Secretion of predicted Inc proteins of Chlamydia pneumoniae by a heterologous type III machinery

Chlamydia spp. are strictly intracellular pathogens that grow inside a vacuole, called an inclusion. They possess genes encoding proteins homologous to components of type III secretion machineries, which, in other bacterial pathogens, are involved in delivery of bacterial proteins within or through the membrane of eukaryotic host cells. Inc proteins are chlamydial proteins that are associated with the inclusion membrane and are characterized by the presence of a large hydrophobic domain in their amino acid sequence. To investigate whether Inc proteins and other proteins exhibiting a similar hydropathic profile might be secreted by a type III system, we used a heterologous secretion system. Chimeras were constructed by fusing the N-terminal part of these proteins with a reporter, the Cya protein of Bordetella pertussis, and these were expressed in various strains of Shigella flexneri. We demonstrate that these hybrid proteins are secreted by the type III secretion system of S. flexneri, thereby providing evidence that IncA, IncB and IncC are secreted by a type III mechanism in chlamydiae. Moreover, we show that three other proteins from Chlamydia pneumoniae, all of which have in common the presence of a large hydrophobic domain, are also secreted by S. flexneri type III secretion machinery.     

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.     

Type III secretion genes identify a putative virulence locus of Chlamydia

Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence-associated type III secretion loci. The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.     

Scc1 (CP0432) and Scc4 (CP0033) function as a type III secretion chaperone for CopN of Chlamydia pneumoniae

The Chlamydia pneumoniae CopN protein is a member of the YopN/TyeA/InvE/MxiC family of secreted proteins that function to regulate the secretion of type III secretion system (T3SS) translocator and effector proteins. In this study, the Scc1 (CP0432) and Scc4 (CP0033) proteins of C. pneumoniae AR-39 were demonstrated to function together as a type III secretion chaperone that binds to an N-terminal region of CopN. The Scc1/Scc4 chaperone promoted the efficient secretion of CopN via a heterologous T3SS, whereas, the Scc3 chaperone, which binds to a C-terminal region of CopN, reduced CopN secretion.     

Analysis of putative Chlamydia trachomatis chaperones Scc2 and Scc3 and their use in the identification of type III secretion substrates

The obligate intracellular pathogen Chlamydia trachomatis expresses a type III secretion system (T3SS) which has the potential to contribute significantly to pathogenesis. Based on a demonstrated role of type III secretion (T3S)-specific chaperones in the secretion of antihost proteins by gram-negative pathogens, we initiated a study of selected putative Chlamydia T3S chaperones in an effort to gain mechanistic insight into the Chlamydia T3SS and to potentially identify Chlamydia-specific secreted products. C. trachomatis Scc2 and Scc3 are homologous to SycD of Yersinia spp. Functional studies of the heterologous Yersinia T3SS indicated that although neither Scc2 nor Scc3 was able to fully complement a sycD null mutant, both have SycD-like characteristics. Both were able to associate with the translocator protein YopD, and Scc3 expression restored limited secretion of YopD in in vitro studies of T3S. CopB (CT578) and CopB2 (CT861) are encoded adjacent to scc2 and scc3, respectively, and have structural similarities with the YopB family of T3S translocators. Either Scc2 or Scc3 coprecipitates with CopB from C. trachomatis extracts. Expression of CopB or CopB2 in Yersinia resulted in their type III-dependent secretion, and localization studies with C. trachomatis-infected cells indicated that both were secreted by Chlamydia.     

Type III protein secretion in Pseudomonas syringae

The type III secretion system is an essential virulence system used by many Gram-negative bacterial pathogens to deliver effector proteins into host cells. This review summarizes recent advancements in the understanding of the type III secretion system of Pseudomonas syringae, including regulation of the type III secretion genes, assembly of the Hrp pilus, secretion signals, the putative type III effectors identified to date, and their virulence action after translocation into plant cells.     

Identification of chromosomal genes in Yersinia pestis that influence type III secretion and delivery of Yops into target cells

Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague.     

Tyrosine-phosphorylated bacterial effector proteins: the enemies within

The tyrosine phosphorylation of proteins has a central role during signal transduction in eukaryotes. Recent progress shows that tyrosine phosphorylation is also a common feature of several effector proteins translocated by bacterial type III and type IV secretion systems. The involvement of these secretion systems in disease development is exemplified by a variety of pathogenic processes: pedestal formation (Tir of EPEC and Citrobacter), cell scattering (CagA of Helicobacter), invasion (Tarp of Chlamydia) and possibly proinflammatory responses and cell proliferation (BepD-F of Bartonella). The discovery that different bacterial pathogens use this common strategy to subvert host-cell function suggests that more examples will soon emerge.     

Identification of Chlamydia trachomatis CT621, a protein delivered through the type III secretion system to the host cell cytoplasm and nucleus

Chlamydiae are obligate intracellular bacteria, developing inside host cells within chlamydial inclusions. From these inclusions, the chlamydiae secrete proteins into the host cell cytoplasm. A pathway through which secreted proteins can be delivered is the type III secretion system (T3SS). The T3SS is common to several gram-negative bacteria and the secreted proteins serve a variety of functions often related to the modulation of host signalling. To identify new potentially secreted proteins, the cytoplasm was extracted from Chlamydia trachomatis L2-infected HeLa cells, and two-dimensional polyacrylamide gel electrophoresis profiles of [³⁵S]-labelled chlamydial proteins from this extract were compared with profiles of chlamydial proteins from the lysate of infected cells. In this way, CT621 was identified. CT621 is a member of a family of proteins containing a domain of unknown function DUF582 that is only found within the genus Chlamydia . Immunofluorescence microscopy and immunoblotting demonstrated that CT621 is secreted late in the chlamydial developmental cycle and that it is the first chlamydial protein found to be localized within both the host cell cytoplasm and the nucleus. To determine whether CT621 is secreted through the T3SS, an inhibitor of this apparatus was added to the infection medium, resulting in retention of the protein inside the chlamydiae. Hence, the so far uncharacterized CT621 is a new type III secretion effector protein.     

The ttsA gene is required for low-calcium-induced type III secretion of Yop proteins and virulence of Yersinia enterocolitica W22703

Pathogenic Yersinia species use a virulence-plasmid encoded type III secretion pathway to escape the innate immune response and to establish infections in lymphoid tissues. At least 22 secretion machinery components are required for type III transport of 14 different Yop proteins, and 10 regulatory factors are responsible for activating this pathway in response to environmental signals. Although the genes for these products are located on the 70-kb virulence plasmid of Yersinia, this extrachromosomal element does not appear to harbor genes that provide for the sensing of environmental signals, such as calcium-, glutamate-, or serum-sensing proteins. To identify such genes, we screened transposon insertion mutants of Y. enterocolitica W22703 for defects in type III secretion and identified ttsA, a chromosomal gene encoding a polytopic membrane protein. ttsA mutant yersiniae synthesize reduced amounts of Yops and display a defect in low-calcium-induced type III secretion of Yop proteins. ttsA mutants are also severely impaired in bacterial motility, a phenotype which is likely due to the reduced expression of flagellar genes. All of these defects were restored by complementation with plasmid-encoded wild-type ttsA. LcrG is a repressor of the Yersinia type III pathway that is activated by an environmental calcium signal. Mutation of the lcrG gene in a ttsA mutant strain restored the type III secretion of Yop proteins, although the double mutant strain secreted Yops in the presence and absence of calcium, similar to the case for mutants that are defective in lcrG gene function alone. To examine the role of ttsA in the establishment of infection, we measured the bacterial dose required to produce an acute lethal disease following intraperitoneal infection of mice. The ttsA insertion caused a greater-than-3-log-unit reduction in virulence compared to that of the parental strain.     

Identification of genistein-inducible and type III-secreted proteins of Bradyrhizobium japonicum

Flagellin is the bulk protein secreted by Bradyrhizobium japonicum. For easier identification of minor protein fractions, the flagellin genes bll6865 and bll6866 were deleted. Extracellular proteins of the corresponding mutant were purified and separated by 2D gel electrophoresis. Several of the protein spots were detectable only after addition of genistein to the growth medium-genistein is an isoflavone secreted by soybean that activates the expression of genes encoding a type III secretion system. These secreted proteins were not present in supernatants of mutants in which conserved genes of the type III secretion system or the regulatory gene ttsI, which is essential for activation of the type III secretion system, are deleted. Out of 22 genistein-inducible protein spots 8 different proteins could be identified by mass spectrometry. One of the proteins, Blr1752, has similarity to NopP of Rhizobium sp. strain NGR234 that is known to be secreted. Another protein is Blr1656 (GunA2) that was shown previously to have endoglucanase activity. Three proteins have similarity to subunits of the flagellar apparatus. Some proteins appeared in several separate spots indicating posttranslational modification. A conserved tts box motif was found in the putative promoter region of six genes encoding secreted proteins.     

Yop fusions to tightly folded protein domains and their effects on Yersinia enterocolitica type III secretion

Yersinia enterocolitica organisms secrete Yop proteins via the type III pathway. Translational fusion of yop genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted. The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops.     

Revisiting the chlamydial type III protein secretion system: clues to the origin of type III protein secretion

The bacterial type III secretion pathway delivers effector proteins into eukaryotic cells. Analysis of the type III system and flagellar export genes in the obligate parasites of the family Chlamydiales suggests that the type III system arose from the flagellar export system in chlamydiae or related bacteria.     

Identification of a Campylobacter jejuni-secreted protein required for maximal invasion of host cells

The food-borne pathogen Campylobacter jejuni is dependent on a functional flagellum for motility and the export of virulence proteins that promote maximal host cell invasion. Both the flagellar and non-flagellar proteins exported via the flagellar type III secretion system contain a sequence within the amino-terminus that directs their export from the bacterial cell. Accordingly, we developed a genetic screen to identify C. jejuni genes that encode a type III secretion amino-terminal sequence that utilizes the flagellar type III secretion system of Yersinia enterocolitica and a phospholipase reporter ( yplA ). We screened a library of 321 C. jejuni genes and identified proteins with putative type III secretion amino-terminal sequences. One gene identified by the screen was Cj1242. We generated a mutation in Cj1242 , and performed growth rate, motility, secretion and INT 407 cell adherence and internalization assays. The C. jejuni Cj1242 mutant was not altered in growth rate or motility when compared with the wild-type strain, but displayed an altered secretion profile and a reduction in host cell internalization. Based on the phenotype of the C. jejuni Cj1242 mutant, we designated the protein Campylobacter invasion antigen C (CiaC). Collectively, our findings indicate that CiaC is a potentially important virulence factor.     

Yersinia enterocolitica type III secretion of YopR requires a structure in its mRNA

Yersinia type III secretion machines transport substrate proteins into the extracellular medium or into the cytoplasm of host cells. Translational hybrids, involving genes that encode substrates as well as reporter proteins that otherwise cannot travel the type III pathway, identified signals that promote transport of effector Yops into host cells. Signals for the secretion of substrates into high calcium media were hitherto unknown. By exploiting attributes of translational hybrids between yopR, whose product is secreted, and genes that encode impassable proteins that jam the secretion machine, we isolated yopR mutations that abolish substrate recognition. Similar to effector Yops, an N-terminal or 5' signal in codons 1-11 is required to initiate YopR into the type III pathway. YopR secretion cannot be completed and translational hybrids cannot impose a block without a second signal, positioned at codons 131-149. Silent mutations in the second signal abrogate function and the phenotype of other mutations can be suppressed by secondary mutations predicted to restore base complementary in a 3' stem-loop structure of the yopR mRNA.     

Regulatory role of PopN and its interacting partners in type III secretion of Pseudomonas aeruginosa

The type III secretion system (T3SS) of Pseudomonas aeruginosa plays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and both popN and pcr1 mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of the popN operon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. Unlike popN and pcr1 mutants, pcr3 and pcr4 mutants are totally defective in type III secretion, while a pcr2 mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.     

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.