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PubMed1989

Chlamydia trachomatis-induced production of interleukin-1 by human monocytes.

Human diseases caused by the intracellular bacterium Chlamydia trachomatis include genital tract infections and blinding trachoma. Chlamydial infections are characterized by chronic inflammation and scarring, and development of such complications is thought to be immunologically mediated. In this study, we show that coculture of C. trachomatis (serovar L2) with human blood monocytes induced the production of interleukin-1 (IL-1), an important mediator of inflammation, tissue remodeling, and scarring. IL-1 was produced in response to UV-inactivated elementary bodies containing from 0.1 to 50 micrograms of protein per ml, with a maximal response at 5 to 10 micrograms/ml. IL-1 activity was detected by 6 h of incubation and was maximal by 24 h. Peak levels were maintained throughout 96 h of incubation. Rabbit antibody to human IL-1(alpha + beta) effectively neutralized the thymocyte-stimulating activity of the supernatants. The apparent molecular weight of chlamydia-induced IL-1 was 16,000, as determined by gel filtration on a Bio-Gel P-60 column. Isoelectric focusing yielded two peaks of activity, with pIs of 5.5 and 6.9. Neutralization studies with antisera against human IL-1 alpha and IL-1 beta showed that the acidic and neutral peaks corresponded to IL-1 alpha and IL-1 beta, respectively, with IL-1 beta predominating. Heat-killed chlamydiae, which are not internalized by monocytes, were effective IL-1 inducers, indicating that phagocytosis was not required for IL-1 induction. Purified C. trachomatis lipopolysaccharide was also an effective IL-1 inducer, suggesting that the response to intact organisms may be largely a response to chlamydial lipopolysaccharide. Finally, purified chlamydial major outer membrane protein induced low but detectable IL-1 activity.

Antibodies↗
PubMed1990

The Chlamydia trachomatis hyp operon is homologous to the groE stress response operon of Escherichia coli.

The Chlamydia trachomatis serovar A hyp operon was cloned, sequenced, and expressed in Escherichia coli. Two cotranscribed open reading frames, hypA and hypB, encoded polypeptides of 17 and 57 kilodaltons, respectively. The deduced amino acid sequences of serovar A HypA and HypB proteins were (respectively) 85 and 94% identical with HypA and HypB proteins of Chlamydia psittaci GPIC, and HypB was greater than 50% identical to 60-kilodalton stress response proteins from other procaryotes and eucaryotes. The sequence should be useful in defining the antigenic structure of the Chlamydia trachomatis HypB protein, a necessary step toward understanding the relationship between the immune response to this protein and the pathogenesis of human chlamydial diseases.

Amino Acid Sequence↗
PubMed1991

Isolation and sequence analysis of the Chlamydia pneumoniae GroE operon.

Chlamydia pneumoniae has emerged as an important human respiratory pathogen. From a lambda gt11 gene bank constructed from C. pneumoniae isolate AR-39 DNA, an immunoreactive plaque containing a 3.0-kb insert was purified. In immunoblots, a 60-kDa protein was recognized by anti-C. pneumoniae rabbit immune serum. The recombinant protein was reactive with a Chlamydia genus-specific monoclonal antibody recognizing a 60-kDa protein found in the Sarkosyl-soluble fraction and with rabbit immune serum prepared against the Chlamydia trachomatis 60-kDa GroEL homolog associated with the delayed-type hypersensitivity response. DNA sequence analysis confirmed that the C. pneumoniae gene product is an analog of the C. trachomatis delayed-type hypersensitivity antigen and the Escherichia coli GroEL heat shock protein.

Amino Acid Sequence↗
PubMed1993

Effects of antibody isotype and host cell type on in vitro neutralization of Chlamydia trachomatis.

Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapping peptides in the variable domain IV of the major outer membrane protein of Chlamydia trachomatis but differ in isotype, were used in a complement-independent (CI) in vitro neutralization assay. These MAbs had previously been shown to neutralize chlamydial infectivity in HeLa 229 cells in a complement-dependent assay. In this report, all three MAbs neutralized chlamydial infectivity in HaK cells in a CI assay. However, when HeLa cells were used as the host cell, MAb E-4 (immunoglobulin G2b [IgG2b]) and MAb DIII A3 (IgG2b) failed to neutralize infectivity, while MAb E-21 (IgG1) neutralized chlamydial infectivity. These findings are consistent with the proposal that because of the presence of Fc gamma RIII receptors, HeLa cells facilitate infectivity and thus block neutralization through the uptake of an IgG2b-chlamydia complex. Since Fc gamma RIII receptors do not bind or bind poorly to IgG1, neutralization of C. trachomatis by MAb E-21 in HeLa cells is also corroborative evidence for the role of Fc gamma RIII receptors in this interaction. A fivefold enhancement of infectivity was seen when 10 and 1 micrograms of MAb E-4 per ml were tested in a CI assay with HeLa cells. In performing CI neutralization synergy studies in HeLa cells with MAbs E-4 and E-21, antagonism between MAbs E-4 and E-21 was observed at MAb E-4 concentrations of 10 and 1 micrograms/ml for all concentrations of MAb E-21 tested (10 to 0.1 micrograms/ml). When HaK cells were used in the same studies, no antagonism between the MAbs was found. In addition, when HeLa cells were used in a CI assay, polyclonal serum raised to a peptide representing variable domain IV of the major outer membrane protein inhibited the neutralizing ability of MAb E-21. The blocking of neutralization and the enhancement of infectivity by chlamydia-specific antibodies seen in this investigation with HeLa cells may have important clinical implications for developing preventive strategies for chlamydial infections.

Amino Acid Sequence↗
PubMed1993

Characterization of a neutralizing monoclonal antibody directed at variable domain I of the major outer membrane protein of Chlamydia trachomatis C-complex serovars.

A monoclonal antibody (MAb), C10, that neutralized in vitro the infectivity of serovars C, I, J, and L3 (members of the C and C-related complexes) of Chlamydia trachomatis was identified. Of the 15 major serovars and the mouse pneumonitis strain of C. trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae, which were used as nontreated and heat-treated (56 degrees C, 30 min) antigens in a dot blot assay, only serovars C, I, J, and L3 were recognized with both the native and treated antigens. Western blot (immunoblot) results showed that MAb C10 recognized the major outer membrane protein of these four serovars. Overlapping hexameric peptides corresponding to variable domains (VDs) I, II, III, and IV of the major outer membrane protein of C. trachomatis serovar C were synthesized, and peptide screening showed that MAb C10 mapped to the VD I amino acid sequence VAGLQNDPT. Results of an in vitro neutralization assay correlated with those of the indirect immunofluorescence assay, Western blot, and dot blot assay in that only serovars C, I, J, and L3 were neutralized by MAb C10. In vitro competitive neutralization experiments, using a peptide representing VD I of serovar C to compete with C. trachomatis serovar C for MAb C10 binding, revealed that both serological and neutralizing activities of MAb C10 were inhibited by the VD I peptide. In an in vivo toxicity/infectivity assay using serovar L3 pretreated with MAb C10, there was 100% survival of mice infected with a lethal dose at 48 h. In contrast, the control group, consisting of mice injected with the same dose of L3 pretreated with a MAb that does not recognize L3, had no survivors during a 48-h observation period. In summary, since the surface-exposed contiguous epitope recognized by MAb C10 binds neutralizing antibodies that are subspecies specific for the C and C-related complexes, it should be considered for inclusion in the development of a chlamydial vaccine.

Amino Acid Sequence↗
PubMed1995

Cytoskeletal requirements in Chlamydia trachomatis infection of host cells.

Infection of genital epithelial cells by the closely related sexually transmitted pathogens Chlamydia trachomatis serovars E and L2 results in different clinical disease manifestations. Following entry into target host cells, individual vesicles containing chlamydiae fuse with one another to form one large inclusion. At the cellular level, the only obvious difference between these serovars is the time until inclusion maturation, which is 48 h for the invasive serovar L2 and 72 h for serovar E. To begin to define the intracellular events of these pathogens, the effect of cytoskeletal disruption on early endosome fusion and inclusion development in epithelial (HEC-1B) and fibroblast (McCoy) cells was analyzed by fluorescence microscopy. Disruption of microfilaments with cytochalasin D markedly reduced serovar E, but not serovar L2, infection of both cell lines. Conversely, microfilament as well as microtubule disruption, with colchicine or nocodazole, had no effect on serovar E inclusion development but resulted in the formation of multiple serovar L2 inclusions per cell during early and mid-development. Later in serovar L2 inclusion development (> 36 h postinfection), vesicles containing chlamydiae fused to form one large inclusion in the absence of an intact cytoskeleton. These results imply that (i) C. trachomatis serovar E may utilize a different pathway for uptake and development from serovar L2; (ii) these differences are consistent in both epithelial cells and fibroblasts; and (iii) the cytoskeleton plays a unique role in the infection of host cells by these two genital pathogens.

Actin Cytoskeleton↗
PubMed1996

Induction of alpha/beta interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine.

The propensity of two Chlamydia trachomatis strains (L2/434/Bu [biovar LGV] and E/DK20/ON [biovar trachoma]) to induce putative host defense responses upon infection of McCoy (mouse) cell cultures was examined. Both strains induced production of alpha/beta interferon and nitric oxide (NO) by McCoy cells. NO synthesis was mediated by the inducible isoform of NO synthase as indicated by the ability of cycloheximide or the arginine analog NG-monomethyl-L-arginine to abolish NO production; the extent of the response was dependent upon the dose of chlamydiae applied. Incubation of McCoy cells with chloramphenicol prior to infection reduced NO production by strain 434 but not by DK20, suggesting that initial chlamydial metabolism was essential to induction by the LGV strain. Antibody inhibition studies indicated that NO synthesis was dependent upon production of alpha/beta interferon and induction via lipopolysaccharide. Overall, our findings show that chlamydiae are capable of the induction of interferon and NO in murine fibroblasts in the absence of exogenous cytokines. However, the role of NO as an antichlamydial effector could not be clearly demonstrated since treatment with an arginine analog, while suppressing NO production, gave no consistent enhancement of infected cell numbers.

Animals↗
PubMed2000

Eukaryotic cell uptake of heparin-coated microspheres: a model of host cell invasion by Chlamydia trachomatis.

Using polystyrene microspheres coated with heparin or heparan sulfate, it was shown that coated microspheres specifically bound eukaryotic cells and were endocytosed by nonprofessional phagocytic cells. Coated microspheres displayed properties of binding to eukaryotic cells that were similar to those of chlamydiae, and the microspheres were competitively inhibited by chlamydial organisms. Endocytosis of heparin-coated beads resulted in the tyrosine phosphorylation of a similar set of host proteins as did endocytosis of chlamydiae; however, unlike viable chlamydial organisms, which prevent phagolysosomal fusion, endocytosed beads were trafficked to a lysosomal compartment. These findings suggest that heparin-coated beads and Chlamydia trachomatis enter eukaryotic cells by similar pathways.

Bacterial Adhesion↗
PubMed2000

Production of basic fibroblast growth factor and interleukin 6 by human smooth muscle cells following infection with Chlamydia pneumoniae.

Chlamydia pneumoniae infection has been associated with asthma and atherosclerosis. Smooth muscle cells represent host cells for chlamydiae during chronic infection. In this study we demonstrated that C. pneumoniae infection of human smooth muscle cells in vitro increased production of interleukin 6 (IL-6) and basic fibroblast growth factor (bFGF) as shown by reverse transcription-PCR, immunoblotting, and enzyme-linked immunosorbent assay. In contrast, levels of platelet-derived growth factor A-chain mRNA were not affected after infection. The stimulation of bFGF and IL-6 production was most effective when viable chlamydiae were used as inoculum. Furthermore, inhibition of bacterial protein synthesis with chloramphenicol prevented up-regulation of IL-6 and bFGF in infected cells. Addition of IL-6 antibody to infected cultures diminished bFGF expression, indicating involvement of produced IL-6. These findings suggest that chlamydial infection of smooth muscle cells elicits a cytokine response that may contribute to structural remodeling of the airway wall in chronic asthma and to fibrous plaque formation in atherosclerosis.

Arteriosclerosis↗
PubMed2001

Infectivity of Chlamydia trachomatis serovar LGV but not E is dependent on host cell heparan sulfate.

The ability of heparan sulfate, heparin, and other glycosaminoglycans to inhibit the infectivity of Chlamydia trachomatis serovars E and LGV was examined using a simple competitive inhibition assay with three cell types from the human female reproductive tract, including primary human endosalpingeal cells. With the majority of the glycosaminoglycans tested, LGV was more significantly inhibited than serovar E. We have compared chlamydial infectivity between a wild-type Chinese hamster ovary cell line and two glycosaminoglycan-deficient cell lines. LGV was shown to be unable to infect heparan sulfate-deficient and GAG-deficient Chinese hamster ovary cell lines, whereas the E serovar infected these cells as efficiently as the control (nondeficient) cells. These two sets of experiments confirmed that serovar LGV is more dependent on a heparan sulfate-related mechanism of infectivity than is serovar E. This is further supported by the fact that attempts to purify a heparan sulfate-like molecule from either serovar cultured in glycosaminoglycan-deficient cell lines were nonproductive. Previous reports have suggested that chlamydia are able to produce a heparan sulfate-like molecule that is important for attachment and infectivity. We have attempted to detect possible binding of a specific heparan sulfate antibody to C. trachomatis by flow cytometry. Results showed no binding of the heparan sulfate antibody to C. trachomatis serovar LGV or E. Our results strongly indicate that chlamydiae do not produce a heparan sulfate-like molecule but rather use host cell heparan sulfate in order to infect cells.

Animals↗
PubMed2002

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.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Chlamydia-infected cells continue to undergo mitosis and resist induction of apoptosis.

Both anti- and proapoptotic activities have been reported to occur during chlamydial infection. To reconcile the apparent controversy, we compared host cell apoptotic responses to infection with 17 different chlamydial serovars and strains. None of the serovars caused any biologically significant apoptosis in the infected host cells. Host cells in chlamydia-infected cultures can continue to undergo DNA synthesis and mitosis. Chlamydia-infected cells are resistant to apoptosis induction, although the extent of the antiapoptotic ability varied between serovars. These observations have demonstrated that an anti- but not proapoptotic activity is the prevailing event in chlamydia-infected cultures.

Apoptosis↗
PubMed2005

Development of secondary inclusions in cells infected by Chlamydia trachomatis.

The chlamydiae are obligate intracellular bacteria that occupy a non-acidified vacuole (the inclusion) during their entire developmental cycle. These bacteria produce a set of proteins (Inc proteins) that localize to the surface of the inclusion within infected cells. Chlamydia trachomatis IncA is also commonly found in long fibers that extend away from the inclusion. We used standard and confocal immunofluorescence microscopy to demonstrate that these fibers extend to newly developed inclusions, termed secondary inclusions, within infected cells. Secondary inclusions observed at early time points postinfection were devoid of chlamydial reticulate bodies. Later in the developmental cycle, secondary inclusions containing variable numbers of reticulate bodies were common. Reticulate bodies were also observed within the IncA-laden fibers connecting primary and secondary inclusions. Quantitative differences in secondary inclusion formation were found among clinical isolates, and these differences were associated with serovar. Isolates of serovar G consistently produced secondary inclusions at the highest frequency (P < 0.0001). Similar quantitative studies demonstrated that secondary inclusion formation was associated with segregation of inclusions to daughter cells following cytokinesis. We conclude that the production of secondary inclusions via IncA-laden fibers allows chlamydiae to generate an expanded intracellular niche in which they can grow and may provide a means for continuous infection within progeny cells following cell division.

Bacterial Proteins↗
PubMed2005

Inhibition of proteolytic activity of a chlamydial proteasome/protease-like activity factor by antibodies from humans infected with Chlamydia trachomatis.

We have previously shown that individuals infected with Chlamydia trachomatis can develop a robust antibody response to a chlamydia-secreted protein (designated chlamydial proteasome/protease-like activity factor [CPAF]). We now report that sera collected from these infected individuals neutralized the proteolytic activity of CPAF. Depletion of the serum sample with CPAF proteins to remove the CPAF-specific antibodies effectively blocked the neutralization, whereas similar depletion with the HSP60 proteins failed to do so. We further demonstrated that the CPAF central region covering residues 200 to 338 was predominantly recognized by the human neutralization antibodies. The significance of the CPAF neutralization antibodies generated in chlamydia-infected individuals is discussed.

Antibodies, Bacterial↗
PubMed2006

Mutational analysis of the promoter recognized by Chlamydia and Escherichia coli sigma(28) RNA polymerase.

sigma(28) RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial sigma(28) promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the sigma(28)-dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a -35 element recognized by chlamydial sigma(28) RNA polymerase that resembles the consensus -35 sequence. Within the -10 element, however, chlamydial sigma(28) RNA polymerase showed a striking preference for a CGA sequence at positions -12 to -10 rather than the longer consensus -10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli sigma(28) RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the -10 promoter element recognized by sigma(28) RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that sigma(28) RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11- or 12-nt spacer equally well. Altogether, we found very similar results for sigma(28) RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred sigma(28) promoter that we defined in the context of the hctB promoter is TAAAGwwy-n(11/12)-ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n(11/12) is a spacer of 11 or 12 nt.

Amino Acid Substitution↗
PubMed2006

Molecular mechanism of tryptophan-dependent transcriptional regulation in Chlamydia trachomatis.

Tryptophan is an essential amino acid that is required for normal development in Chlamydia species, and tryptophan metabolism has been implicated in chlamydial persistence and tissue tropism. The ability to synthesize tryptophan is not universal among the Chlamydiaceae, but species that have a predicted tryptophan biosynthetic pathway also encode an ortholog of TrpR, a regulator of tryptophan metabolism in many gram-negative bacteria. We show that in Chlamydia trachomatis serovar D, TrpR regulates its own gene and trpB and trpA, the genes for the two subunits of tryptophan synthase. These three genes form an operon that is transcribed by the major form of chlamydial RNA polymerase. TrpR acts as a tryptophan-dependent aporepressor that binds specifically to operator sequences upstream of the trpRBA operon. We also found that TrpR repressed in vitro transcription of trpRBA in a promoter-specific manner, and the level of repression was dependent upon the concentrations of TrpR and tryptophan. Our findings provide a mechanism for chlamydiae to sense changes in tryptophan levels and to respond by modulating expression of the tryptophan biosynthesis genes, and we present a unified model that shows how C. trachomatis can combine transcriptional repression and attenuation to regulate intrachlamydial tryptophan levels. In the face of host defense mechanisms that limit tryptophan availability from the infected cell, the ability to maintain homeostatic control of intrachlamydial tryptophan levels is likely to play an important role in chlamydial pathogenesis.

Bacterial Proteins↗
PubMed1970

Lipid composition of Chlamydia psittaci grown in monkey kidney cells in defined medium.

The lipid compositions of (i) monkey kidney (MK-2) cells cultivated in Eagle's minimal essential medium (MEM) with 5% calf serum, (ii) MK-2 cells cultivated in Waymouth medium supplemented with 20 mug of sodium oleate and 2 mg of bovine albumin per ml, (iii) Chlamydia psittaci strain 6BC grown in the latter host system, and (iv) calf serum were compared. Strain 6BC contains 31% phosphatidyl ethanolamine (PE) and 15% phosphatidyl glycerol (PG), whereas the host cell contains almost the same amount of PE (27%) and no PG. A high concentration of total lipid was observed in strain 6BC (29 to 34%), whereas MK-2 cells contain only 9 to 15% and calf serum contains 4.5% total lipid. The fatty acids of the total lipid from strain 6BC contain branched-chain acids. These fatty acids were found mostly in PE (33.0%) and PG (37.0%). No branched-chain fatty acid was found in the MK-2 cells. There was an increase in triglyceride content when MK-2 cells cultivated in MEM (19.2%) were compared with cells cultivated in Waymouth medium (28.0%). A high concentration (62.0%) of octadecenoic acid (C18:1) was found in the triglyceride of MK-2 cells cultivated in Waymouth medium. The level of polyunsaturated fatty acids observed in MK-2 cells cultivated in Waymouth medium (10.8%) and in the chlamydiae grown in these cells (13.3%) was low compared with the level in MK-2 cells (28.8%) cultivated in MEM with 5% calf serum and the level in calf serum itself (50.8%). A higher ratio of sterol ester to free sterol was found in calf serum than in MK-2 cells or in chlamydiae. Host contribution to lipid composition of strain 6BC is discussed.

Animals↗