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.     

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.     

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.     

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'.     

Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.     

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.     

Identification of immunodominant linear B-cell epitopes within the major outer membrane protein of Chlamydia trachomatis

Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. Chlamydial major outer membrane protein (MOMP) can induce strong cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of MOMP was analyzed using computer-assisted techniques to scan B-cell epitopes, and three possible linear B-cell epitopes peptides (VLKTDVNKE, TKDASIDYHE, TRLIDERAAH) with high predicted antigenicity and high conservation were investigated. The DNA coding region for each potential epitope was cloned into pET32a(+) and expressed as Trx-His-tag fusion proteins in Escherichia coli. The fusion proteins were purified by Ni-NTA agarose beads and followed by SDS-PAGE and western blot analysis. We immunized mice with these three fusion proteins. The sera containing anti-epitope antibodies from the immunized mice could recognize C. trachomatis serovars D and E in ELISA. Antisera of these fusion proteins displayed an inhibitory effect on invasion of serovar E by in vitro neutralization assays. In addition, serum samples from convalescent C. trachomatis-infected patients were reactive with the epitope fusion proteins by western blot assay. Our results showed that the epitope sequences selected by bioinformatic analysis are highly conserved C. trachomatis MOMP B-cell epitopes, and could be good candidates for the development of subunit vaccines, which can be used in clinical diagnosis.     

The gene for the S7 ribosomal protein of Chlamydia trachomatis: characterization within the chlamydial sfr operon

The prokaryotic ribosomal operon, str, contains open reading frames for the two elongation factors, elongation factor G (EF-G) and elongation factor Tu (EF-Tu), and ribosomal proteins S7 and S12. The DNA sequence and predicted amino acid sequence for S7 from Chlamydia trachomatis are presented and compared with homologues from other prokaryotes. Also, the relationship of the S7 gene to the open reading frames for ribosomal protein S12 and EF-G is described. Significant amino acid homology is also noted when the amino-terminal sequence of chlamydial EF-G is compared with the cytoplasmic tetracycline resistance factors, tetM and tetO, from streptococci and Campylobacter jejuni. Related findings and possible resistance mechanisms for the newly recognized tetracycline-resistant clinical isolates of C. trachomatis are discussed.     

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.     

Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis

The major outer membrane protein (MOMP) of Chlamydia trachomatis carries serovar-, subspecies-, species- and genus immunodomains, antibodies to which may be protective. We have compared the inferred amino acid sequences for MOMP from different serovars of C. trachomatis and from Chlamydia psittaci to identify the likely locations of these sero-taxonomic epitopes. Overlapping peptides corresponding to each of these regions were synthesized on a solid phase and probed with monoclonal antibodies (MAbs) of appropriate specificities. We describe the primary structures of the binding sites of MAb to each of these four epitopes on C. trachomatis serovar L1 MOMP.     

Cloning and characterization of a secY homolog from Chlamydia trachomatis

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc-α ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50 195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SceY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.     

Nucleotide sequence of DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3

DNA encoding the major outer membrane protein of Chlamydia trachomatis serovar L3 was sequenced following amplification by the polymerase chain reaction. A comparison with the deduced amino acid (aa) sequence of the C. trachomatis serovar L2 showed that the L3 had three extra aa and 55 aa substitutions.     

Core of the partner switching signalling mechanism is conserved in the obligate intracellular pathogen Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that can cause sexually transmitted and ocular diseases in humans. Its biphasic developmental cycle and ability to evade host-cell defences suggest that the organism responds to external signals, but its genome encodes few recognized signalling pathways. One such pathway is predicted to function by a partner switching mechanism, in which key protein interactions are controlled by serine phosphorylation. From genome analysis this mechanism is both ancient and widespread among eubacteria, but it has been experimentally characterized in only a few. C. trachomatis has no system of genetic exchange, so here an in vitro approach was used to establish the activities and interactions of the inferred partner switching components: the RsbW switch protein/kinase and its RsbV antagonists. The C. trachomatis genome encodes two RsbV paralogs, RsbV(1) and RsbV(2). We found that each RsbV protein was specifically phosphorylated by RsbW, and tandem mass spectrometry located the phosphoryl group on a conserved serine residue. Mutant RsbV(1) and RsbV(2) proteins in which this conserved serine was changed to alanine could activate the yeast two-hybrid system when paired with RsbW, whereas mutant proteins bearing a charged aspartate failed to activate. From this we infer that the phosphorylation state of RsbV(1) and RsbV(2) controls their interaction with RsbW in vivo. This experimental demonstration that the core of the partner switching mechanism is conserved in C. trachomatis indicates that its basic features are maintained over a large evolutionary span. Although the molecular target of the C. trachomatis switch remains to be identified, based on the predicted properties of its input phosphatases we propose that the pathway controls an important aspect of the developmental cycle within the host, in response to signals external to the C. trachomatis cytoplasmic membrane.     

A new family of highly variable proteins in the Chlamydophila pneumoniae genome

Chlamydiaceae are obligate intracellular bacterial pathogens characterized by a wide range of vertebrate host, tissue tropism and spectrum of diseases. To get insights into the biological mechanisms involved in these differences, we have put forward a computational and experimental procedure to identify the genome recombination hotspots, as frequent sequence variation allows rapid adaptation to environmental changes. We find a larger potential for recombination in Chlamydophila pneumoniae genomes as compared with Chlamydia trachomatis or Chlamydia muridarum. Such potential is mostly concentrated in a family of seven previously uncharacterized species-specific elements that we named ppp for C.pneumoniae polymorphic protein genes, which have the potential to vary by homologous recombination and slipped-mispair. Experimentally, we show that these sequences are indeed highly polymorphic among a collection of nine C.pneumoniae strains of very diverse geographical and pathological origins, mainly by slippage of a poly(C) tract. We also show that most elements are transcribed during infection. In silico analyses suggest that Ppps correspond to outer membrane proteins. Given their species specificity, their putative location in the outer membrane and their extreme polymorphism, Ppps are most likely to be important in the pathogenesis of C.pneumoniae and could represent targets for future vaccine development.     

Cysteine-rich outer membrane proteins of Chlamydia trachomatis display compensatory sequence changes between biovariants

Two cysteine-rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58,000 (outer membrane protein 2; OMP2) and 15,000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.     

Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins.