Identification and evaluation of a combination of chlamydial antigens to support the diagnosis of severe and invasive Chlamydia trachomatis infections.

Chlamydia trachomatis is the most common sexually transmitted organism in industrialized countries. Nucleic acid amplification testing, using non-invasively collected specimens, is considered to be the method of choice for diagnosis of chlamydial infections of the urethra and the lower genital tract. Serological testing has the potential to circumvent the problem of specimen sampling in invasive C. trachomatis infections of the upper genital tract. However, only a few defined chlamydial antigens have been used in a standardized diagnostic assay format. In this study, we used serological two-dimensional proteomic analysis to broaden the spectrum of diagnostically relevant C. trachomatis proteins. The genes encoding an assortment of already known chlamydial antigens, as well as immunogenic proteins that have not been described before, were cloned, and the recombinant proteins were purified in order to compare their diagnostic usefulness in parallel with a newly developed line immunoassay. With 189 sera collected from patients with and without C. trachomatis infection, recombinant major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), outer membrane protein 2 (OMP2), translocated actin-recruiting protein, and polymorphic membrane protein D (PmpD) showed the highest level of diagnostic sensitivity and specificity. In patients suffering from ascending and invasive C. trachomatis infections, such as pelvic inflammatory disease and lymphogranuloma venereum, the sensitivity reached with these proteins ranged between 71% (PmpD) and 94% (OMP2), and the specificity ranged between 82% (PmpD) and 100% (MOMP and OMP2). Recombinant thio-specific antioxidant peroxidase, ribosomal protein S1 (RpsA) and hypothetical protein 17 showed lower sensitivity but comparably high specificity, ranging from 94% to 100%. The novel line immunoassay based on defined recombinant antigens has promise for improved serodiagnosis in severe and invasive C. trachomatis infections.

Evaluation of WLBU2 peptide and 3-O-octyl-sn-glycerol lipid as active ingredients for a topical microbicide formulation targeting Chlamydia trachomatis.

Topical microbicides for prevention of sexually transmitted diseases (STDs) would be especially useful for women who are not able to persuade their partner(s) to take precautions. Many topical microbicides are in various stages of development, based on a variety of active ingredients. We investigated the in vitro activity of an engineered antimicrobial peptide (WLBU2) and a lipid (3-O-octyl-sn-glycerol [3-OG]) which could potentially be used as active ingredients in such a product. Using commercially available cytotoxicity reagents [Alamar Blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH)], we first determined the toxicity of WLBU2 and 3-OG to the host cells in our assay procedure and excluded toxic concentrations from further testing. To determine activity against Chlamydia trachomatis, we used an assay previously developed by our laboratory in which chlamydial elementary bodies (EBs) were exposed to microbicides prior to contact with epithelial cells: the minimum (microbi)cidal concentration (MCC) assay. To further simulate conditions of transmission, we carried out the same assay in the presence of a simulated vaginal fluid, a simulated seminal fluid, human serum albumin, and a range of pH values which might be found in the human vagina at the time of exposure. Last, we tested WLBU2 and 3-OG in combination to determine if adding them together resulted in synergistic activity. We found that WLBU2 and 3-OG both have excellent activity in vitro against C. trachomatis and significantly more activity when added together. The simulated fluids reduced activity, but the synergy seen is good evidence that they would be effective when combined in a microbicide formulation.

Susceptibility of Chlamydia trachomatis to excipients commonly used in topical microbicide formulations.

Commonly used "inactive" pharmaceutical excipients were tested in a previously developed minimum cidal concentration assay to assess their ability to kill Chlamydia trachomatis topically. Sixteen excipients were evaluated in these studies under various conditions. A range of activities was found among the excipients that could be tested in our assay system.

In vitro microbicidal activities of cecropin peptides D2A21 and D4E1 and gel formulations containing 0.1 to 2% D2A21 against Chlamydia trachomatis.

Topically applied microbicides that eradicate pathogens at the time of initial exposure represent a powerful strategy for the prevention of sexually transmitted infections. To aid in the further development of an effective topical microbicide, we assessed the minimum cidal concentration (MCC) of two cecropin peptides, D2A21 and D4E1, and gel formulations containing 0.1 to 2% D2A21 against Chlamydia trachomatis in vitro. The MCC of peptide D2A21 was 5 microM (18.32 microg/ml), and that of peptide D4E1 was 7.5 microM (21.69 microg/ml). The MCC of gel formulations containing 2% D2A21 was 0.2 mM (0.7 mg/ml), and that of gel formulations containing 0.5% D2A21 was 0.2 mM (0.7 mg/ml). There was no significant variation in the results when two different C. trachomatis strains were tested, and the addition of 10% human blood did not significantly alter the MCCs. pH values above and below 7 reduced the activity of the D2A21 peptide alone, but the MCC of the 2% D2A21 gel formulation was only slightly altered at the various pHs tested. Ultrastructural studies indicated that C. trachomatis membranes were disrupted after D2A21 exposure, resulting in leakage of the cytoplasmic contents. These in vitro results suggest that these cecropin peptides may be an effective topical microbicide against C. trachomatis and support the need for further evaluation.

CD8+ T cells recognize an inclusion membrane-associated protein from the vacuolar pathogen Chlamydia trachomatis.

During infection with Chlamydia trachomatis, CD8(+) T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8(+) T cells recognize antigens processed from cytosolic proteins, the Chlamydia antigens recognized by these CD8(+) T cells very likely have access to the host cell cytoplasm during infection. The identity of these C. trachomatis proteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein from C. trachomatis recognized by protective CD8(+) T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the human C. trachomatis serovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against all C. trachomatis serovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction of C. trachomatis with its host cell.

Gamma interferon production by cytotoxic T lymphocytes is required for resolution of Chlamydia trachomatis infection.

In this study, we used mice in which the gene for gamma interferon (IFN-gamma) has been disrupted (IFN-gamma-/- mice) to study the role of this cytokine in the resolution of Chlamydia trachomatis infection. We show that IFN-gamma-/- mice are impaired in the ability to clear infection with C. trachomatis compared to IFN-gamma+/+ control mice. Activated CD8(+) cytotoxic T lymphocytes (CTL) secrete IFN-gamma in response to intracellular infection, and we have shown previously that a Chlamydia-specific CTL line can reduce C. trachomatis infection when adoptively transferred into infected mice. In the present study, we found that when these IFN-gamma+/+ CTL lines are transferred into Chlamydia-infected IFN-gamma-/- mice, the transferred CTL cannot overcome the immune defect seen in the IFN-gamma-/- mice. We also show that Chlamydia-specific CTL can be cultured from IFN-gamma-deficient mice infected with C. trachomatis; however, the adoptive transfer of IFN-gamma-/- CTL into infected IFN-gamma+/+ mice does not reduce the level of infection. These results suggest that IFN-gamma production by CTL is not sufficient to overcome the defect that IFN-gamma-/- mice have in the resolution of Chlamydia infection, yet IFN-gamma production by CTL is required for the protective effect seen upon adoptive transfer of CTL into IFN-gamma+/+ mice.

Susceptibility of Chlamydia trachomatis to chlorhexidine gluconate gel.

To identify topical antimicrobial preparations which may be effective in preventing the transmission of sexually transmitted diseases, we examined the activity of chlorhexidine gluconate (CHG) against Chlamydia trachomatis. Chlamydial elementary bodies were incubated with dilutions of CHG gel for various times from 0 to 120 min. An aliquot of each dilution was further diluted and was inoculated onto McCoy cell monolayers in individual wells in a 96-well microtiter plate. The cultures were incubated for 48 h, and the chlamydial inclusions were stained and counted. CHG gel diluted fourfold (0.0625% CHG) killed C. trachomatis serovar D, and CHG gel diluted eightfold (0.0313% CHG) killed serovar F immediately upon exposure. CHG gel diluted 16-fold (0.0156% CHG) killed serovar D, and CHG gel diluted 32-fold (0.0078% CHG) killed serovar F after 120 min of exposure. Alteration of the pH over the range of from 4 to 8 did not significantly affect its activity. The addition of 10% whole human blood decreased the CHG gel activity at 0 min but had no significant effect after 120 min of exposure. We conclude that CHG gel may be effective topically against C. trachomatis at concentrations that can be used and under conditions that are found in the female genital tract and that further studies of its antimicrobial efficacy and toxicity in vivo are warranted.

Killing of Chlamydia trachomatis by novel antimicrobial lipids adapted from compounds in human breast milk.

The development of new methods for prevention of sexually transmitted Chlamydia trachomatis infection is a top public health priority. Topical self-administered vaginal microbicides represent one such approach in which the organism is eradicated at the time of initial exposure. To this end, we examined the activity of five synthetic lipids adapted from naturally occurring compounds found in human breast milk. C. trachomatis serovar D or F elementary bodies were added to serial dilutions of the lipids and incubated for various times. Aliquots were then cultured in monolayers of McCoy cells, and inclusions were counted. A 7.5 mM concentration of 2-O-octyl-sn-glycerol completely prevented growth of C. trachomatis after 120 min of contact with the organism. The remaining lipids, 1-O-octyl-, 1-O-heptyl-, 2-O-hexyl-, and 1-O-hexyl-sn-glycerol, showed less activity. On electron microscopic examination, the lipids were shown to have disrupted the chlamydial inner membrane, allowing leakage of the cytoplasmic contents from the cell. Lipid activity was unaffected by the presence of 10% human blood or alterations in pH from 4.0 to 8.0, conditions reflecting those sometimes found in the vagina. Our results suggest that these lipids, especially 2-O-octyl-sn-glycerol, may be effective as topical microbicides in preventing the transmission of C. trachomatis. Further efficacy and toxicity studies with these lipids and assessment of their activity against other sexually transmitted disease pathogens are in progress.

Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera.

We have identified two families of novel Chlamydia trachomatis isolates with amino acid changes within the major outer membrane protein (MOMP) variable domains: one family of Da, D*, and D- and one family of Ia and I-. In order to determine whether these MOMP variants can escape antibody neutralization of infectivity, we tested both the D and I prototype strains and the variants in a complement-independent in vitro neutralization assay. We found that variants can indeed escape neutralization by both monoclonal antibodies and polyclonal human immune sera that neutralize the prototype strain.

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.

Sequence conservation in the major outer membrane protein gene among Chlamydia trachomatis strains isolated from the upper and lower urogenital tract.

To determine the extent of nucleotide sequence variation in the major outer membrane protein (MOMP) gene among 27 clinical isolates of Chlamydia trachomatis, the MOMP gene was amplified from 13 strains isolated from the endometrium of patients with pelvic inflammatory disease and high titers of anti-chlamydial antibodies and from 14 strains isolated from the cervix of patients with presumed first-time chlamydial infection. Amplified MOMP variable domain DNA from these isolates was directly sequenced and compared with previously published results. Very little sequence heterogeneity in the MOMP variable domains was found in all 27 clinical isolates, suggesting that MOMP sequence heterogeneity is not often associated with the spread of C. trachomatis to the upper genital tract and is not common in the chlamydial strains in the patient population studied.

Infection with Chlamydia trachomatis lymphogranuloma venereum serovar L1 in homosexual men with proctitis: molecular analysis of an unusual case cluster.

Among 767 rectal isolates of Chlamydia trachomatis obtained from men over the period 1981-1991, 7 were found to be a rare lymphogranuloma venereum serovar, L1. These isolates were from rectal specimens taken from five male patients in 1982-1983. Six of the seven isolates were available for DNA sequencing studies. All six of these isolates shared the same DNA sequence in the major outer membrane protein (MOMP) gene variable domains and had different MOMP sequences than did the prototype L1, L2, and L3 strains, suggesting a point source outbreak. All five patients infected with serovar L-1 were homosexual men who had symptomatic proctitis characterized by rectal pain, discharge, tenesmus, abnormalities seen on anoscopy, and leukocytes seen on gram stains of rectal specimens. We conclude that an unrecognized point source outbreak of the rare chlamydial L1 serovar occurred among homosexual men in Seattle in 1982-1983, and that the clinical manifestations of L1 infection may be less severe than those of L2 infections.

Protective cytotoxic T lymphocytes are induced during murine infection with Chlamydia trachomatis.

T cell responses are often an important component in immunity to organisms that replicate intracellularly. Cytotoxic T lymphocyte (CTL) recognition of peptide Ag in the context of MHC class I molecules results in lysis of infected cells and the release of cytokines including IFN-gamma. Members of the genus Chlamydia are obligate intracellular pathogens that cause blindness and sexually transmitted disease worldwide. Even though it replicates within a membrane-bound vacuole, Chlamydia trachomatis may elicit a CTL response if Chlamydia Ags are present in the cytoplasmic compartment where they can be processed for presentation and bound by MHC class I. In this study, we characterized a CTL line derived from mice infected with C. trachomatis. This CTL line is specific for, and able to lyse, Chlamydia-infected cells. The peptide epitope recognized by this CTL line is present on infected cells, and is presented to the CTL by the classical MHC class I molecule H-2 Ld. Adoptive transfer of this CTL line into an infected mouse affords protection, and this protection requires the activity of IFN-gamma.

Purification of Chlamydia trachomatis strains in mixed infection by monoclonal antibody neutralization.

A Chlamydia trachomatis D* serovariant strain was found to be mixed with an F serovar strain in a clinical specimen. By using a monoclonal antibody which neutralizes serovar F infectivity in hamster kidney cells, the D* variant strain was enriched until it could be cloned by limiting dilution. This newly described neutralization enrichment procedure can be used to purify a C. trachomatis serovar present in small numbers in a mixed culture or, potentially, to identify nonneutralizable mutants.

Long-term eradication of Chlamydia trachomatis genital infection after antimicrobial therapy. Evidence against persistent infection.

OBJECTIVE: To determine whether Chlamydia trachomatis urogenital infections persist or relapse after antimicrobial therapy by serial measurement of chlamydial-specific DNA using the polymerase chain reaction (PCR), cell cultures, and serological studies. DESIGN: Prospective evaluation of an inception cohort. SETTING: University student health clinic. PARTICIPANTS: Twenty women with culture-proven and PCR-proven C trachomatis urogenital infections. MEASUREMENTS: Incidence of persistent infection as determined by PCR, culture, and serial measurement of local and systemic antibody to C trachomatis for 5 months after doxycycline therapy. RESULTS: Prior to therapy, C trachomatis was isolated in cell culture from the cervix in 19 of 20 women, from the urethra in 13 women, and from the rectum in 13 women. All culture-positive specimens were also PCR-positive. Immediately after completion of antimicrobial therapy, all women had negative cell cultures for chlamydia. Ten of 20 culture-negative cervical specimens and two culture-negative urethral specimens had chlamydial DNA present immediately after treatment. In addition, three women had detectable DNA from cervical specimens 1 week after treatment. The presence of cervicitis (P = .01), high inclusion counts (P = .004), and serological evidence of recent infection (P = .0004) were each significantly associated with PCR positivity after treatment. All 384 subsequent cervical, rectal, and urethral specimens collected over 5 months were negative by both PCR and culture with the exception of one woman who was reinfected. Serum immunoglobulin M (IgM) titers, geometric mean serum immunoglobulin G (IgG) titers, and prevalence of local antibody to chlamydia progressively declined after treatment. CONCLUSIONS: Standard antimicrobial therapy is effective in the long-term microbiologic eradication of uncomplicated C trachomatis urogenital infections. The presence of chlamydial DNA after antimicrobial therapy is of short duration and reflects excretion of nonviable organisms rather than persistent infection.

Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis.

During studies in which we serotyped large numbers of Chlamydia trachomatis clinical isolates by using monoclonal antibodies, three novel serological variants (D-, D*, and I-) were identified. To determine the molecular basis for the altered monoclonal antibody reactions of these strains and other previously identified variants (Da, Ia, and L2a), we determined the nucleotide sequences of the variable domains in their major outer membrane protein genes. Da, D-, and D* variants differed by a single nucleotide and also an amino acid in the carboxy terminus of variable domain IV (VDIV) from the D serovar. The L2a variant also differed from L2 by a single amino acid but in VDII. Ia variants differed in VDI, III, and IV and I- variants differed in all four VDs from the I serovar. These studies demonstrate the potential for using major outer membrane protein VD sequencing as a highly sensitive typing method and further identify immunologically reactive major outer membrane protein epitopes.

Genetic and physical organization of the cloned gyrA and gyrB genes of Bacillus subtilis.

An 8-kilobase fragment already known to contain the gyrA gene of Bacillus subtilis was shown to encode the gyrB gene as well. Plasmids containing this fragment can rescue both B. subtilis gyrA and gyrB mutants and complement Escherichia coli gyrA mutants. Deletion analysis has indicated the gene locations on the cloned fragment. Under low-stringency conditions the cloned E. coli gyrA and gyrB genes each hybridized to the appropriate subfragments, confirming the assignment of the gene locations on the cloned DNA. In E. coli maxicells, proteins of 67,000 (gyrA) and 77,000 (gyrB) Mr were synthesized. Analysis of proteins encoded by various subfragments indicated the direction of transcription. Although the gyrA and gyrB genes are located adjacent to each other on the chromosome, they may be transcribed independently since expression of gyrA protein is not dependent upon the gyrB gene in maxicells.

Cloning of the Bacillus subtilis recF gene.

A cloned DNA fragment from the ori region of the Bacillus subtilis chromosome permits three separate recF mutants to grow in the presence of mitomycin C (MC) and survive after ultraviolet (UV) exposure. The recF gene has been localized to a 2.3-kb EcoRI-SalI restriction fragment on the cloned sequences. This fragment directs expression of a 34.7-kDal protein in Escherichia coli maxicells. Cloned DNA containing the recF gene does not complement or rescue recL, recM or spoOJ B. subtilis mutants. The B. subtilis recF gene also does not complement any of several of the E. coli recombination-deficient (Rec-) mutants tested.

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.

Mutational enzymatic resistance of Enterobacter species to beta-lactam antibiotics.

Mutants with enhanced beta-lactam resistance were selected from strains of Enterobacter cloacae and E. aerogenes by using three antibiotics. High-level beta-lactamase-producing mutants had similar degrees of increased resistance, enzyme substrate profiles, and isoelectric (pI) values irrespective of the selective agent. Reverse mutants from a resistant E. cloacae mutant regained the susceptibility pattern originally exhibited by the wild type, or were of enhanced susceptibility, and no longer expressed increased beta-lactamase production. beta-Lactamases of the mutants were similar in pI values to the wild-type enzyme. The increased resistance of the mutants therefore appeared to be accounted for by increased beta-lactamase production.