沙眼衣原体CT-249基因编码蛋白为一包涵体膜蛋白

使用融合蛋白GST-CT249的抗体对假想蛋白CT249的特性进行研究。使用PCR方法从L2型沙眼衣原体的基因组中扩增编码CT249蛋白的开放读码区基因,限制性内切酶BamHⅠ和NotⅠ消化、T4连接酶连接导入pGEX-6p2载体,进一步把重组质粒pGEX-6p2-CT249转化到XL1-blue细菌,并诱导表达融合蛋白GST-CT249。在融合蛋白GST-CT249免疫小鼠制备抗体后,应用直接免疫荧光技术对衣原体感染细胞内的CT249基因表达的内源性蛋白进行初步定位。成功克隆出沙眼衣原体基因CT249,全长为351bp,并表达了融合蛋白GST-CT249,分子量为38.2kDa。制备了融合蛋白GST-CT249的抗体并初步定位假想蛋白CT249于沙眼衣原体包涵体膜蛋白上。总之,使用融合蛋白GST-CT249的抗体,鉴定假想蛋白CT249为一种新的沙眼衣原体包涵体膜蛋白。该发现将为进一步深入研究衣原体与宿主细胞间某些机制提供了有用的途径。     

Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.     

A secondary structure motif predictive of protein localization to the chlamydial inclusion membrane

Chlamydiae are obligate intracellular pathogens that spend their entire growth phase sequestered in a membrane-bound vacuole called an inclusion. A set of chlamydial proteins, labelled Inc proteins, has been identified in the inclusion membrane (IM). The predicted IncA, IncB and IncC amino acid sequences share very limited similarity, but a common hydrophobicity motif is present within each Inc protein. In an effort to identify a relatively complete catalogue of Chlamydia trachomatis proteins present in the IM of infected cells, we have screened the genome for open reading frames encoding this structural motif. Hydropathy plot analysis was used to screen each translated open reading frame in the C. trachomatis genome database. Forty-six candidate IM proteins (C-lncs) that satisfied the criteria of containing a bilobed hydrophobic domain of at least 50 amino acids were identified. The genome of Chlamydia pneumoniae encodes a larger collection of C-lnc proteins, and only approximately half of the C-lncs are encoded within both genomes. In order to confirm the hydropathy plot screening method as a valid predictor of C-lncs, antisera and/or monoclonal antibodies were prepared against six of the C. trachomatis C-lncs. Immunofluorescence microscopy of C. trachomatis-infected cells probed with these antibodies showed that five out of six C-lncs are present in the chlamydial IM. Antisera were also produced against C. pneumoniae p186, a protein sharing identity with Chlamydia psittaci lncA and carrying a similar bilobed hydrophobic domain. These antisera labelled the inclusion membrane in C. pneumoniae infected cells, confirming that proteins sharing the unique secondary structural characteristic also localize to the inclusion membrane of C. pneumoniae. Sera from patients with high-titre antibodies to C. trachomatis were examined for reactivity with each tested C-lnc protein. Three out of six tested C-lncs were recognized by a majority of these patient sera. Collectively, these studies identify and characterize novel proteins localized to the chlamydial IM and demonstrate the existence of a potential secondary structural targeting motif for localization of chlamydial proteins to this unique intracellular environment.     

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.     

Identification and characterization of a novel Chlamydia trachomatis reticulate body protein

The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'.     

Initial characterization of a chlamydial receptor on mammalian cells

We have examined characteristics of the binding of eukaryotic cells to chlamydial elementary body (EB)-specific proteins. A wide variety of eukaryotic cell lines bound to representatives of both Chlamydia trachomatis lymphogranuloma venereum (LGV) and trachoma biovars and a C. psittaci strain meningopneumonitis (Mn) suggesting the presence of a common host cell receptor. Neither tunicamycin nor neuraminidase treatment of HeLa cells impaired binding to C. trachomatis EB, implying that host cell N-linked carbohydrate domains and sialic acid moieties, respectively, are not involved in attachment. However, trypsinized HeLa cells do not bind to EB, suggestive of a proteinaceous host cell receptor. The trypsin sensitivity of two EB-specific binding proteins Mr = 18,000 and 31,000) was also examined, and the finding that 125I-labeled HeLa cells bind both the 18,000 and 31,000-dalton proteins after chlamydial trypsinization corroborates our earlier observation that these EB binding proteins mediate attachment.     

Mammalian 14-3-3beta associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG

Chlamydiae replicate intracellularly within a vacuole that is modified early in infection to become fusogenic with a subset of exocytic vesicles. We have recently identified four chlamydial inclusion membrane proteins, IncD-G, whose expression is detected within the first 2 h after internalization. To gain a better understanding of how these Inc proteins function, a yeast two-hybrid screen was employed to identify interacting host proteins. One protein, 14-3-3beta, was identified that interacted specifically with IncG. The interaction between 14-3-3beta and IncG was confirmed in infected HeLa cells by indirect immunofluorescence microscopy and interaction with a GFP-14-3-3beta fusion protein. 14-3-3 proteins are phosphoserine-binding proteins. Immunoprecipitation studies with [32P]-orthophosphate-labelled cells demonstrated that IncG is phosphorylated in both chlamydia-infected HeLa cells and in yeast cells expressing IncG. Site-directed mutagenesis of predicted 14-3-3 phosphorylation sites demonstrated that IncG binds to 14-3-3beta via a conserved 14-3-3-binding motif (RS164RS166F). Finally, indirect immunofluorescence demonstrated that 14-3-3beta interacts with Chlamydia trachomatis inclusions but not C. psittaci or C. pneumoniae inclusions. 14-3-3beta is the first eukaryotic protein found to interact with the chlamydial inclusion; however, its unique role in C. trachomatis pathogenesis remains to be determined.     

Characterization and functional analysis of PorB, a Chlamydia porin and neutralizing target

A predicted protein (CT713) with weak sequence similarity to the major outer membrane protein (20.4% identity) in Chlamydia trachomatis was identified by Chlamydia genome analysis. We show that this protein is expressed, surface accessible, localized to the chlamydial outer membrane complex and functions as a porin. This protein, PorB, was highly conserved among different serovars, with nearly identical sequences between serovars D, B, C and L2. Sequence comparison between C. trachomatis and Chlamydia pneumoniae showed less conservation between species with 59.3% identity. Immunofluorescence staining with monospecific antisera to purified PorB revealed antigen localized within chlamydial inclusions and found throughout the developmental cycle. Antibodies to PorB neutralized infectivity of C. trachomatis in an in vitro neutralization assay confirming that PorB is surface exposed. As PorB was found to be in the outer membrane, as well as having weak structural characteristics similar to major outer membrane protein (MOMP) and other porins, a liposome-swelling assay was used to determine whether this protein had pore-forming capabilities. PorB had pore-forming activity and was shown to be different from MOMP porin activity.     

Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.     

Evidence that CT694 is a novel Chlamydia trachomatis T3S substrate capable of functioning during invasion or early cycle development

Chlamydia trachomatis is an obligate intracellular parasite, occupies a membrane-bound vacuole throughout development and is capable of manipulating the eukaryotic host by translocating effector molecules via a type III secretion system (T3SS). The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early cycle events. We present evidence here that the C. trachomatis protein CT694 represents an early cycle-associated effector protein. CT694 is secreted by the Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two-hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. Additionally, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibres that correlated with the ability of CT694 to interact with AHNAK. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis , and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development particular to this species.     

In silico inference of inclusion membrane protein family in obligate intracellular parasites chlamydiae.

Chlamydiae are obligate intracellular pathogens that proliferate only inside a vacuole, called an inclusion. Chlamydial Inc proteins are known to be a major component of the inclusion membrane, but little is known about the gene number and function. The Inc proteins share very low sequence similarity but a similar hydropathy profile among them. Using the hydropathy profile, we computationally searched the open reading frames (ORFs) having a similar profile and predicted 90 and 36 ORFs (Inc-like ORFs) as candidates for Inc proteins in Chlamydia pneumoniae J138 and Chlamydia trachomatis serovar D, respectively. On the other hand, only a few Inc-like ORFs were found in organisms other than chlamydiae, suggesting that the Inc-like ORFs are specific to chlamydiae. Comparative genome analysis also revealed that the Inc-like ORFs have multiplied and diverged as paralogues and orthologues in the chlamydial genomes, and that some Inc-like ORFs lacked the N-terminal portion or encoded the split form. The data suggest that these gene products constitute a large protein family and may play an important role in chlamydial infection, growth and survival in the host cell.     

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia -containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia -containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis .     

CT358蛋白在沙眼衣原体感染细胞中的定位及特性分析

确定沙眼衣原体CT358蛋白在衣原体感染细胞中的位置并初步鉴定其生物学功能．采用PCR方法从D型沙眼衣原体的基因组中扩增CT358基因,并克隆入pGEX和pDSRedC1表达载体中．将重组质粒pGEX-CT358转化到XL1-blue宿主菌,并诱导表达融合蛋白GST-CT358．纯化后的CT358融合蛋白免疫小鼠制备抗体,应用间接免疫荧光技术对CT358蛋白在衣原体感染细胞内的定位及表达模式进行分析．同时,pDSRedC1-CT358重组质粒瞬时转染HeLa细胞,观察CT358蛋白对衣原体感染的影响．实验结果证明CT358蛋白为沙眼衣原体包涵体膜蛋白．该蛋白质在衣原体感染12 h后就表达定位于包涵体膜上,直至持续到整个感染周期,转基因在胞浆表达的CT358融合蛋白不影响其后的衣原体感染．该研究为深入研究衣原体与宿主细胞间相互作用提供了新的线索,并可为衣原体性的治疗、预防提供新方向．     

Secretion of Cpn0796 from Chlamydia pneumoniae into the host cell cytoplasm by an autotransporter mechanism

By comparison of proteome profiles of purified Chlamydia pneumoniae and whole lysates of C. pneumoniae infected HEp-2 cells, an N-terminal fragment of the previously uncharacterized chlamydial protein Cpn0796 was identified as a secreted protein. A 38 kDa cleavage product of Cpn0796 was present in infected cells, whereas only the 65 kDa full-length Cpn0796 could be detected in purified Chlamydia. Confocal immunofluorescence microscopy demonstrated that Cpn0796 was localized in the Chlamydia membrane in young inclusions. However, at 36 h post infection and later Cpn0796 was detected in the cytoplasm of C. pneumoniae infected HEp-2 and BHK cells. Furthermore, Cpn0796 was detected in the cytoplasm of infected cells in the lungs of C. pneumoniae infected C57Bl mice. When cleavage was inhibited, Cpn0796 was retained in the chlamydiae. We propose that Cpn0796 is an autotransporter the N-terminal of which is translocated to the host cell cytoplasm. This is the first example of secretion of a Chlamydia autotransporter passenger domain into the host cell cytoplasm. Cpn0796 is specific for C. pneumoniae, where five homologous proteins are encoded by clustered genes. None of these five proteins were found to be secreted.     

Conservation of the biochemical properties of IncA from Chlamydia trachomatis and Chlamydia caviae: oligomerization of IncA mediates interaction between facing membranes

The developmental cycle of Chlamydiaceae occurs in a membrane compartment called an inclusion. IncA is a member of a family of proteins synthesized and secreted onto the inclusion membrane by bacteria. IncA proteins from different species of Chlamydiaceae show little sequence similarity. We report that the biochemical properties of Chlamydia trachomatis and Chlamydia caviae are conserved. Both proteins self-associate to form multimers. When artificially expressed by the host cell, they localize to the endoplasmic reticulum. Strikingly, heterologous expression of IncA in the endoplasmic reticulum completely inhibits concomitant inclusion development. Using truncated forms of IncA from C. caviae, we show that expression of the C-terminal cytoplasmic domain of the protein at the surface of the endoplasmic reticulum is sufficient to disrupt the bacterial developmental cycle. On the other hand, development of a C. trachomatis strain that does not express IncA is not inhibited by artificial IncA expression, showing that the disruptive effect observed with the wild-type strain requires direct interactions between IncA molecules at the inclusion and on the endoplasmic reticulum. Finally, we modeled IncA tetramers in parallel four helix bundles based on the structure of the SNARE complex, a conserved structure involved in membrane fusion in eukaryotic cells. Both C. trachomatis and C. caviae IncA tetramers were highly stable in this model. In conclusion, we show that the property of IncA proteins to assemble into multimeric structures is conserved between chlamydial species, and we propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion.     

Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae

Whole sequence genome analysis is invaluable in providing complete profiles of related proteins and gene families. The genome sequences of the obligate intracellular bacteria Chlamydia trachomatis and Chlamydia pneumoniae both encode proteins with similarity to several 90-kDa Chlamydia psittaci proteins. These proteins are members of a large superfamily, C. trachomatis with 9 members and C. pneumoniae with 21 members. All polymorphic membrane protein (Pmp) are heterogeneous, both in amino acid sequence and in predicted size. Most proteins have apparent signal peptide leader sequences and hence are predicted to be localized to the outer membrane. The unifying features of all proteins are the conserved amino acid motifs GGAI and FXXN repeated in the N-terminal half of each protein. In both genomes, the pmp genes are clustered at various locations on the chromosome. Phylogenetic analysis suggests six related families, each with at least one C. trachomatis and one C. pneumoniae orthologue. One of these families has seen prolific expansion in C. pneumoniae, resulting in 13 protein paralogues. The maintenance of orthologues from each species suggests specific functions for the proteins in chlamydial biology.     

Autophagy-independent function of MAP-LC3 during intracellular propagation of Chlamydia trachomatis

Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.