The Architecture of the Cytoplasmic Region of Type III Secretion Systems (original) (raw)
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Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported though a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assemble into an extra-membrane helical assembly that likely seeds correct assembly of the rod above. Here we present the structure of an equivalent complex from the more fragile Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher resolution structure reveals further detail and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also reveals details of how the SctS/FliQ subunits sequentially assemble in the complex.
Microbiology, 2014
The type III secretion apparatus (T3SA) is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. The Shigella flexneri T3SA spans the bacterial envelope and its assembly requires the products of~20 mxi and spa genes. Despite progress made in understanding how the T3SA is assembled, the role of several predicted soluble components, such as Spa13, remains elusive. Here, we show that the secretion defect of the spa13 mutant is associated with lack of T3SA assembly which is partly due to the instability of the needle component MxiH. In contrast to its Yersinia counterpart, Spa13 is not a secreted protein. We identified a network of interactions between Spa13 and the ATPase Spa47, the C-ring protein Spa33, and the inner-membrane protein Spa40. Moreover, we revealed a Spa13 interaction with the inner-membrane MxiA and showed that overexpression of the large cytoplasmic domain of MxiA in the WT background shuts off secretion. Lastly, we demonstrated that Spa13 interacts with the cleaved form of Spa40 and with the translocator chaperone IpgC, suggesting that Spa13 intervenes during the secretion hierarchy switch process. Collectively, our results support a dual role of Spa13 as a chaperone escort and as an export gate-activator switch.
Bacterial type III secretion systems are ancient and evolved by multiple horizontal-transfer events
Gene, 2003
Type III secretion systems (TTSS) are unique bacterial mechanisms that mediate elaborate interactions with their hosts. The fact that several of the TTSS proteins are closely related to flagellar export proteins has led to the suggestion that TTSS had evolved from flagella. Here we reconstruct the evolutionary history of four conserved type III secretion proteins and their phylogenetic relationships with flagellar paralogs. Our analysis indicates that the TTSS and the flagellar export mechanism share a common ancestor, but have evolved independently from one another. The suggestion that TTSS genes have evolved from genes encoding flagellar proteins is effectively refuted. A comparison of the species tree, as deduced from 16S rDNA sequences, to the protein phylogenetic trees has led to the identification of several major lateral transfer events involving clusters of TTSS genes. It is hypothesized that horizontal gene transfer has occurred much earlier and more frequently than previously inferred for TTSS genes and is, consequently, a major force shaping the evolution of species that harbor type III secretion systems.
The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H + –protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na + increased it dramatically. Phenamil, a blocker of Na + translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na + concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na + channel. In wild-type cells, however, neither Na + nor phenamil affected protein export, indicating that the Na + channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na + concentration. For construction of the bacterial flagellum beyond the inner and outer membranes, the fla-gellar type III export apparatus transports fourteen flagellar proteins with their copy numbers ranging from a few to tens of thousands to the distal growing end of the flagellar structure. The export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. Here, we show that the export engine of the flagellar type III export apparatus is robust in maintaining its export activity against internal and external perturbations arising from genetic variations and/or environmental changes. When the cytoplasmic ATPase complex is absent, the export gate complex is able to utilize
Nature Structural & Molecular Biology, 2009
Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base bridging both bacterial membranes into which a hollow, external needle is embedded. When isolated, the latter two parts are termed 'needle complex' (NC). Incomplete understanding of NC structure hampers studies of T3SS function. To estimate the stoichiometry of its components, the mass f its sub-domains was measured by scanning transmission electron microscopy (STEM). Subunit symmetries were determined by analysis of top and side views within negatively stained samples in low dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25Å resolution three-dimensional (3D) reconstruction of the NC base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component. T3SS are found in many Gram-negative bacteria, forming molecular injection devices to deliver bacterial effector proteins into target eukaryotic host cells during infection. Understanding their 3D structure is important for design of new broad-spectrum strategies to combat bacterial infection. These macromolecular assemblies are made of ~25 proteins. They are genetically and morphologically related to bacterial flagellar basal bodies (FBBs). Hence, functional parallels with that system have been drawn4. However, FBBs are constitutively active in sequential secretion of axial flagellar proteins while T3SS activation for effector secretion requires direct physical contact with host cells. Shigella flexneri is the causative agent of human bacillary dysentery5. Shigella uses a T3SS for invasion of, and dissemination within, the gut epithelial lining6. In situ, its T3SS is made
BMC Microbiology, 2009
Background: Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a ΔespADB mutant and a ΔescF T3SS mutant.