The entry of Chlamydia trachomatis into McCoy cells (fibroblasts) was studied by transmission electron microscopy. On adsorption of elementary bodies (EBs) to host cells at 37°C, the EBs were bound primarily to preexisting cell-surface microvilli. They were also observed in coated pits located at the bases of the microvilli and along smooth surfaces of the host cells and were internalized within coated vesicles at this temperature. Postembedding immunogold labeling on Lowicryl thin sections with anti-clathrin antibody as the primary reagent revealed the gold marker localized in pits and vesicles containing chlamydiae. Some EBs were present in smooth-surfaced invaginations at or near the bases of microvilli and in vesicles devoid of distinguishable coat material. A similar entry process was observed with centrifugation-assisted inoculation of EBs onto the McCoy cells. Individual EBs were initially internalized into tightly bound endocytic vesicles. However, within 1 to 3 h postinfection, multiple C. trachomatis EBs were observed in large, loosely bound vesicles. Evidence suggests that vesicles containing C. trachomatis may have fused with one another early in the infectious process. These results indicate that chlamydiae can exploit the specific process of adsorptive endocytosis for entry into host cells and for translocation to a given intracellular destination, which may be different for each species.

To study the initial invasion process(es) of genital chlamydiae, a model system consisting of hormonally maintained primary cultures of human endometrial gland epithelial cells (HEGEC), grown in a polarized orientation on collagen-coated filters, was utilized. After Chlamydia trachomatis inoculation of the apical surface of polarized HEGEC, chlamydiae were readily visualized, by transmission electron microscopy, in coated pits and coated vesicles. This was true for HEGEC maintained in physiologic concentrations of estrogen (proliferative phase) and of estrogen plus progesterone (secretory phase), despite the finding that association of chlamydiae with secretory-phase HEGEC is significantly reduced (P = 0.025; A. ). In contrast, chlamydiae were rarely observed in the clathrin-associated structures if the HEGEC were cultured on plastic surfaces. The same pattern of coated pit versus noncoated pit entry was reproducible in HeLa cells. The quantity of coated pits associated with isolated membrane sheets derived from HeLa cells, grown on poly-L-lysine-coated cover slips in medium containing the female hormones, was not significantly different as monitored by radiolabeling studies and by laser scanning microscopy. These data suggest that culture conditions which mimic in vivo cellular organization may enhance entry into coated pits for some obligate intracellular pathogens.Simply stated, our understanding of how Chlamydia spp. enter tissue culture cells is in a state of confusion. Several of the proposed pathways are summarized in Table 1. There are a number of explanations which may account for the various perspectives in different laboratories regarding the method(s) of entry of chlamydiae into nonprofessionally phagocytic cells. We shall focus on two areas, terminology and technology.There are essentially four defined mechanisms for entry of particles into eucaryotic cells (for recent reviews, see references 2, 9, and 30): (i) fluid-phase pinocytosis; (ii) nonspecific adsorptive pinocytosis or high-affinity adsorptive, nonreceptor-mediated endocytosis; (iii) phagocytosis; and (iv) specific absorptive pinocytosis or receptor-mediated endocytosis. As more information emerges on the functional role of clathrin, it appears that the latter endocytic pathway may be subdivided into clathrin-mediated internalization and noncoated-membrane uptake. The difference may hinge on

The in-vitro activity of azithromycin on Chlamydia trachomatis infected human endometrial epithelial cells, both primary and transformed cells growing in a polarized and non-polarized orientation, was analyzed. Addition of azithromycin two hours after adsorption inoculation with continued exposure until 72 h gave an MIC90 and MBC90 of 0.063 and 0.5 mg/L, respectively. In addition, the MBC results were more pronounced in infected cells growing in a polarized orientation. Numerous small fluorescent 'spots' (presumed small abnormal inclusions) were visible in the infected cells exposed to MIC concentrations of azithromycin. Immuno-transmission electron microscopy examination revealed intracellular inclusions filled with chlamydial envelope ghosts. Since standard diagnostic antigen detection methods use anti-envelope antibodies, the aberrant envelope-filled inclusions might be interpreted as viable inclusions by fluorescent microscopy and result in high false positive readings. To simulate treatment of an infected patient, azithromycin was added at 18 h to infected cells containing many reticulate bodies and exposure continued for 54 h after which killing of chlamydiae was seen. The use of polarized human cells may offer a more relevant in-vitro model system for examining the efficacy of antimicrobial action.

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