Comparison of In Vitro Chlamydia muridarum Infection Under Aerobic and Anaerobic Conditions.

Although Chlamydia infects host body regions that are hypoxic to anoxic, standard Chlamydiae culture conditions are in CO2 enriched (5%) atmospheric oxygen (21%). Because of its success in causing disease in principally anaerobic body sites, e.g., vaginal tract, we hypothesize that Chlamydia has an anaerobic life cycle that plays a role in its maintenance in the host. Using a model system developed for the anaerobic culture of mammalian cells, we assessed the anoxic infectious cycle of C. muridarum in anaerobically cultured HeLa 229 cells. In the absence of oxygen, C. muridarum is capable of going through their life cycle, although its cycle is slowed (2 days post-infection anaerobic vs. 1 day aerobic). Interestingly, in addition to a slower rate of replication, there is a reduction in Chlamydia inclusion number and size as compared to aerobic controls. Anaerobic infected host cell physiology also changed with IL-6 and IL-8 production significantly lower (p ≤ 0.05) compared to aerobic infected host cells (day 4 post-infection). These findings demonstrate that Chlamydia are capable of replicating in the absence of oxygen.

A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production.

The center of tumors, stem cell niches and mucosal surfaces all represent areas of the body that are reported to be anoxic. However, long-term study of anoxic cell physiology is hindered by the lack of a sustainable method permitting cell cultivation in the complete absence of oxygen. A novel methodology was developed that enabled anoxic cell cultivation (17d maximum time tested) and cell passage. In the absence of oxygen, cell morphology is significantly altered. All cells tested exhibited morphologic changes, i.e., a combination of tethered (monolayer-like) and runagate (suspension-like) morphologies. Both morphologies replicated (Vero and HeLa cells tested) and could be passaged anaerobically. In the absence of exogenous oxygen, anoxic cells produced reactive oxygen species (ROS). Anaerobic runagate HeLa and Vero cells increased ROS production from day 3 to day 10 by 2- and 3-fold, respectively. In contrast, anoxic tethered HeLa and Vero cells either showed no significant change in ROS production between days 3 and 10 or exhibited a 3-fold decrease in ROS, respectively. Detection of ROS was inversely related to detection of hypoxia-inducible factor-1α (HIF1) mRNA and HIF-1 protein expression which cycled over a 10-day period. This methodology has broad applications for the study of tumor and stem cell physiology as well as gastrointestinal cell-microbiome interactions. In addition, sustainable anaerobic cell culture may lead to the identification of novel pathways and targets for chemotherapeutic drug development.