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PubMed1994

Diagnosis of Chlamydia pneumoniae infection in patients with community-acquired pneumonia by polymerase chain reaction enzyme immunoassay.

We conducted a prospective study of 385 patients who had community-acquired pneumonia with use of a modified polymerase chain reaction (PCR) assay that detects amplified DNA by enzyme immunoassay (EIA). We used PCR-EIA to improve detection of Chlamydia pneumoniae infection and to differentiate C. pneumoniae infection from other chlamydial infections. Cultures of throat swab specimens from four patients yielded Chlamydia species (C. pneumoniae, one patient; Chlamydia species, two patients; and C. psittaci, one patient). C. pneumoniae was repeatedly detected by PCR-EIA for thirteen (3.4%) of these 385 patients. Six of these 13 patients were infected with the human immunodeficiency virus. Ten (76.9%) of the patients who were positive by PCR-EIA had IgG titers of > or = 1:16, and two (15.4%) of the 13 patients had IgG titers of < 1:16; no sera was available in one case. Other pathogens were recovered in eight (61.5%) of the 13 cases in which C. pneumoniae was detected by PCR-EIA. In addition, for 46 (11.9%) of the 385 patients the titers of antibody were considered diagnostic of C. pneumoniae infection; however, as 36 of the 46 patients were infected with the human immunodeficiency virus (which may have affected their serological response to C. pneumoniae), interpretation of these titers was problematic. As PCR-EIA was more sensitive than was culture for detecting C. pneumoniae infection in this study, this method may be a valuable tool for the prompt diagnosis of this infection.

Adult↗
PubMed1991

Typing Chlamydia trachomatis by detection of restriction fragment length polymorphism in the gene encoding the major outer membrane protein.

A method that avoids culture was devised to determine serovars of Chlamydia trachomatis. Polymerase chain reaction was used first to amplify a part of the chlamydial genome that included the leader sequence and all four variable domains of the major outer membrane protein (MOMP) of the 15 serovars of C. trachomatis. The amplified DNA was then digested simultaneously with restriction endonucleases AluI and MspI and the resulting fragments separated on 10% polyacrylamide gels. After silver staining, a total of 13 characteristic patterns were observed for the 15 serovars, leaving two ambiguities that were resolved using alternate enzymes. Analysis of 40 clinical isolates revealed patterns indistinguishable from those of the prototype serovars including, unexpectedly, 5 Ba serovars. The same PCR procedure also allowed amplification of the MOMP gene of two avian Chlamydia psittaci and one Chlamydia pneumoniae isolates.

Bacterial Outer Membrane Proteins↗
PubMed1993

Transmission of Chlamydia trachomatis among sex partners assessed by polymerase chain reaction.

The prevalence of Chlamydia infection in 95 sex partners was determined by both polymerase chain reaction (PCR) and cell culture. Thirty-three (18%) of 186 specimens were positive by culture and 61 (33%) were positive by PCR-EIA. PCR was positive in 75% (21/28) of male partners of PCR-positive women compared with culture, which was positive in only 45% (9/19) of male partners of culture-positive women (P = .053). For female partners of infected men, the difference was less marked. PCR was positive in 58% (21/36) of female partners of infected men versus culture, which was positive in 56% (15/36) of female partners of culture-positive men. The correlation of PCR between partners and sequence analysis of Chlamydia DNA showing the same sequence from sex partners of 7 couples support the accuracy of the assay. These data suggest that PCR is more sensitive than culture for detection of Chlamydia trachomatis, particularly for male partners of infected women.

Adult↗
PubMed1988

Activity of spiramycin against chlamydia, in vitro and in vivo.

In-vitro and in-vivo studies were performed to assess the activity of spiramycin against chlamydiae. In tissue culture spiramycin exhibited very good activity against Chlamydia trachomatis but less against C. psittaci. C. psittaci was also studied in a mouse experimental model. There was 100% clinical cure of the mice with 50 mg/kg, demonstrating excellent in-vivo activity. The livers of mice recovering after therapy showed less chlamydial antigen than controls. These results show good in-vitro and in-vivo activity of spiramycin against chlamydiae, suggesting that clinical trials are warranted.

Animals↗
PubMed1992

Inhibition of Chlamydia trachomatis growth in mouse fibroblasts by liposome-encapsulated tetracycline.

The in-vitro susceptibility of Chlamydia trachomatis to liposome-encapsulated tetracycline was determined and compared with free tetracycline. Anionic, cationic and neutral small unilamellar liposomes were used in this study. Chlamydia-infected mouse fibroblast monolayers were continuously exposed to varying concentrations of antibiotic, incubated for 48 h and Giemsa stained. The minimum inhibitory concentration (MIC) for anionic, cationic and neutral liposomes containing tetracycline were 0.38, 0.08 and 0.04 mg/L, respectively. This was approximately 2, 10, and 20 times more efficient than free tetracycline (MIC, 0.79 mg/L). Neutral liposomes displayed no visible toxic side-effects on the host cells. When compared with free tetracycline, neutral liposomes were the most efficient for the delivery of inhibitory concentrations of tetracycline to chlamydia-infected mouse fibroblast L cell cultures.

Animals↗
PubMed1997

The in-vitro activity of grepafloxacin against Chlamydia spp., Mycoplasma spp., Ureaplasma urealyticum and Legionella spp.

The activity of grepafloxacin, a new orally active fluoroquinolone, was compared with the activities of ofloxacin, clarithromycin and doxycycline against Chlamydia pneumoniae, Chlamydia trachomatis, Mycoplasma pneumoniae, Mycoplasma hominis and Ureaplasma urealyticum, and with the activities of ofloxacin, clarithromycin and rifampicin against Legionella spp. Grepafloxacin (MIC range 0.06-0.12 mg/L) was some 8-16 times more active than ofloxacin against the chlamydiae, showing activity similar to that of doxycycline, and equal or two- to four-fold less active than clarithromycin. Grepafloxacin was four-fold more active than ofloxacin against M. pneumoniae (MIC 0.06-0.5 mg/L) and U. urealyticum (MIC 0.12-1.0 mg/L), but 16 times more active against M. hominis (MIC 0.015-0.05 mg/L). Grepafloxacin was highly active against Legionella spp. (MIC 0.008-0.03 mg/L), showing equivalent activity to ofloxacin, clarithromycin and rifampicin.

Anti-Bacterial Agents↗
PubMed2001

Chlamydia, gonorrhea, and HIV-1 prevalence among five populations of women in the Czech and Slovak Republics.

BACKGROUND: Five populations at risk for sexually transmitted diseases (STDs) in the Czech and Slovak Republics were sampled. GOAL: To estimate prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, and HIV-1 infections. STUDY DESIGN: Urine specimens were collected serially from women at a Prague prenatal clinic (n = 134), a Prague dermatovenerealogy clinic (n = 91), sex workers from northern and central Bohemia (n = 35), students from a northern Bohemian school (n = 217), and Gypsies from Jarovnice, Slovakia (n = 128). These specimens were tested for chlamydia and gonorrhea using a ligase chain reaction pooling algorithm, and for HIV using an enzyme immunoassay confirmed by Western blot. RESULTS: The prevalence of chlamydia was 2.2% (95% CI, 0.4-6.4) in the prenatal clinic, 5.5% (95% CI, 1.8-12.4) in the STD clinic, 22.9% (95% CI, 10.4-40.1) among street sex workers, 8.2% (95% CI, 3.6-15.6) among sexually active female high school students, and 3.9% (95% CI, 1.3-8.9) among Gypsy women. Gonorrhea was found in only two populations: 2.2% (95% CI, 0.3-7.7) in the STD clinic, and 2.9% (95% CI, 0.1-14.9) among sex workers. No HIV-1 infection was detected. CONCLUSIONS: Urine screening was an efficient and accurate method for identifying groups at risk for STDs in the Czech Republic and Slovakia because sample collection was fast and noninvasive, and potential participation bias was reduced by high acceptability.

Adolescent↗
PubMed1981

Polypeptide composition of Chlamydia trachomatis.

Strains of Chlamydia trachomatis representative of 14 serotypes were grown in HeLa 229 cells. HeLa cell susceptibility to chlamydial infection was increased by treating the host cells with DEAE-dextran. Optimal conditions of DEAE-dextran treatment were determined for each serotype of C. trachomatis to maximize chlamydial yields. Chlamydial polypeptides were selectively radiolabelled with 3H-labelled amino acids in the presence of emetine, an inhibitor of HeLa cell protein synthesis. The radiolabelled chlamydiae were purified from host cell components by density gradient centrifugation and their polypeptide composition was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The distribution of the major chlamydial polypeptide of 38 000 to 42 000 daltons amongst the different serotypes correlated closely with the predominant human infections caused by each serotype. Lymphogranuloma venereum agents possessed a unique polypeptide of 118 000 daltons not found amongst trachoma inclusion conjunctivitis strains of chlamydiae. Chlamydial surface polypeptides were selectively radiolabelled with 125I by lactoperoxidase-catalysed oxidation. The major chlamydial polypeptide and polypeptides of 155 000 and 29 000 daltons were thus identified as surface polypeptides of the chlamydial elementary body. It is suggested that the 155 000 dalton polypeptide is a species-specific antigen, the major polypeptide is the principal outer membrane protein, and the 29 000 dalton polypeptide is the type-specific antigen.

Chlamydia trachomatis↗
PubMed1982

Control mechanisms governing the infectivity of Chlamydia trachomatis for hela cells: modulation by cyclic nucleotides, prostaglandins and calcium.

Chlamydia trachomatis causes common infections of the eyes and genital tract in man. The mechanism by which this obligate intracellular bacterium is taken into epithelial cells is unclear. The results described here support the concept that chlamydial infections of HeLa cells is under bidirectional cyclic nucleotide control, with guanosine 3':5'-cyclic monophosphate (cGMP) acting as a stimulator, and adenosine 3':5'-cyclic monophosphate (cAMP) as an inhibitor. Treatment of the HeLa cells with the divalent cation ionophore A23187, with carbamoylcholine, or with prostaglandins known to increase the concentration of endogenous cGMP, also increased host cell susceptibility to chlamydial infection. Cyclic GMP was only effective if added at or before chlamydial inoculation, suggesting that its main effect was on chlamydial uptake. The stimulatory effect of cGMP, but nt antagonism, by cAMP, was abolished if the cells were first treated with any of four different inhibitors of prostaglandin synthesis, suggesting a critical role for endogenous prostaglandin synthesis. Centrifugation of chlamydiae on to host cells was followed by a rapid increase in the mobility of Ca2+ across the cell membrane. The interrelationships of these observations and the possibility that chlamydiae and other intracellular pathogens might evoke alterations in host cell prostaglandin and cyclic nucleotide concentrations to aid their own uptake are discussed.

Bucladesine↗
PubMed1986

Host modification of the adherence properties of Chlamydia trachomatis.

The adherence of Chlamydia trachomatis LGV440(L1) to human HeLa 229 and mouse McCoy cells was stimulated by the lectin wheat germ agglutinin (WGA) and inhibited by the sugars N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and chitobiose, but only when the chlamydiae had been passaged several times in HeLa cells. After passage in McCoy cells, the lectin and the sugars elicited little response. The non-LGV serovar UW-31(K), however, differed from LGV440(L1) in that, regardless of passage, the lectin and sugar effects were observed only in HeLa cells. Affinity chromatography on WGA-agarose confirmed that HeLa-grown LGV440(L1) bound to a significantly greater extent relative to McCoy-grown chlamydiae. In addition, participation of heterogeneous chlamydial ligands was suggested by the observation that the adherence of heated (60 degrees C, 5 min) UW-31(K) to HeLa cells at 37 degrees C was not inhibited at all, but at 5 degrees C, the adherence rate was greatly reduced, indicating the participation of heat-stable as well as heat-labile ligands. These data are interpreted to indicate that the passage history of C. trachomatis results in the acquisition of altered surface components that participate in the initial interaction of the bacterium with the host.

Animals↗
PubMed1987

Chlamydia trachomatis (L2 serovar) can be bound, ingested and destroyed by differentiated but not by undifferentiated human promyelocyte cell line HL-60.

As a model system for analysing interactions between chlamydiae and myeloid cells and their precursors, we have studied binding, ingestion and destruction of Chlamydia trachomatis (L2 serovar) by the human promyelocytic cell line HL-60. HL-60 cells were induced by phorbol myristate acetate (PMA) and dimethyl sulphoxide (DMSO) to differentiate along either the macrophage or the granulocyte pathway, respectively. Using an immunofluorescence assay and electron microscopy, we have shown that induced (differentiated) HL-60 cells, but not uninduced (undifferentiated) HL-60 or other cell lines treated with PMA or DMSO, exhibit increased binding, ingestion and elimination of C. trachomatis; these activities are associated with specific histochemical and antigenic markers of myeloid differentiation. These results suggest that myeloid cells acquire the ability to interact with and kill chlamydiae during cell development.

Cell Differentiation↗
PubMed1990

Extensive heterogeneity of the protein composition of Chlamydia trachomatis following serial passage in two different cell lines.

To determine if the host-modulated adherence characteristics of the intracellular bacterial pathogen Chlamydia trachomatis were due to the acquisition of altered surface-exposed proteins, highly purified chlamydiae grown in two different host cells were analysed. Two serovars, L1 and E, were grown for multiple passages in both HeLa and McCoy host cells. Numerous protein differences in the chlamydial elementary bodies (EB) of each serovar grown in the two different hosts were detected by two-dimensional (2-D) gel electrophoresis and fluorography of radioactively labelled proteins. At least four to six serial passages in the alternative host were necessary before the changes were apparent. Iodination of suspensions of purified chlamydiae and 2-D electrophoresis revealed several surface proteins that were determined by the host cells in which the bacteria had replicated. These iodinated chlamydial proteins were removed by treatment of the iodinated EB with trypsin, indicating their location at the bacterial surface. Two of the major constituents of the outer-membrane complex, the cysteine- and methionine-rich 60 kDa and 40 kDa proteins, remained unchanged in both molecular mass and charge during the host adaptation. Several chlamydial proteins capable of binding iodinated host membrane preparations also exhibited host-dependent alterations. Immunoblotting experiments with a rabbit and a human polyclonal sera indicated that distinct host-specified chlamydial proteins were reactive with the two sera.

Animals↗
PubMed1993

Mapping of a surface-exposed B-cell epitope to the variable sequent 3 of the major outer-membrane protein of Chlamydia trachomatis.

A B-cell epitope, AEFPLDIT, was located to the variable sequent 3 of the major outer-membrane protein (MOMP) using the monoclonal antibody L3-1, raised to the Chlamydia trachomatis serovar L3 MOMP. By Western blot and inclusion immunofluorescence assay the monoclonal antibody recognized all the C complex and C-related complex serovars of C. trachomatis, except serovar C. Dot-blot and ELISA data using native elementary bodies indicated that the epitope was surface exposed. The monoclonal antibody, at concentrations of 10 and 100 micrograms per 10(7) chlamydial inclusion-forming units, was able to neutralize the infectivity of chlamydia in an in vivo assay but did not neutralize chlamydia in vitro or in a mouse toxicity assay. A peptide corresponding to the variable sequent 3 has previously been shown to also elicit a T-cell response; thus, careful consideration should be given to inclusion of this region of the major outer-membrane protein in a subunit vaccine.

Amino Acid Sequence↗
PubMed2001

Substrate-specific diffusion of select dicarboxylates through Chlamydia trachomatis PorB.

Chlamydiae contain two porins, MOMP and PorB, that facilitate diffusion of solutes through the outer membrane. MOMP is a general porin that permits the diffusion of a wide variety of compounds including carbohydrates and amino acids. The relative inefficiency of PorB as a general porin and its low abundance in the outer membrane suggest that it may function as a substrate-specific porin. The tricarboxylic acid (TCA) cycle of chlamydiae is incomplete and to function would require the exogenous acquisition of 2-oxoglutarate or glutamate. A liposome-swelling assay for anions as well as an enzyme-linked liposome assay were used to demonstrate the efficient diffusion of dicarboxylates such as 2-oxoglutarate through PorB. These data demonstrate that PorB is a dicarboxylate-specific porin that may feed the chlamydial TCA cycle and provide chlamydiae with carbon and energy production intermediates.

Bacterial Outer Membrane Proteins↗
PubMed2005

Identification of surface-exposed components of MOMP of Chlamydia trachomatis serovar F.

The identification of surface-exposed components of the major outer membrane protein (MOMP) of Chlamydia is critical for modeling its three-dimensional structure, as well as for understanding the role of MOMP in the pathogenesis of Chlamydia-related diseases. MOMP contains four variable domains (VDs). In this study, VDII and VDIV of Chlamydia trachomatis serovar F were proven to be surface-located by immuno-dot blot assay using monoclonal antibodies (MAbs). Two proteases, trypsin and endoproteinase Glu-C, were applied to digest the intact elementary body of serovar F under native conditions to reveal the surface-located amino acids. The resulting peptides were separated by SDS-PAGE and probed with MAbs against these VDs. N-terminal amino acid sequencing revealed: (1) The Glu-C cleavage sites were located within VDI (at Glu61) and VDIII (at Glu225); (2) the trypsin cleavage sites were found at Lys79 in VDI and at Lys224 in VDIII. The tryptic peptides were then isolated by HPLC and analyzed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer and a quadrupole-orthogonal-TOF mass spectrometer coupled with a capillary liquid chromatograph. Masses and fragmentation patterns that correlated to the peptides cleaved from VDI and VDIII regions, and C-terminal peptides Ser333-Arg358 and Ser333-Lys350 were observed. This result demonstrated that these regions are surface-exposed. Data derived from comparison of nonreduced outer membrane complex proteolytic fragments with their reduced fractions revealed that Cys26, 29, 33, 116, 208, and 337 were involved in disulfide bonds, and Cys26 and 337, and 116 and 208 were paired. Based on these data, a new two-dimensional model is proposed.

Amino Acid Sequence↗
PubMed1998

Differentiation of Chlamydia species by combined use of polymerase chain reaction and restriction endonuclease analysis.

To differentiate Chlamydia spp., a primer pair designed to generate a genus-specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245-259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one-step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.

Chlamydia↗
PubMed2006

Mycobacterium tuberculosis-susceptible I/St mice develop severe disease following infection with taxonomically distant bacteria, Salmonella enterica and Chlamydia pneumoniae.

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. To find out whether tuberculosis (TB)-susceptible I/St mice are susceptible to other intracellular bacteria, we investigated two different taxonomically distant pathogens, Chlamydia pneumoniae and Salmonella enterica serovar Typhimurium. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1(r)) demonstrated that the former are more susceptible to both salmonella and chlamydia, displaying a significantly shortened survival time following challenge. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia, despite their similar ability to control bacterial multiplication. Following infection with salmonella, substantial ( approximately 3 log) but very short (second day post-infection) interstrain differences in bacterial loads were observed, accompanied by higher levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in the peritoneal cavities of I/St mice. I/St macrophages were more permissive for salmonella growth during the first 24 h following infection in vitro. Because the prominent differences in survival time did not correlate with permanent differences in bacterial multiplication, we suggest that both infections trigger fatal pathological processes whose dynamics depend strongly upon the host genetics.

Animals↗
PubMed1993

The genus-specific lipopolysaccharide epitope of Chlamydia is assembled in C. psittaci and C. trachomatis by glycosyltransferases of low homology.

Chlamydiae possess a genus-specific epitope that is located on the lipopolysaccharide (LPS) and is composed of a 3-deoxy-D-manno-octulosonic acid (Kdo) trisaccharide of the sequence alpha Kdo-(2-->8)--alpha Kdo-(2-->4)-alpha Kdo. In Chlamydia trachomatis, this trisaccharide is biosynthetically generated through the action of a multi-functional Kdo-transferase encoded by the gene gseA. gseA of Chlamydia psittaci 6BC was cloned and expressed in a rough mutant (Re chemotype) of Escherichia coli (strain F515) that contains an LPS with only two alpha 2-->4-linked Kdo residues. Recombinant strains were able to add the immunodominant Kdo residue in alpha 2-->8-linkage to the parental LPS, as determined by SDS-PAGE and Western blot analysis using a monoclonal antibody against the genus-specific epitope. The DNA sequence of gseA was determined and aligned to that published recently for C. trachomatis serovar L2. Most surprisingly, the two deduced amino acid sequences shared only an overall homology of 67%. Thus, gseA exhibits species specificity at the DNA level, whereas its gene product results in the synthesis of a carbohydrate antigen with genus specificity.

Amino Acid Sequence↗