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PubMed1998

In vitro inactivation of Chlamydia trachomatis by fatty acids and monoglycerides.

The antichlamydial effects of several fatty acids and monoglycerides were studied by incubating Chlamydia trachomatis bacteria with equal volumes of lipid solutions for 10 min and measuring the reduction in infectivity titer compared with that in a control solution without lipid. Caprylic acid (8:0), monocaprylin (8:0), monolaurin (12:0), myristic acid (14:0), palmitoleic acid (16:1), monopalmitolein (16:1), oleic acid (18:1), and monoolein (18:1) at concentrations of 20 mM (final concentration, 10 mM) had negligible effects on C. trachomatis. In contrast, lauric acid (12:0), capric acid (10:0), and monocaprin (10:0) caused a greater than 10,000-fold (>4-log10) reduction in the infectivity titer. When the fatty acids and monoglycerides were further compared at lower concentrations and with shorter exposure times, lauric acid was more active than capric acid and monocaprin was the most active, causing a greater than 100, 000-fold (>5-log10) inactivation of C. trachomatis at a concentration of 5 mM for 5 min. The high levels of activity of capric and lauric acids and particularly that of monocaprin are notable and suggest that these lipids have specific antichlamydial effects. The mode of action of monocaprin was further studied by removal of the lipid by centrifugation before inoculation of Chlamydia onto host cells and by electron microscopy. The results indicate that the bacteria are killed by the lipid, possibly by disrupting the membrane(s) of the elementary bodies. A 50% effective concentration of 30 microgram/ml was found by incubation of Chlamydia with monocaprin for 2 h. The rapid inactivation of large numbers of C. trachomatis organisms by monocaprin suggests that it may be useful as a microbicidal agent for the prevention of the sexual transmission of C. trachomatis.

Cells, Cultured↗
PubMed2000

Persistence of Chlamydia trachomatis is induced by ciprofloxacin and ofloxacin in vitro.

An in vitro cell culture model was used to investigate the long-term effect of ciprofloxacin and ofloxacin on infection with Chlamydia trachomatis. Standard in vitro susceptibility testing clearly indicated successful suppression of chlamydial growth. To mimic better in vivo infection conditions, extended treatment with the drugs was started after infection in vitro had been well established. Incubation of such established chlamydial cultures with ciprofloxacin and ofloxacin not only failed to eradicate the organism from host cells, but rather induced a state of chlamydial persistence. This state was characterized by the presence of nonculturable, but fully viable, bacteria and the development of aberrant inclusions. In addition chlamydia exhibited altered steady-state levels of key chlamydial antigens, with significantly reduced major outer membrane protein and near constant hsp60 levels. Resumption of overt chlamydial growth occurred after withdrawal of ciprofloxacin, confirming the viability of persisting chlamydia. In vitro ciprofloxacin results are consistent with clinical data, thereby providing an explanation for treatment failures of ciprofloxacin. Parallel in vitro studies with ofloxacin suggest a better correlation between clinical and laboratory-defined efficacy, although the clinical studies on which this assessment is based did not include monitoring of chlamydial persistence. The data presented here clearly demonstrate that under at least some circumstances, standard determination of MICs and minimal bactericidal concentrations for C. trachomatis allows no more than a simple definition of whether an antibiotic has some anti chlamydial activity; however, such testing is not always sufficient to verify that the antibiotic will eliminate the organism in vivo.

Anti-Infective Agents↗
PubMed2005

Rifampin-resistant RNA polymerase mutants of Chlamydia trachomatis remain susceptible to the ansamycin rifalazil.

Stable, homotypic mutants of Chlamydia trachomatis for which MICs of rifampin and rifalazil are elevated were isolated by serial passage at sub-MIC concentrations of these compounds. An alternative approach, in which Chlamydia cells were incubated and subsequently passaged three times, all in the presence of the selective agent at concentrations above the MIC, appeared to be a more effective means of selecting for mutants. In every instance where an elevation in the MIC occurred, one or more mutations in the rpoB gene, encoding the rifampin binding site, were detected. With one exception, all rpoB mutants that contained a single mutation conferred lower levels of resistance than mutants containing multiple mutations. Some rpoB mutations conferred very high levels of resistance to rifampin, up to 512 mug/ml. In all cases, mutants remained susceptible to concentrations of rifalazil at or below 0.064 microg/ml. Thus, rifalazil, a compound that is extremely potent against Chlamydia wild-type cells (MIC of 0.00025 microg/ml), may also protect against the selection of mutants at physiologically achievable concentrations.

Chlamydia trachomatis↗
PubMed1997

Infection of Acanthamoeba castellanii by Chlamydia pneumoniae.

Chlamydia pneumoniae is an intracellular respiratory pathogen, which, similar to Legionella, might have developed mechanisms to escape the intracellular bactericidal activity of both human host cells and amoeba. We therefore investigated the intracellular growth and survival of C. pneumoniae in Acanthamoeba castellanii by using cell culture, immunofluorescence microscopy, and electron microscopy. A castellanii was incubated with purified elementary bodies of C. pneumoniae TW 183 at a concentration of 10(6) inclusion-forming units (IFU)/ml to give a ratio of approximately 1 IFU of C. pneumoniae per amoeba. Quantitative determination of chlamydial growth within A. castellanii revealed viable and infective C. pneumoniae in the range of 10(4) to 10(5) IFU/ml between days 7 and 14 postinfection. Immunofluorescence analysis and transmission electron microscopy with subsequent immunogold staining confirmed evidence of infection of the amoebae by C. Pneumoniae and additionally revealed that C. pneumoniae entered the typical growth cycle. Our results show that amoebae allow the survival of C. pneumoniae, suggesting that amoebae may serve as an additional reservoir for Chlamydia or Chlamydia-related organisms.

Acanthamoeba↗
PubMed1974

Growth of Chlamydia trachomatis in McCoy cells treated with cytochalasin B.

When the fungal metabolite cytochalasin B was added to McCoy cells, multinucleated giant cells developed. Monolayers of these cells proved as efficient as irradiated cells for the growth of three different serotypes of Chlamydia trachomatis and for the primary isolation of chlamydiae from clinical specimens obtained from patients attending a venereal disease clinic. Cytochalasin treatment of McCoy cells provides a convenient alternative to irradiation and should be of value in the isolation of chlamydiae from the eye and genital tract.

Cell Nucleus↗
PubMed1999

Immunization with a peptide corresponding to chlamydial heat shock protein 60 increases the humoral immune response in C3H mice to a peptide representing variable domain 4 of the major outer membrane protein of Chlamydia trachomatis.

C3H (H-2(k)) mice are susceptible to a vaginal challenge with human strains of Chlamydia trachomatis and thus are a useful strain for testing potential Chlamydia vaccine candidates. However, C3H mice are fairly poor responders in terms of the level of antibody resulting from immunization with potential protective peptides representing variable domains (VDs) of the major outer membrane protein (MOMP). C57BL/6 (H-2(b)) mice, on the other hand, are moderately resistant to a vaginal challenge but are good responders to the chlamydial MOMP VDs. Peptides representing universal T-cell helper epitopes were employed to determine whether the antibody response to a peptide representing VD4 of the MOMP, which has been shown to contain neutralizing epitopes, could be enhanced in C3H and C57 mice. Universal T-cell helper peptides from tetanus toxin, the pre-S2 region of hepatitis B virus, and the mouse heat shock protein 60, as well as the corresponding segment of the Chlamydia heat shock protein 60 (hspct), were coadministered with the VD4 peptide. Peptides were coencapsulated in liposomes containing the adjuvant monophosphoryl lipid A and administered by using a combination of mucosal and intramuscular injection. The only T-cell helper peptide that improved the immune response as judged by antibody level, in vitro neutralization assays, and T-cell proliferation was hspct. The response in the C57BL/6 strain was not significantly enhanced with hspct over levels achieved with VD4 alone; however, in C3H mice the levels of serum antibody to C. trachomatis increased to that seen in C57 mice. However, the molecular specificity and immunoglobulin subclass distribution differed from those of the C57 response, and the neutralizing titers and T-cell proliferation responses were lower. In both strains of mice, titers of vaginal antibody to C. trachomatis were low. In summary, of the T-helper peptides used, only hspct significantly enhanced the immune response of C3H mice to the VD4 peptide, but it had only a modest effect on the immune response of C57 mice.

Amino Acid Sequence↗
PubMed1999

Sézary T-cell activating factor is a Chlamydia pneumoniae-associated protein.

We previously identified a protein that was stimulatory for malignant Sézary T cells, termed Sézary T-cell activating factor (SAF). However, the identity of this protein has not been fully elucidated, nor has it's role been determined in the pathogenesis of cutaneous T-cell lymphoma (CTCL). The basis for epidermotropism and proliferation of malignant cells in the skin of patients with CTCL is unknown. Using a monoclonal antibody inhibitory for SAF activity, we demonstrated that SAF is present in the skin of 16 of 27 samples from patients with mycosis fungoides, the predominant form of CTCL. In this report, the SAF determinant is demonstrated to be associated with Chlamydia pneumoniae bacteria by immunohistochemistry, immunoelectron microscopy, and culture analysis. Reactivity of antibodies against an outer membrane protein of C. pneumoniae or against the lipopolysaccharide of Chlamydiae spp. demonstrated that these determinants are coexpressed in 90% of the SAF-positive samples. We confirmed the presence of C. pneumoniae DNA and RNA in the skin by PCR and reverse transcription-PCR and by sequence analysis of the PCR products. The expression of the C. pneumoniae antigens and SAF appears to be associated with active disease in that C. pneumoniae antigens were absent or greatly diminished in the skin of three patients examined after Psoralen and long-wave UVA radiation treatment. Our results suggest that SAF is a Chlamydia-associated protein and that further investigation is warranted to determine whether SAF and C. pneumoniae play a role in the pathogenesis of CTCL.

Antibodies, Monoclonal↗
PubMed2006

Differences in Chlamydia trachomatis serovar E growth rate in polarized endometrial and endocervical epithelial cells grown in three-dimensional culture.

In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells, although higher progeny counts were obtained. In conclusion, this three-dimensional bead culture system represents an improved model for harvesting large quantities of infectious chlamydia progeny from their more natural polarized epithelial host cells.

Animals↗
PubMed1978

Division of single host cells after infection with chlamydiae.

Mouse fibroblasts (L cells) were infected in suspension with Chlamydia psittaci (6BC) and then plated out on a solid substrate at a density of 80 cells per cm2 so that the effect of chlamydial infection on the division of single host cells and their progeny could be determined. Uninfected L cells multiplied with a mean generation time of 15 h. The generation time of single L cells infected with 1.5 50% infectious units (ID50) of C. psittaci was over twice as long. Half of the infected L cells had divided once by day 4 after infection, and the rest had divided more than once. Division of infected cells frequently produced one infected and one uninfected daughter. About half of the L cells infected with 15 ID50 of C. psittaci divided at least once before most of them detached from their substrate before observation on day 3. Less than 10% of the L cells infected with 75 ID50 of C. psittaci divided before they were lost from their substrate by day 2. Comparable results were obtained with single L cells infected with a lymphogranuloma venereum (440L) strain of C. trachomatis and with single HeLa 229 cells infected with C. psittaci. It was concluded that high multiplicities of infection of host cells with chlamydiae quickly bring cell division to a halt, whereas lower multiplicities slow but do not immediately stop the division of host cells. However, indefinitely multiplying clones of chlamydia-infected host cells were not observed. The method used here should be applicable to other studies on the division of cells in culture.

Cell Division↗
PubMed1978

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.

Animals↗
PubMed1980

Persistent infection of mouse fibroblasts (L cells) with Chlamydia psittaci: evidence for a cryptic chlamydial form.

When monolayers of mouse fibroblasts (L cells) were infected with enough Chlamydia psittaci (strain 6BC) to destroy most of the host cells, 1 in every 10(5) to 10(6) originally infected cells gave rise to a colony of L cells persistently infected with strain 6BC. In these populations, the density of L cells and 6BC fluctuated periodically and reciprocally as periods of host cell increase were followed by periods of parasite multiplication. Successive cycles of L-cell and 6BC reproduction were sustained indefinitely by periodic transfer to fresh medium. Isolation of L cells and 6BC from persistent infections provided no evidence that there had been any selection of variants better suited for coexistence. Persistently infected populations consisting mainly of inclusion-free L cells yielded only persistently infected clones, grew more slowly, and cloned less efficiently. They were also almost completely resistant to superinfection with high multiplicities of either 6BC or the lymphogranuloma venereum strain 440L of Chlamydia trachomatis. These properties of persistently infected L cells may be accounted for by assuming that all of the individuals in these populations are cryptically infected with 6BC and that cryptic infection slows the growth of the host cell and makes it immune to infection with exogenous chlamydiae. According to this hypothesis, the fluctuations in host and parasite density occur because some factor periodically sets off the conversion of cryptic chlamydial forms into reticulate bodies that multiply and differentiate into infectious elementary bodies in a conventional chlamydial developmental cycle.

Animals↗
PubMed1981

Factors affecting the rate as which a trachoma strain of Chlamydia trachomatis establishes persistent infections in mouse fibroblasts (McCoy cells).

Chlamydiae from mouse fibroblasts (McCoy cells) persistently infected with a trachoma strain (G17) of Chlamydia trachomatis repeatedly established new persistent infections in wild McCoy cells more rapidly than did wild chlamydiae. The density and nutritional state of the host cell populations may also influence the rate of establishment of persistent infections.

Animals↗
PubMed1982

Toxic effect of human polymorphonuclear leukocytes on Chlamydia trachomatis.

The effect of human polymorphonuclear leukocytes (PMNs) on Chlamydia trachomatis was studied. Both trachoma (B/TW-5/OT) and lymphogranuloma venereum (L2/434/Bu) biotypes were rapidly inactivated by exposure to human PMNs. A decrease of 3 to 3.5 logs in viable count was observed after 60 min of incubation at a chlamydia-to-PMN ratio of 1:10. Both chlamydial biotypes were also rapidly inactivated by the cell-free myeloperoxidase-H2O2-halide system. A decrease in infectivity titer of 4 to 5 logs for TW-5 and complete inactivation of 434 were seen after 30 min of incubation. The microbicidal effect was prevented by the deletion of each component of the system or by the addition of the peroxidase inhibitors cyanide or azide. PMNs from myeloperoxidase-deficient patients inactivated chlamydiae normally, whereas PMNs from patients with chronic granulomatous disease, although strongly chlamydicidal, were less effective than normal PMNs in the activation of TW-5 (2-log drop in viable organisms versus a 3 to 3.5-log drop). The chlamydicidal activity of PMNs from patients with chronic granulomatous disease and normal PMNs were comparable against the 434 biotype. These studies suggest that the myeloperoxidase system, or indeed oxygen-dependent antimicrobial systems, are not essential for the chlamydicidal activity of PMNs.

Animals↗
PubMed1987

The low-molecular-mass, cysteine-rich outer membrane protein of Chlamydia trachomatis possesses both biovar- and species-specific epitopes.

We isolated, by hydroxylapatite high-performance liquid chromatography, 14- and 15-kilodalton (kDa), cysteine-rich outer membrane proteins from Chlamydia trachomatis TW-5/OT (serovar B) and LGV-434 (serovar L2), respectively. Monoclonal antibodies (MAbs) were generated against the purified proteins, and their specificities were determined by immunoblotting. MAb B-14k recognized an epitope located on the 14-kDa cysteine-rich protein of the TW-5/OT strain and was immunoreactive with a comigrating 14-kDa protein that was common to all trachoma biovar strains, but it did not react with the 15-kDa, cysteine-rich protein of LGV biovar strains. In contrast, MAb L2-15k, which recognized an epitope located on the 15-kDa protein of the LGV-434 strain, reacted with the 15- and 14-kDa, cysteine-rich proteins of both LGV and trachoma biovar strains, but did not react with related proteins of two Chlamydia psittaci strains. Thus, the low-molecular-mass, cysteine-rich outer membrane proteins of C. trachomatis possess antigenic determinants that are both biovar and species specific. Neither MAbB-14k nor MAb L2-15k was reactive by dot-blot assay when viable chlamydiae were used as test antigens, indicating that the cysteine-rich proteins are not accessible to antibody on the native chlamydial cell surface.

Antibodies, Bacterial↗
PubMed1989

Cloning and characterization of a Chlamydia trachomatis L3 DNA fragment that codes for an antigenic region of the major outer membrane protein and specifically hybridizes to the C- and C-related-complex serovars.

Chlamydia trachomatis L3 DNA was cloned and expressed in lambda gt11. A recombinant plaque that expressed an antigen that reacted with rabbit polyclonal antichlamydial L3 serum and with two monoclonal antibodies specific for serovars L3 and I was selected from this Chlamydia genomic library. The beta-galactosidase Chlamydia fusion protein was purified by immunoaffinity chromatography and injected into mice to produce monoclonal antibodies. These monoclonal antibodies reacted by Western (immuno-) blot with both the fusion protein and the major outer membrane protein from purified L3 elementary bodies. The chlamydial DNA fragment was shown by DNA sequence analysis to be 168 base pairs in length and to correspond to the constant regions 1 and 2 and the variable segment 1 of the major outer membrane protein gene. The recombinant chlamydial DNA fragment hybridized under stringent conditions by Southern and dot blot analysis exclusively with the DNA from the C- and C-related-complex C. trachomatis serovars.

Amino Acid Sequence↗
PubMed1989

Characterization of gamma interferon-mediated cytotoxicity to chlamydia-infected fibroblasts.

Addition of murine recombinant gamma interferon (IFN-gamma) to mouse fibroblast cultures infected with Chlamydia psittaci was found to induce a cytotoxic response that was dependent on the concentration of IFN-gamma added and the multiplicity of infection given. No cytotoxicity was observed for uninfected cells treated with IFN-gamma, nor did infection alone elicit cytotoxicity. Cytotoxicity was detected only if IFN-gamma was present for at least the first 18 h of a 30-h incubation period. Cytotoxic activity was not observed when infected cells were treated with 50 micrograms of chloramphenicol per ml, a drug which inhibits differentiation of infectious elementary bodies to noninfectious reticulate bodies. Cytotoxic activity was restored if addition of chloramphenicol was delayed until 18 h postinfection. Addition of 100 U of penicillin per ml to infected host cells reduced but did not abolish cytotoxic activity. Treatment of host cells with as little as 0.2 microgram of cycloheximide per ml inhibited cytotoxicity without interfering with chlamydial growth. When addition of cycloheximide was delayed until 12 h after infection and IFN-gamma treatment, cytotoxicity was restored. These data indicate that IFN-gamma functions as a cytotoxic cytokine against chlamydia-infected fibroblasts. Cytotoxicity was found to be dependent on chlamydial multiplicity of infection, differentiation of chlamydiae to the metabolically active form, and host cell protein synthesis.

Animals↗
PubMed1989

Differential modulation of lymphocyte proliferative responses and lymphokine secretion in mice during development of immunity to Chlamydia psittaci.

A murine model was utilized to study immune responses occurring during the period of acquisition of immunity to chlamydial infection. C3H (H-2k) mice were immunized by intramuscular injection of 5 x 10(3) viable Chlamydia psittaci elementary bodies (EBs) by a protocol which permits animals to survive an otherwise lethal intraperitoneal challenge 10 days later with the homologous chlamydial strain. Spleen cells assayed during the 10-day period of development to immunity showed depressed proliferative responses in vitro to the T-cell mitogen, concanavalin A, and also exhibited suppressor cell activity. Spleen cell mitogen responses returned to normal levels by 30 days postimmunization, concomitant with the detectable development in vitro of responses to chlamydia-specific antigen. In marked contrast to the reduced proliferative responses, mitogen-stimulated production of the T-cell-derived lymphokines interleukin-2 and gamma interferon by spleen cells from immunized animals was within the normal range at 10 days postimmunization, and supernatant fluids containing these products had both microbicidal and microbistatic effects on chlamydial organisms in vitro. These results demonstrate that independent regulation of T-cell proliferation and lymphokine production occurs in vivo as part of the development of an antigen-specific protective immune response. These results also suggest that such differential modulation of T-cell responses may contribute to the development of protective immunity to chlamydiae in mice, perhaps through limited T-cell clonal expansion coupled with early or preferential maturation of cytokine-secreting helper T cells.

Animals↗
PubMed1989

Characterization of native and recombinant 75-kilodalton immunogens from Chlamydia trachomatis serovar L2.

A 75-kilodalton (kDa) immunogen from Chlamydia trachomatis serovar L2 was characterized. The 75-kDa protein was localized in the cytoplasm of chlamydiae and was shown to be a protein synthesized early in the developmental cycle of chlamydiae. A gene library was made by the recombinant DNA technique, using the expression vectors pEX1, pEX2, and pEX3. From this library one clone was found which reacted with a monoclonal antibody against the 75-kDa immunogen of C. trachomatis. The 75-kDa protein produced by the recombinant Escherichia coli was expressed independently of the promoter for the hybrid protein cro-betagalactosidase. Thus it is not produced as a fusion protein. Epitope mapping of the 75-kDa protein from C. trachomatis L2 and from the recombinant E. coli performed by Staphylococcus aureus V8 protease digestion showed that the two proteins are identical. Furthermore, patient sera reacted with both proteins.

Animals↗