Differences in Chlamydia trachomatis Serovar E Growth Rate in Polarized Endometrial and Endocervical Epithelial Cells Grown in Three-Dimensional Culture

Abstract

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 collagencoated 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.Chlamydia trachomatis serovars D to K are oculogenital pathogens and the leading cause of bacterial sexually transmitted diseases (41). It is estimated there are 3 to 4 million cases of chlamydial sexually transmitted diseases annually in the United States and some 90 million cases per year worldwide (7). Since the majority of infected individuals are essentially asymptomatic and do not seek medical attention, ascending migration can occur and lead to serious complications, such as prostatitis and epididymitis in men and pelvic inflammatory disease, salpingitis, ectopic pregnancy, and infertility in women (12,14).Chlamydiae are obligate intracellular bacteria and, as such, must be internalized into superficial epithelial cells of the genital mucosa in order to initiate the infectious process. Infection begins with attachment of the infectious elementary bodies (EB) form to the apical surface of columnar epithelial cells, followed by entry via various endocytic mechanisms. The EBcontaining endosomes exit the endocytic pathway to avoid fusion with lysosomes and travel on microtubules to the nuclear hof, where they undergo homotypic fusion with one another, and then the EBs transform into metabolically active reticulate bodies (RB). Since RB divide by binary fission and the number of progeny increases, the expanding endocytic vesicle is termed an inclusion. Eventually, RB mature back into infectious EB, and this devel...

Comparison of Chlamydia trachomatis serovar L2 growth in polarized genital epithelial cells grown in three-dimensional culture with non-polarized cells

“…Suspension cultures of HEC-1B and HeLa cells grown on Cytodex collagen-coated microcarrier beads (Sigma-Aldrich, Inc., St-Louis, MO) were set up and inoculated with chlamydiae, as recently described by Guseva et al ( [13]; 250-ml spinner bottle size). Briefly, after the beads were allowed to settle down and four-fifths of the medium was removed, the cultures of HEC-1B and HeLa cells were inoculated with C. trachomatis serovar L2 at a multiplicity of infection (MOI) of 1.…”

“…Similarly, serial dilutions of uninfected HeLa cell gDNA were used in the MPO-and F8-specific qPCR assays to evaluate the relative host cell gDNA content in each sample. Chlamydial genome as well as euo and omcB transcript levels in experimental samples were finally normalized to host gDNA amounts (averaged MPO and F8 relative amounts), and expressed as copy numbers per ng of host gDNA [13].…”

“…A small aliquot of culture was sampled for both cell lines at 2 and 24 hpi, and processed for qPCR analysis of chlamydial genome numbers (normalized to host cells) to ensure that the two bead cultures received a similar inoculum dose, a factor that could otherwise affect the yield of progeny collected. At 48 hpi, the chlamydial progeny were harvested and Percoll-purified as described by Guseva et al (13), and the final EB pellets were resuspended in a fixed volume of 2-SPG and counted by OD reading at 750 nm. For infectious titer determination, chlamydial progeny suspensions collected from all bead cultures were adjusted to 1 × 10 9 particles/ml and titered in HEC-1B and HeLa cells plated in 24-well plates as described above.…”

“…Of note, infectious titers obtained by these authors for genital serovar L2 in conjunctival cells were lower than those in endocervical HeLa cells, although the drop in infectivity was not nearly as dramatic as that for serovar D. Also, very recently, using polarized genital epithelial cells grown in 3D bead cultures, our group found that C. trachomatis serovar E grows faster in endometrial HEC-1B than in endocervical HeLa cells, which resulted in the recovery of ca. 4 times more chlamydial progeny in the former cell line upon completion of the developmental cycle [13]; other authors reported a 10-fold difference in the number of serovar E infectious progeny recovered between these two cell lines [14]. These differences in recoverable bacterial yields are potentially important, as collecting sufficient numbers of chlamydiae for downstream applications can sometimes be challenging and time-consuming, particularly when studying urogenital strains.…”

“…In this follow-up study to Guseva et al [13], the growth of C. trachomatis serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on microcarrier beads, as well as in the more commonly used HeLa cells plated in tissue culture flasks; chlamydial progeny yields and infectivity were also determined. The objectives were to assess if, as observed for serovar E, the in vitro growth of this popular LGV strain is influenced by the anatomical origin of the genital epithelial cells used, and/or by the polarization state of these cells.…”