genitalium were Selleck AZD1480 detected in the cells (data not shown). Using a color changing unit assay (CCU), high titers of Omipalisib cell line viable intracellular M. genitalium were detected at both 24 h (not shown) and 48 h PI (Figure 3). No M. genitalium viability was detected in supernatants containing gentamicin at either point indicating that the observed titers were due exclusively to intracellular mycoplasmas that were protected from
gentamicin exposure. Extracellular M. genitalium titers, representing organisms that had escaped from infected cells, were quantified from separate wells using supernatants of infected cells. Extracellular titers from culture supernatants (dashed line) were significantly less than intracellular titers (p < 0.05) at the tested time points (48 h shown in Figure 3). These data indicated that, after M. genitalium entry of the cell, more organisms remained inside the cell than egressed to the culture supernatant. Intracellular localization
of M. genitalium in vaginal and cervical ECs also was consistent with electron microscopic analyses (Figure 1 and 2). Figure 3 Intra- and extracellular localization of M. genitalium Compound C in ME-180 cervical epithelial cells. Cervical ECs (ME-180) were inoculated with log-phase cultures of M. genitalium strain G37 (A) or M2300 (B) to determine whether M. genitalium can invade human reproductive tract ECs. To quantify intracellular M. genitalium loads (solid bar), the inoculum was removed following 3 h incubation for attachment and entry and replaced with medium containing gentamicin (200 ug/mL). The ability for M. genitalium to escape infected ECs (open bar) was quantified from culture supernatants in separate wells processed the same way except, following the 3 h incubation, the inoculum was removed and extracellular M. genitalium organisms were killed with gentamicin (2 h exposure). Infected cells then were washed thoroughly and received DOK2 fresh medium without gentamicin allowing escaping M. genitalium to survive. Cell fractions or culture supernatants were collected at 48 h following removal of the inoculum for quantification of bacterial loads using
a color changing unit (CCU) assay. In every case, significant differences between intracellular and extracellular M. genitalium titers were observed (p < 0.05; Student’s t-test). Parallel studies were performed that employed 400 ug/mL gentamicin with similar results (data not shown). M. genitalium elicited pro-inflammatory cytokines from genital epithelial cells Following demonstration of intracellular localization within reproductive tract ECs, we evaluated the host cytokine response from 3 human vaginal (V11I, V12I, and V19I) and 2 cervical EC lines (sA2EN and 3ECI) . Of the tested time points, peak cytokine values were obtained 48 h PI and are presented in Table 1. Vaginal ECs exposed to viable M. genitalium G37 or M2300 (MOI 10) responded with significant secretion of interleukin-6 (IL-6), IL-8 and GM-CSF (p < 0.05 vs.