Cloning and sequence analysis of the major outer membrane protein genes of two Chlamydia psittaci strains.

We cloned and sequenced the gene encoding the major outer membrane protein (MOMP) of two Chlamydia psittaci strains, guinea pig inclusion conjunctivitis (GPIC) strain 1, and meningopneumonitis (Mn) strain Cal-10. Intraspecies alignment of the two C. psittaci MOMP genes revealed 80.6% similarity, and interspecies comparison of C. trachomatis and C. psittaci MOMP genes yielded about 68% similarity. As found previously for C. trachomatis MOMP sequences, stretches of predominantly conserved sequences of GPIC and Mn MOMPs were interrupted by four variable domains whose locations were identical to those of C. trachomatis MOMPs. Seven of eight cysteine residues were found at precisely the same positions in GPIC, Mn, and C. trachomatis MOMPs, emphasizing their importance in structure and function of the protein. Collectively, these results indicate that C. psittaci and C. trachomatis MOMP genes diverged from a common ancestor.     

Comparison of Chlamydia psittaci isolates by DNA restriction endonuclease analysis.

Preparations of DNA from 12 Chlamydia psittaci isolates and one Chlamydia trachomatis strain were compared by restriction endonuclease analysis. Polyacrylamide gel electrophoresis, followed by silver staining, resulted in optimal resolution of fragments generated by digestion. By this technique, four distinct electropherotypes were demonstrated when ovine abortion, ovine arthritis, and avian and Cal10 strains of C. psittaci were examined. Minor profile differences allowed the discrimination of avian isolates derived from psittacine and columbiforme species, and the Cal10 DNA electropherotype was shown to have features in common with these profiles. However, there were no detectable differences in the DNA patterns of eight ovine abortion isolates.     

Immunochemical diversity of the major outer membrane protein of avian and mammalian Chlamydia psittaci

Immunochemical properties of the major outer membrane protein (MOMP) of 16 strains of Chlamydia psittaci isolated from psittacine birds, budgerigars, a pigeon, turkeys, humans, cats, a muskrat, sheep, and cattle and a strain of C. trachomatis, L2/434/Bu, were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblotting analysis with hyperimmunized rabbit antisera to strains of parrot, turkey, feline, and bovine origin. The MOMPs of the strains showed variation in molecular weights and immunological specificities. Fifteen of the C. psittaci strains were classified into two avian and two mammalian types based on immunological specificity of the MOMP, whereas the other strain was not classified in this study. Immunological classification based on specificity of the MOMP by immunoblotting proved to be a valuable method to classify various strains of C. psittaci.     

Lack of allelic polymorphism for the major outer membrane protein gene of the agent of guinea pig inclusion conjunctivitis (Chlamydia psittaci).

The major outer membrane protein gene (omp1) was sequenced for each of six Chlamydia psittaci (guinea pig inclusion conjunctivitis [GPIC]) strains isolated from guinea pigs. Five of the isolates were obtained in the United States during the 1960s and 1970s, including the prototype strain isolated by Murray in 1962. The other isolate was obtained from a guinea pig in England. The nucleotide sequence of the omp1 gene for each strain was identical. The lack of omp1 allelic polymorphism among GPIC isolates suggests that, unlike C. trachomatis, the GPIC agent lacks antigenic variation in the major outer membrane protein.     

High-Level Expression of Chlamydia psittaci Major Outer Membrane Protein in COS Cells and in Skeletal Muscles of Turkeys

The omp1 genes encoding the major outer membrane proteins (MOMPs) of avian Chlamydia psittaci serovar A and D strains were cloned and sequenced. The nucleotide sequences of the avian C. psittaci serovar A and D MOMP genes were found to be 98.9 and 87.8% identical, respectively, to that of the avian C. psittaci serovar A strain 6BC, 84.6 and 99.8% identical to that of the avian C. psittaci serovar D strain NJ1, 79.1 and 81.1% identical to that of the C. psittaci guinea pig inclusion conjunctivitis strain, 60.9 and 62.5% identical to that of the Chlamydia trachomatis L2 strain, and 57.5 and 60.4% identical to that of the Chlamydia pneumoniae IOL-207 strain. The serovar A or D MOMPs were cloned in the mammalian expression plasmid pcDNA1. When pcDNA1/MOMP A or pcDNA1/MOMP D was introduced into COS7 cells, a 40-kDa protein that was identical in size, antigenicity, and electrophoretic mobility to native MOMP was produced. Recombinant MOMP (rMOMP) was located in the cytoplasm of transfected COS7 cells as well as in the plasma membrane and was immunoaccessible. Intramuscular administration of pcDNA1/MOMP in specific-pathogen-free turkeys resulted in local expression of rMOMP in its native conformation, after which anti-MOMP antibodies appeared in the serum.     

Immunological specificity of monoclonal antibodies to Chlamydia psittaci ovine abortion strain

Fifty-one monoclonal antibodies were prepared by two different techniques against Chlamydia psittaci strain A22 isolated from an ovine abortion. These antibodies were tested for reactivity by the indirect immunofluorescent antibody technique with eleven reference Chlamydia strains (nine C. psittaci, one Chlamydia trachomatis and one Chlamydia pneumoniae). Four classes of specificity were recognized for monoclonal antibodies: genus, species, subspecies and type specificity. The type-specific monoclonal antibodies were non-reactive with ovine arthritis isolates. Twenty monoclonal antibodies were specific for two mammalian strains: ovine abortion A22 and K mouse. Some monoclonal antibodies were reactive with C. pneumoniae strains and non-reactive with C. trachomatis strains. All these monoclonal antibodies were very useful for improving the diagnosis of chlamydial infection, the antigenic analysis and the serotyping of C. psittaci.     

Comparison of Chlamydia psittaci isolates by restriction endonuclease and DNA probe analyses.

DNAs from eight Chlamydia psittaci isolates (koala conjunctivitis, avian psittacosis, avian ornithosis, ovine abortion, ovine polyarthritis, sporadic bovine encephalomyelitis, and feline conjunctivitis) and one Chlamydia trachomatis isolate (lymphogranuloma venereum) were compared by restriction endonuclease and DNA probe analyses. Digestion with HindIII yielded a series of discrete fragments which allowed the differentiation of most isolates. A gene probe, pFEN207, which encodes the chlamydia-specific component of the lipopolysaccharide group antigen was used in Southern hybridizations. The probe was chlamydia specific and hybridized to a single BamHI fragment and multiple HindIII fragments in each isolate. The variation in size of the hybridizing fragments allowed easy differentiation of the isolates and may eventually lead to a meaningful subgrouping of the diverse group of disease agents presently included in the species C. psittaci.     

Phylogenetic analyses of Chlamydia psittaci strains from birds based on 16S rRNA gene sequence.

The nucleotide sequences of 16S ribosomal DNA (rDNA) were determined for 39 strains of Chlamydia psittaci (34 from birds and 5 from mammals) and for 4 Chlamydia pecorum strains. The sequences were compared phylogenetically with the gene sequences of nine Chlamydia strains (covering four species of the genus) retrieved from nucleotide databases. In the neighbor-joining tree, C. psittaci strains were more closely related to each other than to the other Chlamydia species, although a feline pneumonitis strain was distinct (983 to 98.6% similarity to other strains) and appeared to form the deepest subline within the species of C. psittaci (bootstrap value, 99%). The other strains of C. psittaci exhibiting similarity values of more than 99% were branched into several subgroups. Two pigeon strains and one turkey strain formed a distinct clade recovered in 97% of the bootstrapped trees. The other pigeon strains seemed to be distinct from the strains from psittacine birds, with 88% of bootstrap value. In the cluster of psittacine strains, three parakeet strains and an ovine abortion strain exhibited a specific association (level of sequence similarity, 99.9% or more; bootstrap value, 95%). These suggest that at least four groups of strains exist within the species C. psittaci. The 16S rDNA sequence is a valuable phylogenetic marker for the taxonomy of chlamydiae, and its analysis is a reliable tool for identification of the organisms.     

Similarity of Chlamydia pneumoniae strains in the variable domain IV region of the major outer membrane protein gene.

DNA was amplified by polymerase chain reaction from the gene encoding the major outer membrane protein (MOMP) of Chlamydia pneumoniae in order to examine the relatedness of strains isolated from diverse geographical regions. Primers for this reaction were chosen to span a 207-bp region comparable to that of the fourth variable segment of the MOMP gene of Chlamydia trachomatis. Among C. trachomatis, sequence heterogeneity is characteristic within variable sequence domain IV (VDIV) and correlates with serovar type. In contrast, sequence analysis of polymerase chain reaction products from 13 C. pneumoniae isolates indicated that all tested strains were identical in this segment of the MOMP gene. The predicted amino acid sequences from the C. pneumoniae VDIV gene products shared only 13.3 to 30% homology with published VDIV regions from serovars of C. trachomatis. Homology of these VDIV amino acid sequences with sequences from strains of C. psittaci ranged from 45.7 to 60%. The sequence conservation of the VDIV region of the MOMP gene indicates that C. pneumoniae strains may be more genetically homogeneous than C. trachomatis or Chlamydia psittaci strains. Future investigations of antigenic diversity among C. pneumoniae strains should be aimed at the evaluation of variation in other regions of the C. pneumoniae genome.     

Sequence analysis of the major outer membrane protein gene of Chlamydia pneumoniae.

Compared with the major outer membrane proteins (MOMPs) of the other chlamydial species, the Chlamydia pneumoniae MOMP appears to be less antigenically complex, and as determined by immunoblot analysis, it does not appear to be the immunodominant antigen recognized during infection. Nucleotide sequence analysis of the C. pneumoniae MOMP gene (ompA) revealed that it consisted of a 1,167-base open reading frame with an inferred 39,344-dalton mature protein of 366 amino acids plus a 23-amino-acid leader sequence. A ribosomal-binding site was located in the 5' upstream region, and two stop codons followed by an 11-base dyad forming a stable stem-loop structure were identified. This sequence shares 68 and 71% DNA sequence homology to the Chlamydia trachomatis serovar L2 and Chlamydia psittaci ovine abortion agent MOMP genes, respectively. Interspecies alignment identified regions, corresponding to the variable domains, which share little sequence similarity with the other chlamydial MOMPs. All seven cysteines conserved in the C. trachomatis and C. psittaci MOMPs, which are involved in the formation of disulfide cross-linkages, are found in the C. pneumoniae MOMP.     

Dissociation of immune determinants of outer membrane proteins of Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

Chlamydia trachomatis is an important human pathogen. Research to develop a Chlamydia vaccine has focused on the major outer membrane protein (MOMP). Determinants of this protein elicit serovar-specific neutralizing antibodies which are thought to play a critical role in protective immunity. MOMP-specific antibody responses are highly variable in the polymorphic population. Genetic factors which might influence the MOMP-specific immune response are consequently of particular interest. The C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) is a natural pathogen of the guinea pig that causes both ocular and genital tract infections that closely resemble those caused by C. trachomatis in humans. As such, it provides an excellent model for disease. In this report, we explore the influence of major histocompatibility complex-linked genes on the MOMP-specific antibody response in mice immunized with either whole GPIC elementary bodies or recombinant GPIC MOMP. Our results indicate that the MOMP-specific antibody response is major histocompatibility complex linked such that mice of the H-2d haplotype are high responders while mice of the H-2k haplotype are low responders. We demonstrate that MOMP-specific B cells are present in H-2k strains which are, however, deficient in MOMP-specific helper T cells. Although immunization of low-MOMP-responder strains with whole chlamydial elementary bodies induces high levels of immunoglobulin G antibody specific for Omp2, the cysteine-rich outer membrane protein, MOMP-specific B cells are unable to receive help from Omp2-specific T cells. The failure of intermolecular help from Omp2-specific T cells and related observations raise important issues regarding the processing and presentation of chlamydial antigens and the design of optimal subunit vaccines.     

Antibody response to epitopes of chlamydial major outer membrane proteins on infectious elementary bodies and of the reduced polyacrylamide gel electrophoresis-separated form.

Approximately 60% of the outer membrane of chlamydial elementary bodies (EBs) consists of the major outer membrane protein (MOMP) that has structural and metabolic functions. The antigenic properties of MOMPs from mammalian strains of serovars 1 and 2 and an avian strain of Chlamydia psittaci were analyzed. Polyclonal-monospecific antisera (PMAs), one monoclonal antibody (MAb), and polyclonal antisera (PAs) were produced against reduced polyacrylamide gel electrophoresis-separated MOMPs and against infectious EBs. Three PMAs and the MAb, which were induced by reduced polyacrylamide gel electrophoresis-separated MOMPs, reacted strongly in Western blot (immunoblot) assays with MOMPs of serovar 1 and 2 strains as well as with that of the avian strain 6BC, and two of these PMAs reacted weakly (dilution, 1:20) with the MOMP of strain LGV-2. The third PMA and the MAb against the MOMP of the serovar 2 strain did not react with the MOMP of LGV-2. Four PAs were produced against infectious EBs of the serovar 1 strain. One of these PAs reacted with the homologous MOMP and that of the avian strain 6BC but did not recognize MOMPs of other chlamydial strains. Three of the PAs reacted with MOMPs of homologous strains only and failed to recognize MOMPs of avian, serovar 2, and LGV-2 strains. Five PAs induced against infectious EBs of the serovar strain 2 reacted only with the MOMPs of the homologous strains and failed to recognize MOMPs of other strains of chlamydiae. Consequently, MOMPs of C. psittaci strains possess genus-, species-, and serovar-specific epitopes whereby the immune response to serovar-specific epitopes of MOMP predominate when infectious EBs are used for immunization.     

Comparison of avian Chlamydia psittaci isolates by restriction endonuclease analysis and serovar-specific monoclonal antibodies.

Avian Chlamydia psittaci isolates were examined by restriction endonuclease analysis and serovar-specific monoclonal antibodies and compared with ovine abortion and polyarthritis isolates. The avian isolates were divided into four serovars (turkey, psittacine, pigeon, and duck) based on their reactivity to the monoclonal antibodies. The DNA digest patterns were similar across the four avian serovars; most bands were identical when the isolates were tested with PstI, BamHI, and EcoRI restriction endonuclease enzymes. The turkey group restriction endonuclease analysis patterns were distinguished from those of the other avian strains by three to four band differences with all enzymes. The duck and pigeon isolates showed only minor DNA pattern differences when compared with the psittacine isolates. Four psittacine isolates from various locations in Texas had an extra band with the EcoRI restriction enzyme, suggesting that they were from a common source; however, they were indistinguishable from the other psittacine isolates when examined with the monoclonal antibodies. The avian isolates were distinctly different from either abortion or polyarthritis isolates by both restriction endonuclease analysis and monoclonal antibody analysis. The data demonstrate that the avian isolates form a distinct group or separate biovar with at least four serovars.     

Identification of genus-specific epitopes on the outer membrane complexes of Chlamydia trachomatis and Chlamydia psittaci immunotypes 1 and 2.

Polyclonal and monoclonal antibodies were used to study the immunogenic and antigenic characteristics of chlamydiae. We focused on the most predominant proteins in the outer membrane complex, the major outer membrane protein (MOMP) and the doublet consisting of proteins of 57 and 62 kilodaltons (57-62 kDa doublet). Immunoblot analyses were performed with chlamydial elementary bodies by using (i) immune sera from sheep which had undergone a recent episode of abortion due to the ovine abortion (OA) strain of C. psittaci, (ii) rabbit hyperimmune anti-C. psittaci (OA) and -C. trachomatis sera, and (iii) monoclonal antibodies to the MOMP of C. trachomatis. The typical pattern of response with polyclonal antisera against heterologous elementary bodies was reactivity with the 57-62 kDa doublet and lipopolysaccharide with weak and sometimes no anti-MOMP activity. Three distinct genus-specific anti-C. trachomatis MOMP monoclonal antibodies showed different patterns of reactivity with the MOMPs of the two immunotypes of C. psittaci and C. trachomatis serovars. Our data confirm the predominance of a genus-specific 57-62 kDa doublet response despite the presence of genus-specific epitopes on the MOMP.     

Detection and strain differentiation of Chlamydia psittaci mediated by a two-step polymerase chain reaction.

Specific and sensitive amplification of major outer membrane protein (MOMP) gene DNA sequences of Chlamydia psittaci was achieved in a two-step polymerase chain reaction. First, oligonucleotide primers specific for 5' and 3' nontranslated regulatory regions of the MOMP gene were used in a polymerase chain reaction to amplify a DNA fragment of approximately 1,400 bp. A portion of this DNA fragment was amplified in a second reaction using a degenerate oligonucleotide primer specific for a DNA sequence contained within the 1,400-bp DNA fragment and one of the first-step primers. This method detected 10 cognate chlamydial genomes. C. psittaci MOMP genes from two avian strains and from mammalian serovars 1, 7, and 8 were amplified and analyzed by restriction endonuclease digestion. MOMP genes from mammalian serovars 2 through 6 and 9 and from strains of C. trachomatis and C. pneumoniae could not be amplified. Restriction endonuclease analysis with HaeIII indicated a close relationship between C. psittaci strains of avian and mammalian serovar 1 lineage, while those of mammalian serovars 7 and 8 exhibited distinct restriction patterns. DNA sequences corresponding to the mammalian serovar 1-wild type parakeet MOMP genotype of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.     

Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmunofluorescence.

A group of twenty-five isolates of Chlamydia psittaci representing at least seven different biotypes of bovine, ovine, caprine, equine, feline, porcine, and guinea pig origin were immunotyped by an indirect microimmunofluorescence test. Different groups of chlamydia-free BALB/c mice received two weekly intravenous inoculations with chicken embryo-propagated, partially purified elementary bodies of each strain. Antisera for immunotyping were collected 4 days after the first inoculation and 3 to 4 days after the second inoculation and tested for antichlamydial immunoglobulin M and immunoglobulin G antibodies by the indirect microimmunofluorescence test with cell culture-propagated, partially purified homologous and heterologous antigens. Nine immunotypes of C. psittaci were distinguished. The correlation between immunotypes and biotypes was close, and a pattern of either disease or host specificity could be associated with each immunotype. Most immunotypes identified induced cross-reacting antibodies against each other, but no significant cross-reactions were observed with elementary bodies of the mouse pneumonitis strain of C. trachomatis. Findings from this study should provide the necessary background for the rational selection of prototype strains of C. psittaci for further antigenic analysis at the molecular level.     

Cell-mediated and humoral immune responses to chlamydial antigens in guinea pigs infected ocularly with the agent of guinea pig inclusion conjunctivitis.

Cell-mediated immune response and humoral response to chlamydial antigens were investigated in guinea pigs infected with the agent of guinea pig inclusion conjunctivitis (GPIC). Pronounced cell-mediated immune response to the homologous antigen, as well as to two other chlamydial antigens, 6BC (Chlamydia psittaci) and LB-1 (C. trachomatis), occurred in all infected animals. Cell-mediated immune response to GPIC, and to a lesser extent to 6BC and LB-1 as well, was enhanced with time after infection even without the re-inoculation of the infectious agent. Extensive cross-reactions among the three chlamydial antigens during the cell-mediated immune response appeared to be due to shared species-specific and group-reactive antigens. Serum antibody response was pronounced and uniform to GPIC; it was less marked to 6BC and LB-1, with fewer cross-reactions than seen in tests for cell-mediated immunity.     

Characterization of the new Chlamydia agent, TWAR, as a unique organism by restriction endonuclease analysis and DNA-DNA hybridization.

Several molecular techniques were used for comparison of the novel Chlamydia agent, TWAR, with Chlamydia trachomatis and Chlamydia psittaci. Unlike all serotypes of C. trachomatis and most strains of C. psittaci, the eight TWAR isolates examined did not contain extrachromosomal DNA. TWAR was readily distinguished from C. trachomatis or C. psittaci by restriction endonuclease analysis, whereas identical or nearly identical restriction patterns were observed among the TWAR isolates. Southern blot analysis with a gene encoding a portion of the C. trachomatis serovar L2 major outer membrane protein as the probe showed that TWAR, like C. psittaci, contained sequences homologous to this gene. However, while the hybridization patterns were identical for all TWAR isolates, they differed from those of any of the other Chlamydia species tested. A PstI gene bank containing TWAR DNA was constructed in pUC19. Random fragments were purified and used for probing Chlamydia chromosomal digests. All of the five probes tested were TWAR specific, with the TWAR isolates showing identical patterns of homology. Qualitative studies of the DNA homology revealed that TWAR did not have significant homology to any of the Chlamydia strains assayed. Collectively, these results demonstrate that the TWAR isolates represent a single strain or closely allied genotypes and are clearly distinct from any of the other chlamydiae tested.     

Role of Bcl-2 Family Members in Caspase-Independent Apoptosis during Chlamydia Infection

Infection with an obligate intracellular bacterium, the Chlamydia trachomatis lymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar of Chlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains of Chlamydia is independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria in C. psittaci-infected cells. C. psittaci-induced apoptosis is inhibited in host cells overexpressing Bax inhibitor-1 and is inhibited through overexpression of Bcl-2, which blocks both caspase-dependent and -independent apoptosis. As Bax and mitochondria are ideally located to sense stress-related metabolic changes emanating from the interior of an infected cell, it is likely that Bax-dependent apoptosis may also be observed in cells infected with other intracellular pathogens.