genitalium by reproductive tract ECs was assessed using the gentamicin invasion assay [26]. The sensitivity of M. genitalium strains G37 and M2300 to gentamicin was established first by inoculation of log-phase organisms into Friis FB medium with gentamicin concentrations ranging from 100–400 ug/mL. No M. genitalium growth was observed at 200 or 400 ug/mL therefore a working concentration of 200 ug/mL was employed in subsequent studies to minimize EC uptake of gentamicin and subsequent killing of intracellular M. genitalium. Confirmatory studies were completed subsequently

using 400 ug/mL gentamicin. As a representative genital EC type, ME-180 cells were seeded into 96-well plates 1d prior to infection at a density of 1 × 105 cells/well. Log-phase M. genitalium was inoculated onto ME-180 cells (MOI of 100) in triplicate.

Following 3 h incubation, CB-839 manufacturer when M. genitalium BVD-523 in vivo appeared to be attached to and invading genital ECs (see Figure 1), the inoculum was removed and replaced with fresh medium containing gentamicin. At 15 min, 24 and 48 h following removal of the inoculum, culture supernatants were removed and the infected cells were washed 3× with sterile PBS. Cells then were removed from the well by scraping into Friis FB medium followed by plating serial 10-fold dilutions prepared in Friis FB medium into a 96-well plate. Outgrowth of M. genitalium from infected ME-180 cells was observed for 14d. The load of viable M. genitalium from each sample was calculated by titration as described above. Figure 1 Cultivation of M. genitalium and ultrastructural analysis of attachment to vaginal epithelial cells. M. genitalium G37 or M2300 were grown to log-phase in Friis FB medium. (A) Light micrograph of attached G37 microcolonies grown in culture flasks containing HSP90 Friis FB medium taken using Variable Relief Contrast (VAREL). (B) TEM micrograph of a single G37 microcolony after 3d growth in Friis FB medium showing highly variable size and morphology. (C) Within M. genitalium G37 microcolonies, an elongated tip-like structure (arrow) was observed. (D) TEM micrograph M. genitalium strain M2300 showing similar variable morphology

compared to G37 and formation of an electron-dense tip structure. Log-phase M. genitalium were harvested from Friis medium and then inoculated onto vaginal EC monolayers for ultrastructural analysis of attachment. (E) SEM micrograph of M. genitalium G37 attached to vaginal ECs (2 h PI). (F) TEM micrograph of M. genitalium G37 attached to vaginal ECs collected 3 h PI. An Z-VAD-FMK nmr electron dense core structure presumably involved in attachment and invasion of vaginal ECs is highlighted by the oval. Similar electron dense cores were observed in some tip structures and can be seen in panel C. The gentamicin invasion assay also was utilized to investigate whether intracellular M. genitalium were able to escape from the infected ECs. For these experiments, ME-180 cervical ECs were infected with M.