Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation

To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype.     

Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.     

A rapid method for staining inclusions of Chlamydia psittaci and Chlamydia trachomatis.

A new staining method was developed for the detection of inclusions of Chlamydia psittaci and Chlamydia trachomatis inclusions in cell cultures. Using a combination of methyl green and neutral red stains and washing at pH 5.0, inclusions were stained red while cell cytoplasm was pale pink and cell nuclei were pale green. The method was significantly better than Giemsa staining and comparable to immunofluorescence for detecting C psittaci inclusions. Its sensitivity for detection C trachomatis inclusions by dark field microscopy was similar to that of Giemsa staining.     

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.     

Detection of Chlamydia trachomatis inclusions in Mccoy cell cultures with fluorescein-conjugated monoclonal antibodies.

We compared two methods for identification of Chlamydia trachomatis inclusions in McCoy cell monolayers: conventional iodine staining and immunofluorescence staining with monoclonal antibodies against the species-specific major outer membrane protein antigen of C. trachomatis. Among 878 urethral and cervical specimens tested in parallel, the immunofluorescence method detected eightfold more inclusions per monolayer, identified a higher proportion of positive specimens on first passage (98 versus 62% by iodine staining; P less than 0.01), and improved overall sensitivity (98% of total positive specimens detected versus 84% by iodine staining; P less than 0.01). Improved sensitivity was most evident in specimens with low numbers of inclusions. Compared with conventional iodine staining, immunofluorescence staining with monoclonal antibodies improves sensitivity and offers more rapid detection of chlamydial inclusions in cell culture.     

Indirect immunofluorescence staining of Chlamydia trachomatis inclusions in microculture plates with monoclonal antibodies.

Indirect immunofluorescence (IF) staining, using a monoclonal antibody, detected two- to fourfold more inclusions than did iodine staining. Of 274 clinical specimens, 53 (19.3%) were positive by IF on passage 1 as compared with 33 (12%) by iodine staining (P less than 0.005). IF-stained inclusions in McCoy cells in the bottom of microculture wells were readily viewed with a long-focal-length objective at a magnification of 250 X.     

Seroreactivity to Chlamydia trachomatis Hsp10 correlates with severity of human genital tract disease

We have identified the chlamydial heat shock protein Hsp10 as a potential correlate to the immunopathogenic process in women with tubal factor infertility (TFI). The human serologic response to chlamydial Hsp10, Hsp60, and major outer membrane protein (MOMP) was measured by enzyme-linked immunosorbent assay. Three populations of women were studied: uninfected controls (CU), acutely infected (AI) women, and women with TFI. Sera from women in the AI and TFI groups both recognized Hsp10 more frequently and at a higher overall level than sera from healthy uninfected controls. Moreover, the infertile women had significantly greater Hsp10 seroreactivity than acutely infected women, indicating a concomitant increase of Hsp10 recognition in populations with increasing levels of disease severity. Hsp60 reactivity showed a similar correlation in these populations, while MOMP reactivity peaked at the same level in both AI and TFI populations but did not increase with disease severity. Test populations were standardized by level of reactivity to formalin-fixed Chlamydia trachomatis elementary bodies (EBs) to address whether these associations were reflections of increased overall chlamydial exposure rather than a property specific to Hsp10. Associations between Hsp10 seropositivity and TFI were greater in the EB(+) subgroup while associations among the EB(-) subgroup were diminished. When restricted to the EB(+) subgroups, Hsp60 and MOMP responses in the TFI population did not increase significantly over the level of AI group responses. Thus, among women with similar exposure to chlamydiae, the serologic response to Hsp10 exhibited a stronger correlation with TFI than did the response to Hsp60 or MOMP. These findings support the hypothesis that the serological response to C. trachomatis heat shock proteins is associated with the severity of disease and identifies Hsp10 as an antigen recognized by a significant proportion of women with TFI.     

Sensitivity of immunofluorescence with monoclonal antibodies for detection of Chlamydia trachomatis inclusions in cell culture.

Monoclonal antibodies which recognize the species-specific major outer membrane protein antigen of Chlamydia trachomatis were used for immunofluorescence staining of chlamydial inclusions in cell culture. A total of 115 clinical specimens were inoculated onto replicate HeLa 229 cell monolayers and assayed for chlamydial inclusions by immunofluorescence staining and Giemsa staining. Of the isolates, 38 were detected by immunofluorescence staining on passage 1 and 1 was detected on passage 2; 23 isolates on passage 1 and 13 isolates on passage 2 were detected by Giemsa staining. Immunofluorescence staining was significantly more sensitive than Giemsa staining for detecting chlamydial inclusions, particularly from specimens containing low titers of Chlamydia.     

Inhibition of growth of Chlamydia trachomatis by human gamma interferon.

Treatment of HEp-2 cell cultures with highly purified human gamma interferon before infection resulted in the reduction of Chlamydia trachomatis (L2/434/Bu) infectious particle yield. Electron microscope studies showed that interferon did not affect chlamydial conversion to reticulate bodies but influenced the extent of maturation to elementary bodies. High interferon concentrations (greater than 350 IU/ml) inhibited inclusion body formation and resulted in the appearance of aberrant reticulate bodies.     

Inhibition of Chlamydia trachomatis growth by recombinant tumor necrosis factor.

Purified human recombinant tumor necrosis factor (rTNF) alpha inhibited the growth of Chlamydia trachomatis (L2/434/Bu) in HEp-2 cell cultures. The inhibition of C. trachomatis yield could be achieved even when the rTNF alpha (200 ng/ml) was added up to 12 h after infection. The effect of rTNF alpha on chlamydial infection was synergistic with that of gamma interferon.     

The Rab6 effector Bicaudal D1 associates with Chlamydia trachomatis inclusions in a biovar-specific manner

Chlamydia species are obligate intracellular bacteria that replicate within a membrane-bound vacuole, the inclusion, which is trafficked to the peri-Golgi region by processes that are dependent on early chlamydial gene expression. Although neither the host nor the chlamydial proteins that regulate the intracellular trafficking have been clearly defined, several enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, including Rab6, are recruited to Chlamydia trachomatis inclusions. To further characterize the association of Rab6 with C. trachomatis inclusions, we examined the intracellular localization of guanine nucleotide-binding mutants of Rab6 and demonstrated that only active GTP-bound and not inactive GDP-bound EGFP-Rab6 mutants were recruited to the inclusion, suggesting that EGFP-Rab6 interacts with the inclusion via a host Rab6 effector or a chlamydial protein that mimics a Rab6 effector. Using EGFP-tagged fusion proteins, we also demonstrated that the Rab6 effector Bicaudal D1 (BICD1) localized to C. trachomatis inclusions in a biovar-specific manner. In addition, we demonstrated that EGFP-Rab6 and its effector EGFP-BICD1 are recruited to the inclusion in a microtubule- and Golgi apparatus-independent but chlamydial gene expression-dependent mechanism. Finally, in contrast to the Rab6-dependent Golgi apparatus localization of endogenous BICD1, EGFP-BICD1 was recruited to the inclusion by a Rab6-independent mechanism. Collectively, these data demonstrate that neither Rab6 nor BICD1 is trafficked to the inclusion via a Golgi apparatus-localized intermediate, suggesting that each protein is trafficked to the C. trachomatis serovar L2 inclusion by a unique, but as-yet-undefined, mechanism.     

Serotyping of Chlamydia trachomatis by indirect fluorescent-antibody staining of inclusions in cell culture with monoclonal antibodies.

A new method for determining the serovars of Chlamydia trachomatis isolates by utilizing fluorescent-antibody staining of inclusions in cell culture is described. Monoclonal antibodies which have been successfully used previously for serotyping in the microimmunofluorescence test were employed. The cell culture method offers two advantages over the microimmunofluorescence test for many laboratories. It requires less antigen of the new isolate, about 10% cell culture infectivity versus 50% for the microimmunofluorescence test. Although fewer isolates can be typed at one time in cell culture, the technical requirements of the test are less rigorous.     

Fusion of inclusions following superinfection of HeLa cells by two serovars of Chlamydia trachomatis.

We used a double-label immunofluorescence assay to examine the ability of Chlamydia trachomatis serovar F to infect and develop within HeLa 229 cells previously infected with serovar E. No exclusion to superinfection occurred for up to 24 h following infection by serovar E. The percentage of HeLa cells infected in cultures inoculated with both strains was identical to that of cells in cultures inoculated with one strain as a control. Organisms of both serovars were located within the same intracellular inclusion in 88 to 95% of HeLa cells infected with both serovars. The proportion of superinfected HeLa cells containing both strains in separate inclusions increased when there was exposure to inhibitors of cytoskeletal structure and transport. We used this inhibition to demonstrate that fusion of C. trachomatis phagosomes occurs throughout the developmental cycle.     

Inhibition of PCR in Genital and Urine Specimens Submitted for Chlamydia trachomatis Testing

We determined the frequency of PCR inhibition in genital and urine specimens submitted for Chlamydia trachomatis testing using the internal control DNA provided with the COBAS AMPLICOR C. trachomatis test and assessed methods to remove it. Inhibition occurred in 65 of 906 (7%) cervical swabs, 23 of 51 (45%) urethral swabs, and 2 of 175 (1.1%) urine samples. Overall, inhibition was eliminated in processed specimens after storage at 4°C in 77 of 90 specimens (86%), freezing at −70°C in 59 of 82 specimens (72%), storage at 4°C followed by either 1:100 dilution in 37 of 43 specimens (86%) or 1:10 dilution in 42 of 47 specimens (89%), and phenol-chloroform extraction in 79 of 80 specimens (99%). No positive specimens were missed due to inhibition. We conclude that PCR inhibition is rare with urine specimens and infrequent with endocervical swabs but occurs frequently with urethral swabs. The frequency of PCR inhibition may be significantly reduced by methods which can be easily incorporated into the processing of specimens.     

Influence of iron restriction on Chlamydia pneumoniae and C. trachomatis

Iron is an essential metabolite for pathogenic bacteria, and the specificity exhibited by bacteria for host-iron chelates may be correlated with host and tissue tropism. The effect of iron restriction on Chlamydia pneumoniae and C. trachomatis was studied by use of the iron-chelating compound deferoxamine. Growth of C. pneumoniae was inhibited much more than that of C. trachomatis and the effect of iron restriction largely depended on the cell line used for propagation. This might reflect differences in tissue tropism of the two chlamydial species. As iron levels are usually higher in men than in women, this might also be connected with the higher prevalence rate of C. pneumoniae antibodies in males, observed in all populations studied so far.     

Detection of Chlamydia trachomatis with fluorescent monoclonal antibody

Commercial kits of fluorescein-labelled monoclonal antibody against Chlamydia trachomatis have been evaluated (1) as an alternative to Giemsa staining for detection of chlamydia inclusions in cell culture and (2) for direct detection of chlamydia in conjunctival, urethral and cervical smears. The inclusion-detection kit (Micro Trak Culture Confirmation Test) was tested on 270 cultures and was found to be highly sensitive, detecting all 16 Giemsa-positive specimens plus an additional 3 that were negative by Giemsa. It was also superior to Giemsa-staining in terms of simplicity of use, ease of detection, and readability with toxic specimens. The direct detection kit (Micro Trak Direct Specimen Test) gave results which agreed with the culture result for all 33 genital tract specimens and for 32 of 34 conjunctival specimens tested in parallel. The kit is considered to be a valuable test for diagnosis of chlamydial infection by laboratories lacking adequate tissue-culture facilities.     

Incidence of Chlamydia trachomatis in patients with sterility

Eighty six women were studied at the Hospital Chiquinquirá in order to determine the role of Chlamydia Trachomatis in infertility. Endocervical samples were taken in 45 infertile women (IG) who concurred to our Gynecologic Endocrinology Clinic and in 41 (CG) who sought medical attention at our General Gynecology Clinic for different reasons. The Chlamydiazyme Test (CT) was performed in all of them. We obtained 12 positive Chlamydiazymes (13.95%), four of them were in the IG (8.89%) and 8 (19.5%) in the CG; this was not statistically significant (p = 0.3). We also found no correlations when positive tests were correlated with sterility (p = 0.5, tube pathology history (p = 0.2), HSG (p = 0.2) and laparoscopic findings (p = 0.1). We did not find an increase in the occurrence of CT in our infertile women as reported by other authors therefore its significance in our study group at this time is uncertain.     

Inhibition of phagolysosome fusion is localized to Chlamydia psittaci-laden vacuoles.

Intracellular survival of Chlamydia psittaci is in part dependent on the ability of the organism to thwart phagolysosome formation. Circumvention of phagolysosome fusion could be either localized to chlamydia-laden vacuoles or generalized to all phagosomes in the host cell. To determine which of these modes is in operation the ability of chlamydia elementary and reticulate bodies to protect Saccharomyces cerevisiae from degradation in macrophage phagolysosomes was examined via acridine orange and Giemsa staining. No statistically significant difference was evident between the amount of fusion observed in coinfected macrophages and those infected with yeast cells alone. This was ot dependent on some unique interaction between the chlamydia and the yeast cells since viable count studies to determine the protection of a second organism, Escherichia coli, also failed to show significantly different amounts of inactivation of the bacteria by macrophages in the presence of C. psittaci. Therefore, the inhibition of phagolysosome fusion is localized to chlamydia-laden phagosomes.