Integrative structural analysis of the type III secretion system needle complex from Shigella flexneri

Abstract

The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram‐negative bacteria to transport virulence factors into the host cell during infection. Understanding the structure of T3SSs is crucial for future developments of therapeutics that could target this system. However, much of the knowledge about the structure of T3SS is available only for Salmonella, and it is unclear how this large assembly is conserved across species. Here, we combined cryo‐electron microscopy, cross‐linking mass spectrometry, and integrative modeling to determine the structure of the T3SS needle complex from Shigella flexneri. We show that the Shigella T3SS exhibits unique features distinguishing it from other structurally characterized T3SSs. The secretin pore complex adopts a new fold of its C‐terminal S domain and the pilotin MxiM[SctG] locates around the outer surface of the pore. The export apparatus structure exhibits a conserved pseudohelical arrangement but includes the N‐terminal domain of the SpaS[SctU] subunit, which was not present in any of the previously published virulence‐related T3SS structures. Similar to other T3SSs, however, the apparatus is anchored within the needle complex by a network of flexible linkers that either adjust conformation to connect to equivalent patches on the secretin oligomer or bind distinct surface patches at the same height of the export apparatus. The conserved and unique features delineated by our analysis highlight the necessity to analyze T3SS in a species‐specific manner, in order to fully understand the underlying molecular mechanisms of these systems. The structure of the type III secretion system from Shigella flexneri delineates conserved and unique features, which could be used for the development of broad‐range therapeutics.

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

“…The pilotin is mobile at this stage and may additionally contribute to efficient secretion by interacting with other partners, including PTS sugar transporters, which could provide the necessary energy for secretion. Such species-specific functions would be in line with the observed differences between species in localization of the pilotin protein after T3SS assembly (Flacht et al, 2023;Hu et al, 2015Hu et al, , 2017Hu et al, , 2018Miletic et al, 2021). Challenging this model in future studies will lead to a better understanding of the role of pilotins in both assembly and function of the T3SS.…”

“…For T2SS secretins, such attachment was initially proposed, but later rejected (Chami et al., 2005; Nouwen et al., 1999); recent analyses of purified secretin rings found pilotins attached stoichiometrically outside the main ring (Chernyatina & Low, 2019; Yin et al., 2018). In situ structures of the T3SS showed electron densities around the secretin, possibly corresponding to pilotin proteins, in Shigella flexneri (Flacht et al., 2023; Hu et al., 2015), but not in Salmonella enterica (Hu et al., 2017, 2018; Miletic et al., 2021). In spite of the importance of pilotins for the nucleation and localization of the central secretin ring structure (which also prevents ion leakage caused by secretin misassembly in the inner membrane (Burghout et al., 2004; Collin et al., 2011; Majewski et al., 2021)), protein secretion by the T3SS was found to be strongly impaired, but not completely prevented in absence of the pilotin in different organisms (Allaoui et al., 1995; Burghout et al., 2004; Perdu et al., 2015).…”

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

“…The pilotin is mobile at this stage and may additionally support this function by interacting with other partners, including PTS sugar transporters, which could provide the necessary energy for secretion. Such species-specific functions would be in line with the observed differences between species in localization of the pilotin protein after T3SS assembly [33][34][35][36][37]. Challenging this model in future studies will lead to a better understanding of the role of pilotins in both assembly and function of the T3SS.…”

“…For T2SS secretins, such attachment was initially proposed, but later rejected [17,30]; recent analyses of purified secretin rings found pilotins attached stoichiometrically outside the main ring [31,32]. In situ structures of the T3SS showed electron densities around the secretin, possibly corresponding to pilotin proteins, in Shigella flexneri [33,34], but not in Salmonella enterica [35][36][37]. In spite of the importance of pilotins for the localization of the central secretin ring structure, protein secretion by the T3SS was found to be strongly impaired, but not completely prevented in absence of the pilotin in different organisms [15,19,24].…”

The sorting platform in the type III secretion pathway: From assembly to function

“…Therefore, significant progress has been made in unraveling its structural intricacies over the last two decades, culminating in atomic-level resolution structures of the entire NC. [16][17][18][19] However, our understanding of the architecture of the SP has been limited as this substructure is lost during the NC preparation, most likely due to its weak association with the NC base. This structural "labileness" makes challenging the direct application of single-particle cryo-EM analysis to study the SP.…”