Structure of a bacterial type III secretion system in contact with a host membrane in situ

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform– ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking ‘pump-action’ conformational changes that underpin effector injection

The chlamydial type III secretion mechanism: Revealing cracks in a tough nut

Present-day members of the Chlamydiaceae contain parasitic bacteria that have been co-evolving with their eukaryotic hosts over hundreds of millions of years. Likewise, a type III secretion system encoded within all genomes has been refined to complement the unique obligate intracellular niche colonized so successfully by Chlamydia spp. All this adaptation has occurred in the apparent absence of the horizontal gene transfer responsible for creating the wide range of diversity in other Gram-negative, type III-expressing bacteria. The result is a system that is, in many ways, uniquely chlamydial. A critical mass of information has been amassed that sheds significant light on how the chlamydial secretion system functions and contributes to an obligate intracellular lifestyle. Although the overall mechanism is certainly similar to homologous systems, an image has emerged where the chlamydial secretion system is essential for both survival and virulence. Numerous differences, some subtle and some profound, differentiate chlamydial type III secretion from others. Herein, we provide a comprehensive review of the current state of knowledge regarding the Chlamydia type III secretion mechanism. We focus on the aspects that are distinctly chlamydial and comment on how this important system influences chlamydial pathogenesis. Gaining a grasp on this fascinating system has been challenging in the absence of a tractable genetic system. However, the surface of this tough nut has been scored and the future promises to be fruitful and revealing

The extended structure of the periplasmic region of CdsD, a structural protein of the type III secretion system of Chlamydia trachomatis

The type III secretion system (T3SS) is required for the virulence of many gram-negative bacterial human pathogens. It is composed of several structural proteins, forming the secretion needle and its basis, the basal body. In Chlamydia spp., the T3SS inner membrane ring (IM-ring) of the basal body is formed by the periplasmic part of CdsD (outer ring) and CdsJ (inner ring). Here we describe the crystal structure of the C-terminal, periplasmic part of CdsD, not including the last 60 residues. Two crystal forms were obtained, grown in three different crystallization conditions. In both crystal forms there is one molecule per asymmetric unit adopting a similar extended structure. The structures consist of three periplasmic domains (PDs) of similar αββαβ topology as seen also in the structures of the homologous PrgH (Salmonella typhimurium) and YscD (Yersinia enterocolitica). Only in the C2 crystal form, there is a C-terminal additional helix after the PD3 domain. The relative orientation of the three subsequent CdsD PD domains with respect to each other is more extended than in PrgH but less extended than in YscD. Small-angle X-ray scattering data show that also in solution this CdsD construct adopts the same elongated shape. In both crystal forms the CdsD molecules are packed in a parallel fashion, using translational crystallographic symmetry. The most extensive crystal contacts are preserved in both crystal forms, suggesting a possible mode of assembly of the CdsD periplasmic part into a 24-mer complex forming the outer ring of the IM-ring of the T3SS

Disulfide Bonding within Components of the Chlamydia Type III Secretion Apparatus Correlates with Development ▿

Chlamydia spp. exhibit a unique biphasic developmental cycle whereby infectious elementary bodies (EBs) invade host epithelial cells and differentiate into noninfectious, metabolically active reticulate bodies (RBs). EBs posses a unique outer envelope where rigidity is achieved by disulfide bonding among cysteine-rich envelope-associated proteins. Conversely, these disulfide bonds become reduced in RBs to accommodate vegetative growth, thereby linking the redox status of cysteine-rich envelope proteins with progression of the developmental cycle. We investigated the potential role of disulfide bonding within the chlamydial type III secretion system (T3SS), since activity of this system is also closely linked to development. We focused on structural components of the T3S apparatus that contain an unusually high number of cysteine residues compared to orthologs in other secretion systems. Nonreducing SDS-PAGE revealed that EB-localized apparatus proteins such as CdsF, CdsD, and CdsC form higher-order complexes mediated by disulfide bonding. The most dramatic alterations were detected for the needle protein CdsF. Significantly, disulfide bonding patterns shifted during differentiation of developmental forms and were completely reduced in RBs. Furthermore, at later time points during infection following RB to EB conversion, we found that CdsF is reoxidized into higher-order complexes. Overall, we conclude that the redox status of specific T3SS apparatus proteins is intimately linked to the developmental cycle and constitutes a newly appreciated aspect of functionally significant alterations within proteins of the chlamydial envelope

Early expression of the type III secretion system of Parachlamydia acanthamoebae during a replicative cycle within its natural host cell Acanthamoeba castellanii.

The type three secretion system (T3SS) operons of Chlamydiales bacteria are distributed in different clusters along their chromosomes and are conserved at both the level of sequence and genetic organization. A complete characterization of the temporal expression of multiple T3SS components at the transcriptional and protein levels has been performed in Parachlamydia acanthamoebae, replicating in its natural host cell Acanthamoeba castellanii. The T3SS components were classified in four different temporal clusters depending on their pattern of expression during the early, mid- and late phases of the infectious cycle. The putative T3SS transcription units predicted in Parachlamydia are similar to those described in Chlamydia trachomatis, suggesting that T3SS units of transcriptional expression are highly conserved among Chlamydiales bacteria. The maximal expression and activation of the T3SS of Parachlamydia occurred during the early to mid-phase of the infectious cycle corresponding to a critical phase during which the intracellular bacterium has (1) to evade and/or block the lytic pathway of the amoeba, (2) to differentiate from elementary bodies (EBs) to reticulate bodies (RBs), and (3) to modulate the maturation of its vacuole to create a replicative niche able to sustain efficient bacterial growth