FIG. 5.

SenX3 is required for the regrowth of Mtb in response to the restoration of ambient oxygen levels. (A) Wild-type, senX3 mutant, and senX3 complementing strains were found to have similar growth profiles in the Wayne model. (B) No significant differences in colony-forming units between wild-type, mutant, and complementing strains were observed during hypoxic adaptation after days 10, 20, 30, and 40. After 10 days (C), 20 days (D), 30 days (E), and 40 days (F) of hypoxic adaptation, the wild-type, SenX3 mutant, and SenX3 complementing strains were exposed to O₂ and allowed to resume growth. (G) The reactivation of the wild-type, ΔsenX3 mutant, and complemented strains was assessed based on the time required for the development of a detectable growth signal by the BACTEC MGIT 960 system. A consistent and statistically significant delay in regrowth after the restoration of O₂ was observed in all cultures of the ΔsenX3 mutant (at 20, 30, 40, and 130 days) compared with the wild type. However, episomal expression of SenX3 in the mutant strain led to a decrease in that attenuation. WT, SKO, and SCO indicate the wild-type, senX3 knockout Mtb, and senX3 complementing Mtb, respectively. (H, I) The results of the kinase assay were further validated in the Wayne model by exposing the hypoxic culture at day 40 to 200 μM of NO (generated by DETA NONOate) (H) and 50 μM of CO (released by CORM-2) (I) in the presence of ambient oxygen. Exposure of the wild-type and complemented strains to NO and CO leads to an arrest in regrowth in response to ambient oxygen similar to that observed in the SenX3 mutant strain. *p<0.05. Statistical significance was determined using Student's t-test. These figures (A–I) are representative of at least three independent experiments performed in triplicate. Mtb, Mycobacterium tuberculosis; SCO, Senx3 complementing strain of Mtb; SKO, SenX3 knockout strain of Mtb. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars