Outer membrane complex proteins of Chlamydia pneumoniae

Abstract The protein composition of the outer membrane complex (OMC) of Chlamydia pneumoniae strain AR-39 was analyzed by metabolic labeling with [³⁵S]methionine and [³⁵S]cysteine. Cysteine-rich proteins with molecular masses of 98, 60 doublet, 39.5 (MOMP) and 15.5 kDa were found in the OMC of C. pneumoniae . The cysteine-rich proteins of the OMCs of the threee Chlamydia species showed specific reaction patterns by immunoassay and autoradiography to rabbit or turkey immune sera. Recognition of the MOMP and 60-kDa proteins of the three species was cross-reactive. However, the C. pneumoniae 98-kDa protein was recognized by anti- C. pneumoniae (AR-39) and anti- C. psittaci (TT3) immune sera. None of the immunee sera recognized the 12-kDa cysteine-rich complex.     

Plasmids of Chlamydia psittaci: Cloning and comparison of isolates by Southern hybridisation

Abstract A chlamydial plasmid, 6.2 kb in size, was isolated from an avian strain of Chlamydia psittaci and cloned into the Eco RI site of pUC13. A restriction enzyme cleavage map of the resultant clone, pAP¹p, was very similar to the published map of the plasmid cloned from the C. psittaci meningopneumonitis strain Cal-10. Southern hybridisation analyses using pAP¹p as a probe, revealed the presence of plasmids with homologous DNA sequences in avian psittacosis, avian ornithosis, ovine polyarthritis and sporadic bovine encephalomyelitis strains of C. psittaci , as well as the LGV strain of Chlamydia trachomatis . Plasmid was not detected in koala conjunctivitis, ovine abortion or feline conjunctivitis isolates. The plasmid-containing isolates could be grouped according to size (6.2 or 7.2–7.3 kb) and restriction endonuclease pattern. These three plasmid categories correlate with previously reported C. psittaci biotypes, immunotypes and serotypes. The absence of plasmid from three infectious, pathogenic strains of C. psittaci suggests that, in this species at least, plasmid-encoded genes are not essential for survival, infectivity or virulence of the parasite.     

衣原体质粒的研究进展

衣原体质粒是一个分子量约为7.5 kb,基因序列高度保守,非整合性的DNA分子,广泛存在于沙眼衣原体的各个血清型中,鼠衣原体和鹦鹉热衣原体也携带该质粒.近年来,人们发现衣原体质粒是一种毒力因子,可以导致小鼠输卵管积水.动物实验显示质粒缺失株可作为减毒活疫苗来预防衣原体感染所致的生殖道和眼睛的病变.不仅如此,衣原体质粒还是一种有效的基因操纵工具,可用于沙眼衣原体致病机制的研究.因此,开展对衣原体质粒的研究具有重要的意义.     

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.     

Plasmid diversity within the genus Chlamydia

Examination of 12 Chlamydia psittaci strains recovered from nine different host species (three avian and six mammalian) revealed the presence of a 7.5 kb plasmid in all isolates except two ovine abortion strains, the human strain IOL207 and the Cal 10 strain. Restriction mapping analysis distinguished four different plasmids that were associated with avian, feline, equine and guinea-pig C. psittaci isolates, respectively. The restriction maps of these four C. psittaci plasmid types all differed from that of the plasmid recovered from C. trachomatis L2/434. Despite this plasmid diversity, which is likely to be of taxonomic importance, all four plasmids identified within the species C. psittaci were found to share some sequence homology, which was mapped to two separate regions in the plasmid molecules. One region, which showed a high degree of homology between C. psittaci plasmids and also detectable homology with the C. trachomatis plasmid, may represent a common replication control region for plasmids of this genus.     

Identification of a multigene family coding for the 90 kDa proteins of the ovine abortion subtype of Chlamydia psittaci

Abstract While attempting to identify genes and their corresponding antigens that could be used to improve the current methods of diagnosing Chlamydia psittaci infection which causes enzootic abortion in ewes, two candidate clones were isolated from a λgt11 genomic DNA expression library of ovine abortion subtype (strain S26/3) C. psittaci . These clones contained fragments of a gene coding for a group of three chlamydial proteins of approximately 90 kDa which appeared as major immunogens by immunoblotting experiments, indicating their potential as diagnostic or possibly protective antigens. Southern blotting of S26/3 genomic DNA using the two clones as probes identified a family of three or four genes. These represent the first example of protein gene duplication reported in Chlamydia .     

Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many Gram-negative bacteria

In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.     

Detection of Chlamydia psittaci by Immunofluorescence

A direct fluorescent-antibody (FA) test was developed to detect Chlamydia psittaci in dural impressions from specimen-inoculated mice. Technical procedures for the test were compared. C. psittaci was found in mice after infection as early by the FA technique as it was by cytochemical staining methods usually used. The lymphogranuloma venereum organism was also stained by conjugated antibody to C. psittaci. A distinctive advantage of the described FA test is that organisms are identified immunologically as members of the genus Chlamydia simultaneously with their detection.     

Analysis of partial 16S rRNA nucleotide sequences of Chlamydia pecorum and C. psittaci

Partial 16S nucleotide sequences of Chlamydia psittaci isolates S26/3 (abortion), P94/1 (pigeon) and Chlamydia pecorum isolates W73 (enteric) and E58 (encephalomyelitis) were determined. Analysis of these data indicates very high levels of interspecies sequence conservation, with C. psittaci being more closely related to C. pecorum than to C. pneumoniae or C. trachomatis. Restriction enzyme analysis of nucleotide sequences indicated that BslI can be used to clearly distinguish C. psittaci and C. pecorum isolates. Psittacine and non-psittacine (pigeon) avian isolates of C. psittaci were distinguished using MaeI.     

Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants.

Chlamydia pecorum sp. nov. is proposed as the fourth species of the genus Chlamydia on the basis of the results of a genetic analysis of Chlamydia strains that were isolated from cattle and sheep which had various diseases, including sporadic encephalitis, infectious polyarthritis, pneumonia, and diarrhea. The levels of DNA-DNA homology between C. pecorum and strains of C. psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis were less than 10%. Several DNA probes were used to identify C. pecorum. The C. pecorum strains were distinguished from C. psittaci strains by the results of immunological assays, including an immunofluorescence antibody assay performed with monoclonal antibodies and an immunoblot analysis of the immunological specificity of the major outer membrane protein. Species identification was based on results obtained from DNA analyses and serology. The type strain of C. pecorum is strain ATCC VR628.     

Restriction pattern of the major outer-membrane protein gene provides evidence for a homogeneous invasive group among ruminant isolates of Chlamydia psittaci.

Thirty-six ruminant isolates of Chlamydia psittaci, previously classified as invasive or non-invasive in a mouse model of virulence, were compared by analysing AluI restriction patterns of the major outer-membrane protein (MOMP) gene after DNA amplification by the polymerase chain reaction. The 24 invasive isolates, although from various origins, all belonged to serotype 1 and represented a strictly homogeneous group sharing a specific MOMP-gene restriction pattern that was not observed in the non-invasive strains. On the other hand, the 12 non-invasive strains, although all belonging to serotype 2, constituted a heterogeneous group with eight distinct MOMP-gene restriction patterns. However, all eight patterns shared a 180 bp fragment or the corresponding restricted fragments of 110 and 70 bp. MOMP-gene restriction patterns also clearly distinguished the ruminant strains from an avian C. psittaci isolate, a C. pneumoniae isolate and two C. trachomatis isolates which were studied for comparison. The homogeneous character of the invasive C. psittaci strains argues strongly for their genetic relatedness. Our results illustrate the usefulness of the MOMP-gene restriction mapping in typing chlamydiae.     

Distribution of plasmid sequences in avian and mammalian strains of Chlamydia psittaci

Plasmid DNA from an avian strain of Chlamydia psittaci was purified and estimated to be 7.9 kb in size using restriction endonuclease analysis. A 5.9 kb fragment of this plasmid was cloned, mapped and used to screen a range of chlamydial strains. Hybridizing DNA was absent from ovine abortion and arthritis isolates and also from the Cal 10 strain but related sequences were detected in C. psittaci strains of feline pneumonitis, guinea-pig inclusion conjunctivitis, ovine conjunctivitis and C. trachomatis serovar L2. The plasmid DNA from the feline strain was shown to have a distinct restriction endonuclease profile. Similar plasmid sequences were detected in all avian isolates tested: thus the clone may have a useful diagnostic role for the detection of the pathogen in its natural host and in zoonotic episodes.     

Diversity in nucleotide acquisition by antigenically similar Chlamydia psittaci of avian origin

Abstract Two different nucleic acid precursor utilization patterns were obtained for five avian isolates of Chlamydia psittaci . Three of the isolates bahaved in a manner similar to that previously described, showing total dependency on the host cell for ribonucleoside triphosphates and being unable to utilize medium-supplied thymidine. In contrast, the other two isolates were incapable of taking pyrimidine ribonucleotides from the host cell and they could efficiently utilize medium-supplied thymidine. These unusual isolates were resistant to 5-fluorouridine while the other three isolates were sensitive. Of the five isolates only 6BC was sensitive to sulfonamides. The five isolates were divided into two groups by comparing the Alu I restriction endonuclease patterns obtained following digestion of the major outer membrane protein (OMP1) gene, amplified by the polymerase chain reaction. The OMP1 genotyping results were confirmed by serotyping.     

Identification of a major envelope protein in Chlamydia spp.

A major cell envelope protein of Chlamydia psittaci with a molecular weight of approximately 43,000 was identified and partially characterized. It was present at all stages of the C. psittaci developmental cycle. A major protein with a similar molecular weight was also observed in two Chlamydia trachomatis strains.     

Genetic diversity of avian and mammalian Chlamydia psittaci strains and relation to host origin.

Genetic relationships were reported for Chlamydia psittaci derived from psittacine birds, pigeons, turkeys, humans, cats, muskrats, cattle, and sheep and for C. trachomatis, including representative strains of the three biovars, through physical analysis of genomic DNA including DNA fingerprinting with restriction endonuclease SalI, DNA-DNA hybridization in solution with S1 nuclease, and Southern analysis with genomic DNA probes. A total of 26 strains were divided into four groups of C. psittaci and two groups of C. trachomatis, on the basis of DNA fingerprints. The six groups of Chlamydia spp. were related to host origin: two avian groups (Av1 and Av2), one feline and muskrat group (Fe1), one ruminant group (Ru1), one C. trachomatis biovars trachoma and lymphogranuloma group (CtHu), and one C. trachomatis mouse biovar group (CtMo), although an ovine abortion strain belonged to the avian group Av2. DNA-DNA hybridization assay and Southern analysis with genomic DNA probes indicated three DNA homology groups in the genus Chlamydia: an avian-feline group (groups Av1, Av2, and Fe1), a ruminant group (group Ru1), and a C. trachomatis group (groups CtHu and CtMo). Furthermore, the Southern analysis indicated that the homologous sequences (DNA homology of at least 14%) within the avian-feline group were distributed along the whole genome, whereas the homologous sequences (DNA homology of less than 24%) among the three DNA homology groups were localized in distinct regions of the genome DNA. These results suggest that Chlamydia spp. are derived from a common ancestor and have diverged into various groups showing restricted host ranges as a natural characteristic and that the species C. psittaci should be differentiated into groups related to host origin and DNA homology.     

Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.     

Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis.

The disulfide bond cross-linked major outer membrane protein (MOMP) of the extracellular elementary bodies (EBs) of Chlamydia psittaci was reduced to its monomeric form within 1 h of entry of EBs into host cells by a process which was inhibited by chloramphenicol, while monomeric forms of three cross-linked cysteine-rich proteins could not be detected in Sarkosyl outer membrane complexes at any time in either extracellular or intracellular forms of C. psittaci. Synthesis and incorporation of the MOMP into outer membrane complexes were detected early in the infection cycle (12 h postinfection), while synthesis and incorporation of the cysteine-rich proteins were not observed until reticulate bodies had begun to reorganize into EBs at 20 to 22 h postinfection. By 46 h postinfection, the intracellular population of C. psittaci consisted mainly of EBs, the outer membrane complexes of which were replete with monomeric MOMP and cross-linked cysteine-rich proteins. Upon lysis of infected cells at 46 h, the MOMP was rapidly cross-linked, and infectious EBs were released. The status of the MOMP of intracellular Chlamydia trachomatis was similar to the status of the MOMP of C. psittaci in that the MOMP was largely uncross-linked at 24 and 48 h postinfection, but formed interpeptide disulfide bonds when it was exposed to an extracellular environment late in the developmental cycle. In contrast to C. psittaci, only a fraction of the cross-linked MOMP of infecting EBs of C. trachomatis was reduced by 4 h postinfection, and reduction of the MOMP was not inhibited by chloramphenicol. Exposure of extracellular EBs of C. trachomatis and C. psittaci to dithiothreitol reduced the MOMP but failed to stimulate metabolic activities normally associated with reticulate bodies.     

Sequence analysis of the major outer membrane protein gene of an ovine abortion strain of Chlamydia psittaci

The major outer membrane protein (MOMP) gene from an ovine abortion strain of Chlamydia psittaci (S26/3) has been cloned and sequenced. The gene shows the features of other chlamydial MOMPs but comparison with the previously reported sequence for the ovine abortion isolate A22/M has revealed substantial sequence divergence which is clustered into the same four intramolecular regions as the sequence variation found between C. trachomatis serovars. Subsequent restriction enzyme analysis of A22/M DNA has shown that it has an avian-type genomic profile and thus the comparison is between types rather than between strains.     

鹦鹉热衣原体主要外膜蛋白基因序列的扩增、克隆和原核表达

主要外膜蛋白在鹦鹉热衣原体感染过程中起主要作用。扩增了主要外膜蛋白基因,克隆入pGEM-T和pET32a( ),经PCR筛选和酶切鉴定,进行诱导表达和重组蛋白的纯化与复性研究,为进一步进行鹦鹉热衣原体的诊断试剂和疫苗研究创造了条件。