Ptr/CTL0175 Is Required for the Efficient Recovery of Chlamydia trachomatis From Stress Induced by Gamma-Interferon

In this study, we carried out a screen to identify C. trachomatis genes important for recovery from IFNγ- and/or penicillin-induced stress. We found that ptr, encoding a poorly characterized secreted protease, is required for rapid recovery from IFNγ- but not penicillin-induced stress. Materials and Methods Cell Culture, Infection and C. trachomatis Strains HeLa (CCL-2, ATCC) and Vero cells (CCL-81, ATCC) were cultured in high glucose Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FBS (Natocor) without antibiotics in 5% CO2 at 37°C in a humidified atmosphere. Cells were routinely checked for Mycoplasma contamination using PCR, as described previously (van Kuppeveld et al., 1992). Infections were done by adding a previously titered suspension of purified EBs at the specified multiplicity of infection (MOI) in culture media to cell monolayers, followed by centrifugation at 2,500 ×g, 10°C, 30 min. Then infected cells were transferred to a tissue-culture incubator for the indicated hours post infection (hpi). EB purifications were carried out as described in Saka et al. (2011). EB stocks were titered by assessing the number of Inclusion Forming Units (IFUs) per microliter as described in Nguyen and Valdivia (2012), with minor modifications. Briefly, serial dilutions of EBs were added to Vero or HeLa cell monolayers seeded onto 96-well plates. At 24–30 hpi, cells were fixed with ice-cold methanol (15 min)...
Source: Frontiers in Microbiology - Category: Microbiology Source Type: research