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.     

The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion

Chlamydiae replicate within an intracellular vacuole, termed an inclusion, that is non-fusogenic with vesicles of the endosomal or lysosomal compartments. Instead, the inclusion appears to intersect an exocytic pathway from which chlamydiae intercept sphingomyelin en route from the Golgi apparatus to the plasma membrane. Chlamydial protein synthesis is required to establish this interaction. In an effort to identify those chlamydial proteins controlling vesicle fusion, we have prepared polyclonal antibodies against several Chlamydia trachomatis inclusion membrane proteins. Microinjection of polyclonal antibodies against three C. trachomatis inclusion membrane proteins, IncA, F and G, into the cytosol of cells infected with C. trachomatis demonstrates reactivity with antigens on the cytoplasmic face of the inclusion membrane, without apparent inhibition of chlamydial multiplication. Microinjection of antibodies against the C. trachomatis IncA protein, however, results in the development of an aberrant multilobed inclusion structure remarkably similar to that of C. psittaci GPIC. These results suggest that the C. trachomatis IncA protein is involved in homotypic vesicle fusion and/or septation of the inclusion membrane that is believed to accompany bacterial cell division in C. psittaci . This proposal is corroborated by the expression of C. trachomatis and C. psittaci IncA in a yeast two-hybrid system to demonstrate C. trachomatis , but not C. psittaci , IncA interactions. Despite the inhibition of homotypic fusion of C. trachomatis inclusions, fusion of sphingomyelin-containing vesicles with the inclusion was not suppressed.     

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.     

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。     

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.     

Influence of the Chlamydia pneumoniae AR39 bacteriophage ϕCPAR39 on chlamydial inclusion morphology

The human respiratory tract pathogen Chlamydia pneumoniae AR39 is naturally infected by the bacteriophage ?CPAR39. The phage genome encodes six ORFs, [ORF8, ORF4, ORF5, and viral protein (VP) 1, VP2 and VP3]. To study the growth of the phage, antibodies were generated to VP1 and used to investigate the ?CPAR39 infection. Using immunofluorescence laser confocal microscopy and two-dimensional gel electrophoresis, we investigated the ?CPAR39 infection of C. pneumoniae AR39. It was observed that ?CPAR39 infection differentially suppressed the C. pneumoniae protein synthesis as the polymorphic membrane protein 10 and the secreted chlamydial protein Cpn0796 was hardly expressed while the secreted chlamydial protein Cpaf was expressed, but not secreted. The inclusion membrane protein, IncA, was demonstrated to surround the phage-infected abnormal reticulate bodies (RB) as well as being located in the inclusion membrane. As IncA is secreted by the type 3 secretion (T3S) system, it is likely that the T3S is disrupted in the phage-infected chlamydiae such that it accumulates around the infected RB.     

A directed screen for chlamydial proteins secreted by a type III mechanism identifies a translocated protein and numerous other new candidates

Chlamydiae are strict intracellular parasites that induce their internalization upon contact with the host cell and grow inside an intracellular compartment called an inclusion. They possess a type III secretion (TTS) apparatus, which allows for the translocation of specific proteins in the host cell cytosol. In particular, chlamydial proteins of the Inc family are secreted to the inclusion membrane by a TTS mechanism; other TTS substrates are mostly unknown. Using a secretion assay based on the recognition of TTS signals in Shigella flexneri, we searched for TTS signals in the proteins of unknown function, conserved between three different chlamydial species, Chlamydia pneumoniae, C. trachomatis and C. caviae. We identified 24 new candidate proteins which did not belong to the Inc family. Four of these proteins were also secreted as full-length proteins by a TTS mechanism in S. flexneri, indicating that their translocation does not require other chlamydial proteins. One of these proteins was detected in the cytosol of infected cells using specific antibodies, directly demonstrating that it is translocated in the host cell during bacterial proliferation. More generally, this work represents the first directed search for TTS effectors not based on genetic information or sequence similarity. It reveals the abundance of proteins secreted in the host cell by chlamydiae.     

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.     

肺炎嗜衣原体蛋白酶样活性因子免疫优势区基因 重组蛋白在早期诊断中的应用

[目的]克隆和表达肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)蛋白酶样活性因子(CPAF)免疫优势区基因,评价重组蛋白在早期感染诊断中的应用价值.[方法]挑选并克隆出Cpn CPAF免疫优势区基因,构建原核表达载体,诱导表达并纯化重组蛋白,分析其抗原特异性;间接ELISA法检测Cpn参考血清、临床血清标本中的特异性IgM抗体,以及呼吸道感染患者痰咽拭子中的Cpn抗原;检测沙眼衣原体(Chlamydia trachomatis,Ct)临床阳性血清和泌尿生殖道分泌物.[结果]高效表达和纯化出一相对分子量约51.3kDa的重组蛋白;Western blot证明其只与人抗Cpn抗血清发生特异性反应;间接ELISA法检测40份Cpn IgM参考血清,阴性和阳性结果的一致率均为100%(40/40);与"金标准"方法MIF对照,检测300例临床血清标本中的IgM抗体,符合率为98.3%;与PCR试剂对照,检测120份呼吸道感染患者痰咽拭子中的Cpn抗原,符合率为88.3%;检测Ct阳性血清和泌尿生殖道分泌物,与Ct没有交叉反应.[结论]制备的CPAF免疫优势区基因重组蛋白具有良好的抗原性,在Cpn感染早期诊断中具有较高的利用价值.     

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.     

Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes.

Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L2 were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. Our data suggest that these putative chlamydial adhesins play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.     

Nucleotide sequence and taxonomic value of the major outer membrane protein gene of Chlamydia pneumoniae IOL-207

Chlamydia pneumoniae IOL-207 genomic DNA was hybridized with a 1.5 kb labelled DNA probe containing the 3' region of the coding sequence for the major outer membrane protein (MOMP) of C. trachomatis serovar L1. An 8.5 kb Bg/II fragment containing the complete MOMP gene was cloned into lambda EMBL3. Two hybridizing EcoRI fragments were sub-cloned into the lambda ZAP II cloning vector and the resulting plasmids were used as templates for sequencing both strands of the C. pneumoniae MOMP gene. Computer taxonomic studies using the nucleotide and inferred amino acid sequence of the MOMP of C. pneumoniae IOL-207 and all known chlamydial MOMP sequences supported the designation of C. pneumoniae as a new species, but electron microscope studies suggested that the presence of pear-shaped elementary bodies (EBs) may not be a reliable taxonomic criterion.     

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 .     

Sequence analysis and lipid modification of the cysteine-rich envelope proteins of Chlamydia psittaci 6BC.

The envelopes of elementary bodies of Chlamydia spp. consist largely of disulfide-cross-linked major outer membrane protein (MOMP) and two cysteine-rich proteins (CRPs). The MOMP gene of Chlamydia psittaci 6BC has been sequenced previously, and the genes encoding the small and large CRPs from this strain were cloned and sequenced in this study. The CRP genes were found to be tandemly arranged on the chlamydial chromosome but could be independently expressed in Escherichia coli. The deduced 87-amino-acid sequence of the small-CRP gene (envA) contains 15 cysteine residues, a potential signal peptide, and a potential signal peptidase II-lipid modification site. Hydropathy plot and conformation analysis of the small-CRP amino acid sequence indicated that the protein was unlikely to be associated with a membrane. However, the small CRP was specifically labeled in host cells incubated with [3H]palmitic acid and may therefore be associated with a membrane through a covalently attached lipid portion of the molecule. The deduced 557-amino-acid sequence of the large-CRP gene (envB) contains 37 cysteine residues and a single putative signal peptidase I cleavage site. In one recombinant clone the large CRP appeared to be posttranslationally cleaved at two sites, forming a doublet in a manner similar to the large-CRP doublet made in native C. psittaci 6BC. Comparison of the deduced amino acid sequences of the CRPs from chlamydial strains indicated that the small CRP is moderately conserved, with 54% identity between C. psittaci 6BC and Chlamydia trachomatis, and the large CRP is highly conserved, with 71% identity between C. psittaci and C. trachomatis and 85% identity between C. psittaci 6BC and Chlamydia pneumoniae. The positions of the cysteine residues in both CRPs are highly conserved in Chlamydia spp. From the number of cysteine residues in the MOMP and the CRPs and the relative incorporation of [35S]cysteine into these proteins, it was calculated that the molar ratio of C. psittaci 6BC elementary body envelope proteins is about one large-CRP molecule to two small-CRP molecules to five MOMP molecules.     

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.     

Characterization of a hypervariable region in the genome of Chlamydophila pneumoniae

Chlamydophila pneumoniae displays surprisingly little genomic variation, as seen by comparisons of the published genomes from three different isolates and sequencing of four different genes from different isolates. We have in the present study, however, demonstrated genomic variation between 10 C. pneumoniae isolates in the 11690-bp region between the two outer membrane protein genes pmp1 and pmp2. This region of the C. pneumoniae CWL-029 isolate contains seven C. pneumoniae-specific open reading frames (hb1-7, encoding hydrophobic beta-sheet-containing proteins). We identified additionally 12 open reading frames in the C. pneumoniae CWL-029 genome encoding hypothetical proteins with similarity to the seven hypothetical Hb-proteins. Compared to other isolates, genomic variation is seen to cause frame-shifting of three of the 19 hb-open reading frames, which are proposed to be three full-length genes and eight frame-shifted pseudogenes. The hypothetical proteins encoded by these proposed genes contain an N-terminally located highly hydrophobic stretch of 50-60 residues. A similar motif is found in all identified Chlamydia inclusion membrane proteins and therefore the Hb-proteins are candidate inclusion proteins.     

Characterization of in vitro chlamydial cultures in low-oxygen atmospheres

To mimic in vivo conditions during chlamydial infections, Chlamydia trachomatis serovar D and Chlamydia pneumoniae CWL029 were cultured in low-oxygen atmospheres containing 4% O(2), with parallel controls cultured in atmospheric air. Both were enriched with 5% CO(2). The results showed a dramatic increase in the growth of C. pneumoniae but not of C. trachomatis.