Chlamydia psittaci ewe abortion agent: complete nucleotide sequence of the major outer membrane protein gene

The complete nucleotide sequence encoding the major outer membrane protein (MOMP) of Chlamydia psittaci strain A22/M, responsible for enzootic abortion of ewes (EAE), has been determined. An 800bp Eco RI/ Xba I fragment containing a portion of the MOMP coding sequence from C. trachomatis serovar L1 was used to probe a λL47.1 genomic library constructed from DNA obtained from C. psittaci EAE A22/M. The recombinant L47.1/EA1 was selected and contained the entire C. psittaci MOMP gene within a 7.5 kb Bam HI fragment. The DNA sequence revealed an open reading frame encoding 402 amino acids, including a 22 amino acid signal peptide, which exhibited 17/22 conservation with the signal peptide of C. trachomatis MOMP. The calculated molecular mass of the C. psittaci MOMP was 43 kDa. A comparison of the MOMP genes of C. psittaci and C. trachomatis revealed only 34% nucleotide sequence homology, but 65% amino acid homology.     

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.     

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.     

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues.     

Cloning and expression in Escherichia coli of a species-specific Chlamydia trachomatis outer membrane antigen

Abstract A gene library of Chlamydia trachomatis serovar L₂ (strain 434) was constructed in Escherichia coli using plasmid pBR322. Amongst 200 recombinants we have identified and characterized a recombinant E. coli that expresses a protein antigen of M _r 74 000 similar in size to an outer membrane antigen produced by elementary bodies of C. trachomatis . Immunologically, the molecule synthesised by E. coli has the same specificity as the protein encoded by serovar L₂. A 1.8 kb DNA fragment from the recombinant insert, used as a hybridization probe, confirmed the species specificity of this clone at the gene level.     

Analysis and detection of chlamydial DNA

Elementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6.7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.     

Chlamydia trachomatis Mip-like protein

A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described. In this paper the entire chlamydia Mip-like sequence of C. trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins. The chlamydial mip-like gene was detected by polymerase chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein failed to demonstrate surface-exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno-gold electron microscopy. However, a complement-dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N-terminal fragment of the Mip-like protein suggesting that antibody-accessible epitopes are present on infectious EBs.     

Comparison of Chlamydia trachomatis serovar L2 growth in polarized genital epithelial cells grown in three-dimensional culture with non-polarized cells

A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.     

Genetic differences in the Chlamydia trachomatis tryptophan synthase alpha-subunit can explain variations in serovar pathogenesis

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses of approximately 30 and 40 kDa was observed on 2D-gels. The 30-kDa protein from C. trachomatis L2 migrated with a significantly lower molecular weight in C. trachomatis A. In this paper we include C. trachomatis B, C and D in our investigations and identify the proteins as alpha- and beta-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the TrpA activity, thus elucidating a possible molecular mechanism behind variations in the pathogenesis of C. trachomatis serovars.     

Isolation and molecular characterization of the ribosomal protein L6 homolog from Chlamydia trachomatis.

The cloning of a Chlamydia trachomatis eukaryotic cell-binding protein reported earlier from our laboratory (R. Kaul, K. L. Roy, and W. M. Wenman, J. Bacteriol. 169:5152-5156, 1987) represents an artifact generated by nonspecific recombination of chromosomal DNA fragments. However, the amino terminus of this plasmid-encoded fusion product demonstrated significant homology to Escherichia coli ribosomal protein L6. By using a 458-bp PstI-HindIII fragment of recombinant pCT161/18 (representing the 5' end of the cloned gene), we isolated and characterized a C. trachomatis homolog of the ribosomal protein L6 gene of E. coli. Sequence analysis of an 1,194-bp EcoRI-SacI fragment that encodes chlamydial L6 (designated CtaL6e) revealed a 552-bp open reading frame comprising 183 amino acids and encodes a protein with a molecular weight of 19,839. Interestingly, complete gene homology between C. trachomatis serovars L2 and J, each of which exists as a single copy per genome, was observed. Expression of a plasmid-encoded gene product is dependent on the lac promoter, since no product was obtained if the open reading frame was oriented in opposition to the lac promoter. Immunoblotting of purified ribosomes revealed functional, as well as antigenic, homology between the E. coli and C. trachomatis ribosomal L6 proteins.     

Interspecies structural diversity among chlamydial genes encoding histone H1.

Recently, a eukaryotic histone H1-like protein has been detected in Chlamydia trachomatis serovar L2 [Hackstadt et al., Proc. Natl. Acad. Sci. USA 88 (1991) 3937-3941; Tao et al., J. Bacteriol. 173 (1991) 2818-2822]. We have cloned the corresponding gene from C. trachomatis serovar J and the Chlamydia psittaci strain mn. Sequencing demonstrated absolute gene identity between the two C. trachomatis serovars L2 and J, but divergence in the C. psittaci strain mn. These differences resulted in altered aa residues (in particular no cysteines) and a smaller molecular mass for H1 from C. psittaci strain mn. The amino acid (aa) sequence comparisons with other histone proteins show best alignment to sea urchin H1, notably in the C terminus, for both C. trachomatis and C. psittaci histones. Chlamydial interspecies aa homology, however, is most conserved at the N terminus, suggestive of a bi-functional role for these unique histone proteins.     

PCR amplification and cycle DNA sequencing analysis of the Chlamydia trachomatis elongation factor Tu gene

Based on the elongation factor Tu (EF-Tu) gene of Chlamydia trachomatis, a pair of oligonucleotide primers CTUFU and CTUFD, were designed to amplify a specific target fragment of 931bp. The PCR assay could detect C. trachomatis in cervical smear specimens obtained from sex workers undergoing routine examination in an STD clinic. Distinct target bands were also amplified from at least 10ng of positive control DNA samples from cultured cells infected with C. trachomatis. PCR with these primers could differentiate C. trachomatis from eight non-chlamydial bacterial species. Further verification could be obtained from the non-digestion of C. trachomatis PCR products by MspA1I restriction endonuclease, in contrast to the digestion of the non-specific PCR products of Klebsiella and Bacillus. Direct cycle DNA sequencing of 450bp of the PCR products of four C. trachomatis isolates revealed complete identity of one isolate with the known sequence of serovar F, while the other three isolates harboured three phenotypically silent point mutations at codons 96, 305 and 312 of the EF-Tu gene. The sequence analyses confirm the authenticity of the target bands, reiterate the conservation and role of the EF-Tu gene in protein biosynthesis, and indicate the utility of the primers for the rapid detection of C. trachomatis.     

PCR amplification and cycle DNA sequencing analysis of the Chlamydia trachomatis elongation factor Tu gene

Based on the elongation factor Tu (EF-Tu) gene of Chlamydia trachomatis, a pair of oligonucleotide primers CTUFU and CTUFD, were designed to amplify a specific target fragment of 931bp. The PCR assay could detect C. trachomatis in cervical smear specimens obtained from sex workers undergoing routine examination in an STD clinic. Distinct target bands were also amplified from at least 10ng of positive control DNA samples from cultured cells infected with C. trachomatis. PCR with these primers could differentiate C. trachomatis from eight non-chlamydial bacterial species. Further verification could be obtained from the non-digestion of C. trachomatis PCR products by MspA1I restriction endonuclease, in contrast to the digestion of the non-specific PCR products of Klebsiella and Bacillus. Direct cycle DNA sequencing of ～450bp of the PCR products of four C. trachomatis isolates revealed complete identity of one isolate with the known sequence of serovar F, while the other three isolates harboured three phenotypically silent point mutations at codons 96, 305 and 312 of the EF-Tu gene. The sequence analyses confirm the authenticity of the target bands, reiterate the conservation and role of the EF-Tu gene in protein biosynthesis, and indicate the utility of the primers for the rapid detection of C. trachomatis.     

Identification by sequence analysis of two-site posttranslational processing of the cysteine-rich outer membrane protein 2 of Chlamydia trachomatis serovar L2.

The 60,000-molecular-weight cysteine-rich outer membrane protein (OMP2) from Chlamydia trachomatis participates in the disulfide-mediated outer-membrane organization unique to this organism. In addition, this protein is a primary focus of the host immune response. We cloned and sequenced the gene for OMP2 from C. trachomatis serovar L2. A lambda gt11 recombinant that expressed an antigenic portion of this protein was selected by antibody screening and provided a probe for the selection in lambda 1059 of a clone containing the entire gene. DNA sequencing of this clone identified one open reading frame of 1,641 base pairs, starting with a methionine codon and coding for a polypeptide with a molecular weight of 58,792. Amino-terminal protein sequencing and analysis of the translated DNA sequence demonstrated that processing at alternate signal peptide cleavage sites accounts for the molecular-weight polymorphism of this protein. The mature proteins had a net positive charge and contained 24 cysteine residues.     

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。     

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.     

Biochemical evidence for the existence of thymidylate synthase in the obligate intracellular parasite Chlamydia trachomatis.

Since eucaryotic cell-derived thymidine or thymidine nucleotides are not incorporated into Chlamydia trachomatis DNA, we hypothesized that C. trachomatis must obtain dTTP for DNA synthesis by converting dUMP to dTMP. In most cells, this reaction is catalyzed by thymidylate synthase (TS) and requires 5,10-methylenetetrahydrofolate as a cofactor. We used C. trachomatis serovar L2 and a mutant CHO K1 cell line with a genetic deficiency in folate metabolism as a host for chlamydial growth. This cell line lacks a functional dihydrofolate reductase (DHFR) gene and, as a result, is unable to carry out de novo synthesis of dTTP. C. trachomatis inclusions form normally when DHFR- cells are starved for thymidine 24 h prior to and during the course of infection. When [6-3H]uridine is used as a precursor to label C. trachomatis-infected CHO DHFR- cells, radiolabel is readily incorporated into chlamydia-specific DNA. When DNA from [6-3H]uridine-labelled infected cultures is acid hydrolyzed and subjected to high-performance liquid chromatography analysis, radiolabel is detected in thymine and cytosine nucleobases. By using the DHFR- cell line as a host and [5-3H]uridine as a precursor, we could monitor intracellular C. trachomatis TS activity simply by following the formation of tritiated water. There is a good correlation between in situ TS activity and DNA synthesis activity during the chlamydial growth cycle. In addition, both C. trachomatis-specific DNA synthesis and 3H2O release are inhibited by exogenously added 5-fluorouridine but not by 5-fluorodeoxyuridine. Finally, we demonstrated in vitro TS activity in crude extracts prepared from highly purified C. trachomatis reticulate bodies. The activity is dependent on the presence of methylenetetrahydrofolic acid and can be inhibited with 5-fluoro-dUMP. Taken together, these results indicate that C. trachomatis contains a TS for the synthesis of dTMP.     

Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis

OBJECTIVE: The aim of this study was to compare and evaluate three methods of DNA extraction for the amplification of Chlamydia trachomatis in uterine cervical samples collected in PreservCyt solution. ThinPrep is the trade name for the slide preparation. METHODS: Thirty-eight samples collected in LCx buffer medium, which were identified as C. trachomatis infected by ligase chain reaction (LCR), were selected for this study. DNA from the PreservCyt samples was extracted by three methods: (i) QIAamp kit, (ii) boiling in Tris-EDTA buffer with Chelex purification, and (iii) Proteinase K digestion with Chelex purification. Sample DNA was tested for the presence of C. trachomatis by PCR using cryptic plasmid research (CTP) primers and major outer membrane protein research momp gene (MOMP) primers. Real-time (LightCycler) PCR for relative C. trachomatis quantification following DNA extraction was performed using primers (Hsp 60) for the 60 kDa heat-shock protein hsp60 gene. RESULTS: Amplification using CTP primers was the most successful with each of the extraction protocols. Boiling in buffer was the least successful extraction method. QIAamp was the best extraction method, yielding the most positives with both the CTP and MOMP primers. Proteinase K-Chelex extraction gave similar sensitivity to QIAamp extraction with CTP primers but lower for MOMP primers. CONCLUSIONS: The DNA extraction method must be carefully selected to ensure that larger PCR amplicons can be successfully produced by PCR and to ensure high sensitivity of detection of C. trachomatis. In this study it was found that the QIAamp extraction method followed by PCR with the CTP primers was the most successful for amplification of C. trachomatis DNA.