substructures and components of T3SS are highly conserved in diverse pathogens.In the past two decades,the assembly,structure and function of T3SS nanomachines have been widely studied.However,limited information about the core component SctV family restricts the understanding of energy transformation and substrate secretion of T3SS,which further impedes the development of new anti-infection drugs targeting T3SSs.SctV family proteins are located at the core parts of T3SS.They consist of a transmembrane domain with eight transmembrane helices(SctVTM)and a～40 kDa cytoplasmic domain(SctVC).SctV family proteins are the first identified component of T3SS in 1989,they have beed widely studied due to their central role in the cirulence.Most research on the structure of SctV cytoplasmic domain(SctVC)suggests that it form a nonameric ring embedded in the bacterial inner membranes.Except for being a part of the secretion channel,the transmembrane domain of SctV(SctVTM)may act as an energy source for subsequent secretion in T3SS.And the conformational change of SctVC affected by the linker region(SctVL)connecting the SctVC and SctVTM is perceived as involving in substrate recognition and secretion.However,the molecular mechanism of SctV in the protein secretion process of T3SS is poorly understood.This study mainly focuses on the SctV family proteins InvA from S.Typhimurium SPI-1 and SsaV from S.Typhimurium SPI-2.We first designed multiple strategies to overcome the low-level expression of InvA and the other purification difficulties.We then generated a chimeric protein consisting of the transmembrane region of InvA and the cytoplasmic region of SsaV(InvATM-SsaVc),to produce a full-length chimeric SctV in a high-order oligomeric state for structural study using single-particle cryo-EM.Finally,we obtained the 2.11 (?) nonameric ring structure of SsaVc with a semi-open state of each monomer.By comparison with SctVC homologous proteins,we found that the SsaVc ring assembly relies primarily on electrostatic interactions,and hydrophobic interactions.SctVL is involved in the hydrophobic interactions,and the peptides of SctVL have great diversity in different homologous proteins,which is one of the reasons for the different oligomeric states of SctVC in vitro.Therefore,the linker region plays an essential role in the structural stability and function of SsaV.Although the structures of the transmembrane region and linker region were unsolved due to their flexibility,we observed two major states of the linker region with different prientation relative to SsaVc in the fixed position by two separate mehtods.Further,the in vitro secretion assay of S.Typhimurium confirmed that the conserved amino acids in the transmembrane domain of SctV are essential for the secretion of T3SS effectors.Combining the structure of SctVL and the functional analysis of SctVTM,we proposed a potential model coupling energy transformation and substrate transport of T3SS.Based on the structure and function analysis above,we screened the compounds and macrocyclic peptides targeting InvAC and SsaVc by RaPID screening system and virtual screening.Combining with secretion assay and bacterial invasion assay,the compounds C4 and C5 could significantly inhibit the secretion of T3SS effectors.C5 may downregulate the expression of the regulator hilD to inhibit the secretion of T3SS effectors,and further hinder the infection of S.Typhimurium into NCM460 cells.While how C4 inhibits the secretion of T3SS effectors is unclear.Additionally,we identified macrocyclic peptide InvAc-L1 may bind to InvA and InvAc-L1 exhibited strong inhibition on the secretion of T3SS effectors without affecting S.Typhimurium growth at concentrations up to 100 μM and thus prevented S.Typhimurium from invading NCM460 cells.Together,this work provides a structural basis for understanding the molecular mechanisms of SctV involved secretion process of T3SS,which provides new targets and ideas for the development of novel anti-infective drugs targeting T3SS.