mosR , a Novel Transcriptional Regulator of Hypoxia and Virulence in Mycobacterium tuberculosis

Abstract

Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of Mycobacterium tuberculosis revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (mce1) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in Mycobacterium smegmatis, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (tgs1)] and intracellular survival (e.g., mce1), among other genes. Interestingly, the colonization level of the ⌬mosR mutant strain was significantly lower than that of the wild-type strain of M. tuberculosis, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed mosR for regulator of mycobacterial operons of survival.

Characterization of a Novel Heat Shock Protein (Hsp22.5) Involved in the Pathogenesis of Mycobacterium tuberculosis

“…Following 4 to 6 weeks of incubation at 37°C, hygromycin-resistant colonies were selected for further analysis. PCR and Southern blot analyses were used to verify the mutant genotypes as described previously by our laboratory (1). For complementation experiments, the coding sequence of rv0990c was amplified, cloned into pGEM-T vector, and verified by DNA sequencing.…”

“…At the designated time points, lungs and spleens from sacrificed animals were removed aseptically, weighed, and examined. One lung from each animal was homogenized for CFU enumeration, and the other was fixed in 10% buffered formaldehyde for histopathology as previously described (1). Normalized organ weights were calculated by multiplying the organ weight on the day of sacrifice by the ratio of the body weight on day 1/body weight on day of sacrifice.…”

“…The lacZ assays were performed in the soluble fractions of recombinant M. smegmatis in triplicates and repeated at least twice (1).…”

“…The infection of the monolayer-adhering cells with the M. tuberculosis H37Rv strain was performed to achieve an MOI of 1:10 (cell/bacteria). After 3 h of infection, cells were washed with sterile PBS buffer and incubated at 37°C in 5% CO 2 , with RPMI medium supplemented with 5% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) for 24 h. Cells were lysed with 0.05% sodium dodecyl sulfate (SDS), and bacteria were collected and processed for RNA extraction using the Trizol method (1).…”

“…Several in vitro and in vivo models have been developed in order to elucidate the mechanisms employed by the pathogen to survive and persist inside the host (5, 12, 25, 30-33). Such studies have identified genes activated during M. tuberculosis survival inside macrophages or under hostile stress conditions, including transcriptional regulators which control the expression of a large number of genes (1,22). One such gene is rv0990c, which was preferentially activated in murine lungs among a group of 32 genes sharing the same genomic locus, termed the in vivo-expressed genomic island, iVEGI (31).…”