SummaryUnlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/ DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alphacrystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis .

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis α-crystallin ( acr ) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

BackgroundA significant body of evidence accumulated over the last century suggests a link between hypoxic microenvironments within the infected host and the latent phase of tuberculosis. Studies to test this correlation have identified the M. tuberculosis initial hypoxic response, controlled by the two-component response regulator DosR. The initial hypoxic response is completely blocked in a dosR deletion mutant.Methodology/Principal FindingsWe show here that a dosR deletion mutant enters bacteriostasis in response to in vitro hypoxia with only a relatively mild decrease in viability. In the murine infection model, the phenotype of the mutant was indistinguishable from that of the parent strain. These results suggested that additional genes may be essential for entry into and maintenance of bacteriostasis. Detailed microarray analysis of oxygen starved cultures revealed that DosR regulon induction is transient, with induction of nearly half the genes returning to baseline within 24 hours. In addition, a larger, sustained wave of gene expression follows the DosR-mediated initial hypoxic response. This Enduring Hypoxic Response (EHR) consists of 230 genes significantly induced at four and seven days of hypoxia but not at initial time points. These genes include a surprising number of transcriptional regulators that could control the program of bacteriostasis. We found that the EHR is independent of the DosR-mediated initial hypoxic response, as EHR expression is virtually unaltered in the dosR mutant.Conclusions/SignificanceOur results suggest a reassessment of the role of DosR and the initial hypoxic response in MTB physiology. Instead of a primary role in survival of hypoxia induced bacteriostasis, DosR may regulate a response that is largely optional in vitro and in mouse infections. Analysis of the EHR should help elucidate the key regulatory factors and enzymatic machinery exploited by M. tuberculosis for long-term bacteriostasis in the face of oxygen deprivation.

The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) is a live attenuated organism, but the mutation responsible for its attenuation has never been defined. Recent genetic studies identified a single DNA region of difference, RD1, which is absent in all BCG strains and present in all Mycobacterium tuberculosis (MTB) strains. The 9 open-reading frames predicted within this 9.5-kb region are of unknown function, although they include the TB-specific immunodominant antigens ESAT-6 and CFP-10. In this study, RD1 was deleted from MTB strain H37Rv, and virulence of H37Rv:DeltaRD1 was assessed after infections of the human macrophage-like cell line THP-1, human peripheral blood monocyte-derived macrophages, and C57BL/6 mice. In each of these systems, the H37Rv:DeltaRD1 strain was strikingly less virulent than MTB and was very similar to BCG controls. Therefore, it was concluded that genes within or controlled by RD1 are essential for MTB virulence and that loss of RD1 was important in BCG attenuation.