Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early-cycle development

The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole termed an inclusion. During its intracellular developmental cycle, C. trachomatis maintains this intracellular niche, presumably by expressing a type III secretion system, which deploys a set of host cell-interactive proteins including inclusion membrane-localized proteins termed Incs. Some Incs are expressed and secreted by 2 h (early cycle) after infection, whereas the expression of type III-specific genes is not detectable until 6-12 h (mid-cycle). To resolve this paradox, we investigated the presence of a type III apparatus on elementary bodies (EBs) that might function early in infection. We demonstrate the existence of the type III secretory apparatus by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and immunoblot analyses of purified EB extracts. Immunoblots using polyclonal antibodies specific for the core apparatus component CdsJ identified this protein in both EB and reticulate body (RB) extracts. Furthermore, CdsJ-specific signals were detected by immunoblot of whole infected-culture extracts and by indirect immunofluorescence of infected monolayers at times before the detection of cdsJ-specific message. Finally, expression of IncC, expressed by 2 h after infection during C. trachomatis infections, in Yersinia pseudotuberculosis resulted in its secretion via the Yersinia type III apparatus. Based on these data, we propose a model in which type III secretion pores are present on EBs and mediate secretion of early Incs and possible additional effectors. Mid-cycle expression of type III genes would then replenish secretion apparatus on vegetative RBs and serve as a source of secretion pores for subsequently formed EBs.     

Interactions between type III secretion apparatus components from Yersinia pestis detected using the yeast two-hybrid system

Interactions among the Yersinia secretion (Ysc) proteins of Yersinia pestis were explored using the yeast two-hybrid system. Various pairwise combinations of the yscEFGHIKLN and Q genes fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast, and expression of a reporter gene encoding beta-galactosidase was detected. Combinations of yscN and yscL, yscL and yscQ, and yscQ and yscK resulted in high levels of reporter gene activation. These results suggest that YscL interacts with both YscN and YscQ, and that YscQ interacts with both YscL and YscK. Three-hybrid analyses using plasmid pDELA to target a third hybrid protein to the yeast nucleus was used to detect the formation of ternary protein complexes. Using the three-hybrid system, YscQ expressed from plasmid pDELA was able to bring together the YscK and YscL fusion proteins. In a similar manner, YscL expressed from plasmid pDELA was able to bring together the YscN and YscQ fusion proteins. Together, these results suggest that a complex composed of YscN, YscQ, YscK and YscL is involved in the assembly and/or function of the Y. pestis type III secretion apparatus.     

Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.     

Interaction between components of the type III secretion system of Chlamydiaceae

Members of the family Chlamydiaceae possess at least 13 genes, distributed throughout the chromosome, that are homologous with genes of known type III secretion systems (TTS). The aim of this study was to use putative TTS proteins of Chlamydophila pneumoniae, whose equivalents in other bacterial TTS function as chaperones, to identify interactions between chlamydial proteins. Using the BacterioMatch Two-Hybrid Vector system (Stratagene, La Jolla, Calif.), lcrH-2 and sycE, positions 1021 and 0325, respectively, from C. pneumoniae CM-1 were used as "bait" to identify target genes (positions 0324, 0705, 0708, 0808 to 0810, 1016 to 1020, and 1022) in close proximity on the chromosome. Interaction between the products of the lcrH-2 (1021) and lcrE (copN) (0324) genes was detected and confirmed by pull-down experiments and enzyme immunoassays using recombinant LcrH-2 and LcrE. As further confirmation of this interaction, the homologous genes from Chlamydia trachomatis, serovar E, and Chlamydophila psittaci, Texas turkey, were also cloned in the two-hybrid system to determine if LcrH-2 and LcrE would interact with their orthologs in other species. Consistent with their genetic relatedness, LcrH-2 from C. pneumoniae interacted with LcrE produced from the three species of Chlamydiaceae; LcrH-2 from C. psittaci reacted with LcrE from C. pneumoniae but not from C. trachomatis; and C. trachomatis LcrH-2 did not react with LcrE from the other two species. Deletions from the N and C termini of LcrE from C. pneumoniae identified the 50 C-terminal amino acids as essential for the interaction with LcrH-2. Thus, it appears that in the Chlamydiaceae TTS, LcrH-2 interacts with LcrE, and therefore it may serve as a chaperone for this protein.     

Mechanism of protein folding : III. Disulfide bonding

It was shown in lysozyme and phospholipase, and generally in proteins with disulfide bonds, that after the formation of secondary structures the hydrophobic interactions between the key pairs responsible for folding tertiary structures bring several cysteine residues close together. Among the possible combination of cysteine residues some definite pairs are realized in the tertiary structure. In the Appendix to this paper an algebraic relation is given which must be satisfied for two cysteine residues to make a disulficle bond. This relation is too strict to be applied to real problems, where the two cysteines come close together, but the distance is still too great to yield a disulfide bond. In this case the two residues can attract each other by disuifide formation potential. A geometrical graphic representation is given for the locus of the H atom of the SH group in the cysteine residue. This looks like a lampshade and provides us with a guide to select the correct choice among cysteine pairs. This method is applied to lysozyme and phospholipase to supplement the discussion of the preceding paper (T. Yoshimura, H. Noguchi, T. Inoue and N. Saitô, Biophys. Chem. 40 (1991) 277).     

Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells

Background  

Chlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB) were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness.     

IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus

Type III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to inject virulence proteins into animal and plant host cells. The core of the T3S apparatus, known as the needle complex, is composed of a basal body transversing both bacterial membranes and a needle protruding above the bacterial surface. In Shigella flexneri, IpaD is required to inhibit the activity of the T3S apparatus prior to contact of bacteria with host and has been proposed to assist translocation of bacterial proteins into host cells. We investigated the localization of IpaD by electron microscopy analysis of cross-linked bacteria and mildly purified needle complexes. This analysis revealed the presence of a distinct density at the needle tip. A combination of single particle analysis, immuno-labeling and biochemical analysis, demonstrated that IpaD forms part of the structure at the needle tip. Anti-IpaD antibodies were shown to block entry of bacteria into epithelial cells.     

Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle

Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.     

YopK regulates the Yersinia pestis type III secretion system from within host cells

The pathogenic Yersinia species share a conserved type III secretion system, which delivers cytotoxic effectors known as Yops into target mammalian cells. In all three species, YopK (also called YopQ) plays an important role in regulating this process. In cell culture infections, yopK mutants inject higher levels of Yops, leading to increase cytotoxicity; however, in vivo the same mutants are highly attenuated. In this work, we investigate the mechanism behind this paradox. Using a β-lactamase reporter assay to directly measure the effect of YopK on translocation, we demonstrated that YopK controls the rate of Yop injection. Furthermore, we find that YopK cannot regulate effector Yop translocation from within the bacterial cytosol. YopE is also injected into host cells and was previously shown to contribute to regulation of the injectisome. In this work we show that YopK and YopE work at different steps to regulate Yop injection, with YopK functioning independently of YopE. Finally, by expressing YopK within tissue culture cells, we confirm that YopK regulates translocation from inside the host cell, and we show that cells pre-loaded with YopK are resistant to Yop injection. These results suggest a novel role for YopK in controlling the Yersinia type III secretion system.     

The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus

Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to transfer bacterial proteins into host cells. Two translocators, IpaB and IpaC, are first inserted with the aid of IpaD by Shigella into the host cell membrane. Then at least two supplementary effectors of cell invasion, IpaA and IpgD, are transferred into the host cytoplasm. How TTSSs are induced to secrete is unknown, but their activation appears to require direct contact of the external distal tip of the apparatus with the host cell. The extracellular domain of the TTSS is a hollow needle protruding 60 nm beyond the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a superhelical assembly. To probe the role of the needle in the activation of the TTSS for secretion, we examined the structure-function relationship of MxiH by mutagenesis. Most point mutations led to normal needle assembly, but some led to polymerization or possible length control defects. In other mutants, secretion was constitutively turned "on." In a further set, it was "constitutively on" but experimentally "uninducible." Finally, upon induction of secretion, some mutants released only the translocators and not the effectors. Most types of mutants were defective in interactions with host cells. Together, these data indicate that the needle directly controls the activity of the TTSS and suggest that it may be used to "sense" host cells.     

IpaD is localized at the tip of the Shigella flexneri type III secretion apparatus

Type III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to inject virulence proteins into animal and plant host cells. The core of the T3S apparatus, known as the needle complex, is composed of a basal body transversing both bacterial membranes and a needle protruding above the bacterial surface. In Shigella flexneri, IpaD is required to inhibit the activity of the T3S apparatus prior to contact of bacteria with host and has been proposed to assist translocation of bacterial proteins into host cells. We investigated the localization of IpaD by electron microscopy analysis of cross-linked bacteria and mildly purified needle complexes. This analysis revealed the presence of a distinct density at the needle tip. A combination of single particle analysis, immuno-labeling and biochemical analysis, demonstrated that IpaD forms part of the structure at the needle tip. Anti-IpaD antibodies were shown to block entry of bacteria into epithelial cells.     

Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli

Few interactions have been reported between effectors and components of the type III secretion apparatus, although many interactions have been demonstrated between type III effectors and their cognate chaperones. It is thought that chaperones may play a role in directing effectors to the type III secretion apparatus. The ATPase FliI in the flagellar assembly apparatus plays a pivotal role in interacting with other components of the apparatus and with substrates of the flagellar system. We performed experiments to determine if there were any interactions between the effector Tir and its chaperone CesT and the type III secretion apparatus of enteropathogenic Escherichia coli (EPEC). Specifically, based on analogies with the flagella system, we examined Tir-CesT interactions with the putative ATPase EscN. We showed by affinity chromatography that EscN and Tir bind CesT specifically. Tir is not necessary for CesT and EscN interactions, and EscN binds Tir specifically without its chaperone CesT. Moreover, Tir directly binds EscN, as shown via gel overlay and enzyme-linked immunosorbent assay, and coimmunoprecipitation experiments revealed that Tir interacts with EscN inside EPEC. These data provide evidence for direct interactions between a chaperone, effector, and type III component in the pathogenic type III secretion system and suggest a model for Tir translocation whereby its chaperone, CesT, brings Tir to the type III secretion apparatus by specifically interacting with the type III ATPase EscN.     

Bioinformatic and biochemical evidence for the identification of the type III secretion system needle protein of Chlamydia trachomatis

Chlamydia spp. express a functional type III secretion system (T3SS) necessary for pathogenesis and intracellular growth. However, certain essential components of the secretion apparatus have diverged to such a degree as to preclude their identification by standard homology searches of primary protein sequences. One example is the needle subunit protein. Electron micrographs indicate that chlamydiae possess needle filaments, and yet database searches fail to identify a SctF homologue. We used a bioinformatics approach to identify a likely needle subunit protein for Chlamydia. Experimental evidence indicates that this protein, designated CdsF, has properties consistent with it being the major needle subunit protein. CdsF is concentrated in the outer membrane of elementary bodies and is surface exposed as a component of an extracellular needle-like projection. During infection CdsF is detectible by indirect immunofluorescence in the inclusion membrane with a punctuate distribution adjacent to membrane-associated reticulate bodies. Biochemical cross-linking studies revealed that, like other SctF proteins, CdsF is able to polymerize into multisubunit complexes. Furthermore, we identified two chaperones for CdsF, termed CdsE and CdsG, which have many characteristics of the Pseudomonas spp. needle chaperones PscE and PscG, respectively. In aggregate, our data are consistent with CdsF representing at least one component of the extended Chlamydia T3SS injectisome. The identification of this secretion system component is essential for studies involving ectopic reconstitution of the Chlamydia T3SS. Moreover, we anticipate that CdsF could serve as an efficacious target for anti-Chlamydia neutralizing antibodies.