DNA fingerprinting of Chlamydia trachomatis by use of ribosomal RNA, oligonucleotide and randomly cloned DNA probes.

DNA fingerprinting of 15 reference strains and 24 clinical isolates of Chlamydia trachomatis, 2 strains of C. psittaci and one strain of C. pneumoniae was studied by use of universal 16 + 23S RNA from Escherichia coli, 16S rDNA-directed oligonucleotide and randomly cloned chlamydial DNA probes. The rRNA-gene restriction patterns (ribotypes) enabled the differentiation of chlamydial species. Following DNA cleavage by restriction endonuclease PvuII, lymphogranuloma venereum and trachoma biovars of C. trachomatis could be differentiated. An oligonucleotide, designed to hybridize the C. trachomatis 16S rDNA, also allowed for both species-specific identification and biovar typing of C. trachomatis human strains. Molecular typing system using 3 lambda clones containing C. trachomatis serotype E random DNA inserts, combined to ribotyping, revealed 12 groups of variable banding patterns within C. trachomatis, and could provide an alternative epidemiological tool.     

Restriction endonuclease analysis of DNA from Chlamydia trachomatis biovars.

DNA from a total of 60 Chlamydia trachomatis isolates was examined by restriction endonuclease analysis. Strains from all established biovars and serovars were tested. There was great diversity between the mouse biovar and the lymphogranuloma venereum (LGV) and trachoma biovars. The LGV and trachoma biovar isolates generated similar fragment patterns; however, distinct fragments appeared to be unique to both biovars, thus allowing differentiation of these two major groups. In most cases, strains of the same serovar could be differentiated from one another when a battery of restriction enzymes was used. In addition, in some cases, certain restriction fragments appeared to be characteristic of strains from a particular geographical location. The DNA patterns generated by all C. trachomatis isolates differed greatly from the DNA patterns generated from the Chlamydia psittaci isolates tested, including TWAR, a human C. psittaci strain.     

Differences in outer membrane proteins of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis.

The lymphogranuloma venereum (LGV) and trachoma biovars of Chlamydia trachomatis exhibit differences in biological properties both in vivo and in vitro. To identify analogous biochemical differences, we studied the molecular charges of chlamydial outer membrane proteins (OMPs) by means of isoelectric focusing and nonequilibrium pH gradient electrophoresis. Analysis of proteins of whole elementary bodies biosynthetically labeled with L-[35S]cysteine revealed that most chlamydial proteins were neutral or acidic. The major OMPs (MOMPs) of all strains tested were acidic and had apparent isoelectric points (pIs) that varied within narrow limits (approximately 5.3 to 5.5) despite differences in molecular mass of up to 3,000 daltons (Da). However, a low-molecular-mass cysteine-rich OMP analogous to that previously described for Chlamydia psittaci varied consistently in molecular mass (12,500 versus 12,000 Da) and pI (5.4 versus 6.9) between LGV strains and trachoma strains, respectively. OMPs with a molecular mass of 60,000 Da in the trachoma biovar strains had pIs in the 7.3 to 7.7 range. However, analogous OMPs in the LGV strains existed as a doublet with a molecular mass of about 60,000 Da. Both members of the doublet were basic (pIs greater than 8.5). Both proteins of this basic doublet in LGV strains and the neutral analog in trachoma strains bound a species-specific monoclonal antibody in an immunoblot assay. These data indicate substantial differences in biochemical characteristics of analogous OMPs in the LGV and trachoma biovars. Such differences are the first structural differences described between LGV and trachoma strains which support their distinction into separate biovars and may be related to some of their biological differences.     

Systemic Chlamydia trachomatis infection in mice: a comparison of lymphogranuloma venereum and trachoma biovars.

We developed a murine model of systemic infection with Chlamydia trachomatis biovar lymphogranuloma venereum (LGV). The pathological features of this infection resemble those of human LGV infection since both are characterized by granuloma formation. Mice developed resistance to reinfection with LGV, and this resistance was based on cellular immune mechanisms since it was transferable with immune spleen cells but not with immune serum. Resistance required viable organisms for induction. We compared LGV biovar infection with trachoma biovar infection. Trachoma biovar produced similar but less marked microbiological and pathological features. Cross-immunity was less apparent between serovars from trachoma and LGV biovars than it was between serovars within the same biovar. This model of systemic C. trachomatis infection will be useful in exploring virulence features of LGV.     

Chlamydial infection induces pathobiotype-specific protein tyrosine phosphorylation in epithelial cells

Members of the genus Chlamydia are strict obligate intracellular pathogens that exhibit marked differences in host range and tissue tropism despite sharing a remarkable level of genomic synteny. These pathobiotype differences among chlamydiae are also mirrored in their early interactions with cultured mammalian host cells. Chlamydial attachment and entry is known to trigger protein tyrosine phosphorylation. In this study, we examined the kinetics and pattern of protein tyrosine phosphorylation induced by infection with a comprehensive collection of chlamydial strains exhibiting diversity in host, tissue, and disease tropisms. We report new findings showing that protein tyrosine phosphorylation patterns induced by infection directly correlate with the pathobiotype of the infecting organism. Patterns of protein tyrosine phosphorylation were induced following early infection that unambiguously categorized chlamydial pathobiotypes into four distinct groups: (i) Chlamydia trachomatis trachoma biovars (serovars A to H), (ii) C. trachomatis lymphogranuloma venereum biovars (serovars L1 to L3), (iii) C. muridarum, and (iv) C. pneumoniae and C. caviae. Notably, chlamydia-infected murine and human epithelial cells exhibited the same protein tyrosine phosphorylation patterns; this is indirect evidence suggesting that the phosphorylated protein(s) is of chlamydial origin. If our hypothesis is correct, these heretofore-uncharacterized proteins may represent a novel class of bacterial molecules that influence pathogen-host range or tissue tropism.     

Degradation of Chlamydia trachomatis in human polymorphonuclear leukocytes: an ultrastructural study of peroxidase-positive phagolysosomes.

We have previously shown that human polymorphonuclear leukocytes (PMNs) killed organisms belonging to both human biovars of Chlamydia trachomatis. However, the mechanism of destruction was still unclear. We therefore conducted an ultrastructural and cytochemical study to investigate the mechanism of chlamydial degradation. PMNs were inoculated with the trachoma serovar B (B/TW-5/OT) or with the lymphogranuloma venereum serovar L2 (L2/434/Bu) for 15, 30, 60, or 120 min and then fixed and processed for transmission electron microscopy. Diaminobenzidine, a cytochemical marker, was used to demonstrate the localization of intracellular peroxidase. Ultrastructural evidence is presented showing the progressive degradation of chlamydiae over a 2-h period within peroxidase-positive phagolysosomes. Pretreatment of organisms with normal or immune serum was not required for the process of degradation.     

Laboratory diagnosis of human chlamydial infections.

Chlamydia trachomatis is a human pathogen that causes ocular disease (trachoma and inclusion conjunctivitis), genital disease (cervicitis, urethritis, salpingitis, and lymphogranuloma venereum), and respiratory disease (infant pneumonitis). Respiratory chlamydioses also occur with infection by avian strains of C. psittaci or infection by the newly described TWAR agent. Diagnosis of most acute C. trachomatis infections relies on detection of the infecting agent by cell culture, fluorescent antibody, immunoassay, cytopathologic, or nucleic acid hybridization methods. Individual non-culture tests for C. trachomatis are less sensitive and specific than the best chlamydial cell culture system but offer the advantages of reduced technology and simple transport of clinical specimens. Currently available nonculture tests for C. trachomatis perform adequately as screening tests in populations in which the prevalence of infection is greater than 10%. A negative culture or nonculture test for C. trachomatis does not, however, exclude infection. The predictive value of a positive nonculture test may be unsatisfactory when populations of low infection prevalence are tested. Tests that detect antibody responses to chlamydial infection have limited utility in diagnosis of acute chlamydial infection because of the high prevalence of persistent antibody in healthy adults and the cross-reactivity due to infection by the highly prevalent C. trachomatis and TWAR agents. Assays for changes in antibody titer to the chlamydial genus antigen are used for the diagnosis of respiratory chlamydioses. A single serum sample that is negative for chlamydial antibody excludes the diagnosis of lymphogranuloma venereum.     

Genetic diversity and identification of human infection by amplification of the chlamydial 60-kilodalton cysteine-rich outer membrane protein gene.

The 60-kDa cysteine-rich outer membrane protein genes of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis have very different 5' ends, but two areas flanking this variable region show absolute sequence conservation. This observation permitted differentiation of the three species of Chlamydia by the polymerase chain reaction (PCR), forming the basis of a diagnostic test for chlamydial infections. The PCR product containing the variable region of the respective 60-kDa CrP genes was also subjected to restriction endonuclease digestion, enabling differentiation of individual type strains of C. psittaci. Differentiation was possible between lymphogranuloma venereum and trachoma isolates of C. trachomatis. The PCR-based diagnostic test was successful with all strains of chlamydiae studied. The PCR primers showed high specificity and did not produce any product with common bacterial pathogens that may share the same sites of infection.     

Hemorrhagic proctitis due to lymphogranuloma venereum serogroup L2. Diagnosis by fluorescent monoclonal antibody

Definitive diagnosis of lymphogranuloma venereum is impeded by difficulty in culturing the causative agent and by serologic cross-reactivity between Chlamydia trachomatis L1, L2, and L3, which can cause the disease, and the many other serotypes of C. trachomatis, which do not. In a 23-year-old man with massive rectal bleeding, an exudative rectal ulcer, and inguinal lymphadenopathy, serologic findings were compatible with a recent lymphogranuloma venereum infection, but stains and cultures of lymph-node aspirates were negative, and biopsy specimens of the rectum and lymph nodes showed only nonspecific inflammatory changes. A diagnosis of lymphogranuloma venereum was made when intracellular organisms and inclusion bodies were demonstrated in rectal submucosal tissue by fluorescein-tagged monoclonal antibodies directed against both chlamydial group antigens and L2 serotype antigen. This technique was of particular value in this patient because it specifically identified an unusual cause of severe gastrointestinal bleeding.     

Physicochemical surface properties of elementary bodies from different serotypes of chlamydia trachomatis and their interaction with mouse fibroblasts.

Aqueous biphasic partitioning, hydrophobic interaction chromatography, and ion-exchange chromatography were used to characterize the surface properties of Renografin-purified elementary bodies of Chlamydia trachomatis serotypes E and L1. The two serotypes differed with respect to liability to hydrophobic interaction and negative surface charge. Furthermore, the mutual relative magnitude of these parameters differed between the two serotypes, depending on the chromatographic technique used. This indicates that these chromatographic techniques register different aspects of charge and hydrophobicity on the chlamydial surface. DEAE-dextran and dextran sulfate affected association of, penetration, and intracellular development of C. trachomatis in mouse fibroblasts (McCoy cells). DEAE-dextran affected the association of C. trachomatis serotype E with McCoy cells mainly by charge-dependent forces, whereas both DEAE-dextran and dextran sulfate influenced the association of C. trachomatis serotype L1 mainly by charge-independent forces. These results indicate that the numerous biological differences between lymphogranuloma venereum and non-lymphogranuloma venereum strains of C. trachomatis may be assigned to differences in surface properties between the two strains.     

Comparative genomic analysis of Chlamydia trachomatis oculotropic and genitotropic strains

Chlamydia trachomatis infection is an important cause of preventable blindness and sexually transmitted disease (STD) in humans. C. trachomatis exists as multiple serovariants that exhibit distinct organotropism for the eye or urogenital tract. We previously reported tissue-tropic correlations with the presence or absence of a functional tryptophan synthase and a putative GTPase-inactivating domain of the chlamydial toxin gene. This suggested that these genes may be the primary factors responsible for chlamydial disease organotropism. To test this hypothesis, the genome of an oculotropic trachoma isolate (A/HAR-13) was sequenced and compared to the genome of a genitotropic (D/UW-3) isolate. Remarkably, the genomes share 99.6% identity, supporting the conclusion that a functional tryptophan synthase enzyme and toxin might be the principal virulence factors underlying disease organotropism. Tarp (translocated actin-recruiting phosphoprotein) was identified to have variable numbers of repeat units within the N and C portions of the protein. A correlation exists between lymphogranuloma venereum serovars and the number of N-terminal repeats. Single-nucleotide polymorphism (SNP) analysis between the two genomes highlighted the minimal genetic variation. A disproportionate number of SNPs were observed within some members of the polymorphic membrane protein (pmp) autotransporter gene family that corresponded to predicted T-cell epitopes that bind HLA class I and II alleles. These results implicate Pmps as novel immune targets, which could advance future chlamydial vaccine strategies. Lastly, a novel target for PCR diagnostics was discovered that can discriminate between ocular and genital strains. This discovery will enhance epidemiological investigations in nations where both trachoma and chlamydial STD are endemic.     

Amino acid transport into cultured McCoy cells infected with Chlamydia trachomatis

Amino acid transport into McCoy cells infected with strains representative of the two major biovars of Chlamydia trachomatis has been studied to determine if uptake is increased during infection. Preliminary work suggested that the transport systems L, A/ASC (for neutral amino acid transport), N (for transport of Asn, Gln, and His) and y+ (for cationic amino acids) were present in McCoy cells. With lymphogranuloma venereum biovar strain 434, little difference in the influx of representative amino acids Trp, His, and Lys or the analogue 2-aminoisobutyric acid (AIB) was observed during infection. With trachoma biovar strain DK20, a small increase in the initial entry rate and equilibrium concentration of each amino acid was found. McCoy cells appear to have great capacity for concentrating amino acids, which might obviate the need for transport induction by chlamydiae under conditions favoring the growth of infectious organisms.     

Chlamydia trachomatisglycosaminoglycan-dependent and independent attachment to eukaryotic cells

Chlamydia trachomatisconsists of two biovars, lymphogranuloma venereum (LGV) and trachoma, that differ in their infectivityin vivoandin vitro. Although addition of exogenous heparin or heparan sulfatein vitroeffectively inhibits infectivity of both biovars and inhibits LGV biovar attachment to host cells, trachoma biovar attachment was only modestly inhibited (30%) by exogenous heparin. To dissect the relationship of heparin inhibition of attachment and infectivity, a heparan sulfate lyase (heparitinase) was used to treat organisms and evaluated for changes in attachment and infectivity. In contrast to heparitinase-treated LGV biovar organisms that lose their ability to attach and infect, treatment of trachoma biovar organisms with a concentration of heparitinase sufficient to reduce trachoma biovar infectivity by >90%, only inhibited attachment to host cells by 40%. Significantly, attachment could be fully restored for heparitinase-treated organisms of both biovars with exogenous heparan sulfate; however, the coating of the trachoma biovar organisms with heparan sulfate rendered the trachoma biovar similar to the phenotype of the LGV biovar by >90% sensitivity to heparin inhibition of attachment. These data suggest that the LGV biovar used predominantly a heparin-inhibitable mechanism for attaching to host cells, whereas the trachoma biovar used a heparin-independent means in addition to a heparin-dependent mechanism to adhere to host cells. Once attached, the trachoma biovar, nevertheless, relied on the heparin-dependent pathway to enter host cells.     

Modification of the microimmunofluorescence test to provide a routine serodiagnostic test for chlamydial infection.

A modification of the microimmunofluorescence test to provide a practicable routine serodiagnostic test for detecting and characterising chlamydial infection is described which uses four antigen pools, one of which corresponds with each of the four main clinical and epidemiological types of chlamydial infection. The three subgroup A Chlamydia (Chlamydia trachomatis) pools are: pool 1, hyperendemic trachoma TRIC agent serotypes A, B, and C; pool 2, paratrachoma TRIC agent serotypes D, E, F, G, H, I, and K; pool 3, lymphogranuloma venereum (LGV) agent serotypes L1, L2, and L3. Pool 4 contained four representative isolates of subgroup B Chlamydia (Chlamydia psittaci). For routine purposes sera need be screened only against these four representative antigen pools. This will detect antibody and indicate which clinical and epidemiological type of chlamydial infection is implicated, thereby clearly distinguishing those infections that are due to C. psittaci. The pattern of the cross-reactions may indicate the individual serotype involved, and further titration requiring a maximum of four individual antigens is sufficient to determine the serotype. The slight loss in sensitivity (twofold) is more than compensated for by the reduction in cost and the tenfold increase in the total number of sera which can be examined.     

Interaction between a trachoma strain of Chlamydia trachomatis and mouse fibroblasts (McCoy cells) in the absence of centrifugation.

A system was devised for studying the interaction of a trachoma strain of Chlamydia trachomatis (G17) and mouse fibroblasts (McCoy cells) in the absence of centrifugation, which is usually employed to enhance the infection of cell cultures with non-lymphogranuloma venereum human strains of C. trachomatis. In this system, the conditions of infection more closely approached those encountered in natural infections, and the entry of G17 into host cells could be compared with the previously described entry of C. psittaci 6BC and a lymphogranuloma venereum strain (440L) of C. trachomatis. McCoy cells were infected by shaking at 37 degrees C with inocula suspended in 0.01 M phosphate buffer, pH 7.2, containing 0.2 M sucrose. The efficiency of infection (inclusion counts without centrifugation/inclusion counts with centrifugation) was 1.5% for monolayers and 7.5% for suspensions. When measured either by inclusion counts or by host cell-associated 14C-amino acid-labeled G17, association was proportional to G17 concentration and increased linearly for 60 min. Pretreatment of host cells with diethylaminoethyl-dextran (30 micrograms/ml, 30 min) raised the efficiency of infection to about 13% for both monolayers and suspensions. Host cells treated with cytochalasin B (2 x 10(-5) M, 90 min) or trypsin (50 micrograms/ml, 60 min) associated with G17 at undiminished rates. 14C-labeled G17 inactivated by heat (60 degrees C, 3 min) or ultraviolet light (1,800 ergs per mm2) associated with McCoy cells at the same rate as live G17. Comparison of these results with those previously reported for strains 6BC and 440L showed that strain G17 exhibited some, but not all, of the host cell association properties of the other two chlamydial strains.     

Immediate cytotoxicity of Chlamydia trachomatis for mouse peritoneal macrophages.

The toxicity of Chlamydia trachomatis was studied with mouse peritoneal macrophage culture. Inoculation of 30 inclusion-forming units of trachoma B/TW-5/OT organisms and 250 inclusion-forming units of lymphogranuloma venereum L2/434/Bu organisms per cell caused immediated toxicity, with the killing of 40 to 90% of the macrophages within 6 h after inoculation. Inhibition of phagocytosis by adsorption at 0 degrees C or by NaF pretreatment of macrophages prevented the toxicity, indicating that chlamydiae must be phagocytized to induce toxicity. Infectivity and toxicity could be dissociated, since ultraviolet-inactivated chlamydiae were still toxic. However, the toxicity was destroyed by heating the organisms at 56 degrees C for 10 min. Tetracycline, and antichlamydial drug, did not prevent toxicity, indicating that multiplication of the organisms was not required to induce toxicity. Toxicity was not prevented by treatment of macrophages with hydrocortisone. The toxicity of trachoma TW-5 was reduced by the rabbit immune serum of trachoma TW-5 but not by the rabbit immune serum of psittacosis meningopneumonitis.     

Murine cytotoxic T lymphocytes induced following Chlamydia trachomatis intraperitoneal or genital tract infection respond to cells infected with multiple serovars.

The obligate intracellular bacterium Chlamydia trachomatis is associated with human diseases ranging from blinding trachoma to sexually acquired genital infections and the systemic disease lymphogranuloma venereum (LGV). We have previously reported the isolation and culture of protective murine cytotoxic T lymphocytes (CTL) following intraperitoneal infection with C. trachomatis serovar L2, a serotype associated with human LGV. In this report, we now demonstrate that CTL can also be primed following introduction of C. trachomatis serovar L2 into the uterus or ovarian bursa of mice. We also describe Chlamydia-specific CTL lines isolated following murine infection with a typical human urogenital isolate of C. trachomatis (serovar D) and show that such CTL can be primed by intraperitoneal, intrauterine, or intrabursal infection. Last, we demonstrate that these murine CTL lines respond to multiple serovars, recognizing and lysing cells infected with C. trachomatis serovars B, C, D, F, J, K, L2, and L3, representative of organisms causing blinding trachoma, genital infection, and LGV.     

Isolation and characterization of a mutant Chinese hamster ovary cell line that is resistant to Chlamydia trachomatis infection at a novel step in the attachment process

Host factors involved in Chlamydia trachomatis pathogenesis were investigated by random chemical mutagenesis of Chinese hamster ovary (CHO-K1) cells followed by selection for clones resistant to chlamydial infection. A clonal mutant cell line, D4.1-3, refractory to infection by the C. trachomatis L2 serovar was isolated. The D4.1-3 cell line appears to be lacking in a previously undescribed temperature-dependent and heparin-resistant binding step that occurs subsequent to engagement of cell surface heparan sulfate by L2 elementary bodies. This novel binding step differentiates the lymphogranuloma venereum (LGV) serovar from other serovars and may contribute the different pathologies associated with LGV and non-LGV strains.     

Differences in susceptibilities of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis to neutralization by immune sera.

Sera from seven patients from whom a C. trachomatis serovar L2 strain was isolated were tested in vitro for their ability to neutralize the infectivity of this organism. In one patient an inguinal lymph node was culture positive, whereas the remaining six patients had positive rectal biopsies. Sera from four of the patients, including the patient with the lymph node isolate, failed to neutralize serovar L2(434). In addition, the homologous strain recovered from the inguinal lymph node was available and was resistant to neutralization by the homologous sera. However, the same sera effectively neutralized a trachoma serovar, E(Bour). All four sera had inclusion immunofluorescent-antibody titers to C. trachomatis serovar L2 of 2,048 to 16,384 and microimmunofluorescent-antibody titers to the lymphogranuloma venereum biovar were equal or higher in all cases than to the 12 serovars of the trachoma biovar. The three remaining sera, while neutralizing the infectivity of the L2 strains tested, neutralized serovar E to a greater extent. These sera had the same inclusion immunofluorescent antibody titers as the sera that failed to neutralize serovar L2. To see whether this difference in the sensitivity of the biovars toward neutralization could be characterized, sera were obtained from mice immunized with different doses of both serovars L2 and E. Sera obtained from mice immunized with serovar E were able to effectively neutralize the homologous strain. In contrast, neutralization of the immunizing strain, L2(UCI-20), was not seen with sera obtained on days 7, 14, and 21 after immunization from animals receiving 8 x 10(5) and 8 x 10(4) inclusion-forming units of L2(UCI-20); however, these same sera neutralized serovar E. However, with a higher immunizing dose of L2 (10(7) IFUs), both E and L2 were neutralized with sera obtained 7 and 14 days after immunization. Therefore, the relative resistance to neutralization by serovar L2 compared with that of serovar E in the mouse model was inoculum dependent.     

Functional and structural mapping of Chlamydia trachomatis species-specific major outer membrane protein epitopes by use of neutralizing monoclonal antibodies.

Three monoclonal antibodies (MAbs), E4, L1-4, and L1-24, to the major outer membrane protein (MOMP) of Chlamydia trachomatis were identified that neutralized in vitro the infectivity of members of the B- and C-related complex as well as the mouse pneumonitis strain. MAbs L1-4, L1-24, and E4 gave a strong signal in an indirect immunofluorescence assay and/or Western immunoblot with all serovars of the lymphogranuloma venereum and trachoma biovars and a weak signal with the mouse biovar. In addition, C. psittaci and C. pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by the technique of pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by by the technique of overlapping peptides, all three MAbs showed reactivity to variable domain (VD) IV of MOMP. While all three MAbs had different recognition patterns, all strongly bound to the peptides TLNPTI and LNPTIA within the species-conserved region of VD IV. MAb E4 also recognized the peptide SATAIF in the subspecies region of VD IV. Peptides corresponding to VD IV of MOMP were synthesized and used in competitive inhibition experiments to determine the functional location of the epitope recognized by these three MAbs. Both the serological and neutralizing activities of MAb E4 were inhibited by the peptides ATAIFDTTTLNPTIAG and FDTTTLNPTIAG; however, none of the peptides made to the VD IV region blocked the neutralizing activity of MAbs L1-4 and L1-24. Therefore, the neutralizable domain of the epitope recognized by MAb E4 is contiguous and may be an important candidate for inclusion in a subunit vaccine.