The enduring hypoxic response of Mycobacterium tuberculosis

Abstract

Background: A 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 findings: We 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/significance: Our 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.

Figure 1. Survival of H37Rv and H37Rv:ΔdosR in hypoxia-induced bacteriostasis in vitro.

Colony forming units per ml of parent strain H37Rv (white bar) and H37Rv:ΔdosR (black bar) were counted at each time point in the (A) standing culture and (B) Wayne models of hypoxia. Data are from one representative experiment of 3 (standing culture) or 5 (Wayne model). Each bar represents a minimum of 3 biological replicates.

Figure 2. The phenotype of H37Rv:ΔdosR in mouse model is indistinguishable from wild type.

Bacterial burdens of H37Rv (squares) and H37Rv:ΔdosR (triangles) in the (A) lungs and (B) spleens of C57BL/6 mice over the course of infection. Each point represents the average of three experiments with 3–5 animals per time point in each experiment. Error bars represent standard deviation. (C&D) Disregulation of the DosR regulon in a dosR mutant during murine infection was verified by quantitative real-time PCR of sentinel genes Rv0081 (C) and Rv2031c (D) from H37Rv (white) and H37Rv:ΔdosR (black) mRNA isolated from mouse lungs at 4 and 6 weeks post infection. Values were normalized to housekeeping gene SigA and shown as the ratio of normalized in vivo expression to log phase in vitro levels of expression.

Figure 3. Enduring hypoxic response (EHR) dominates the hypoxic time course.

Numbers of genes significantly induced over log phase (>two fold, FDR<0.4%) were tabulated at each time point over a hypoxic time course. Each bar represents the total number of genes (gray) and genes from the EHR (black).

Figure 4. Downstream hypoxic response independent of DosR regulon.

Each bar represents the number of genes induced in H37Rv at each point during a short hypoxic time course (>two fold in all three arrays). The DosR regulon represents a dwindling fraction of the genes induced over time (gray). Very few genes were induced in H37Rv and not in the dosR mutant (diagonal lines). The overwhelming majority of genes induced in the parent strain at later hypoxic time points were also induced in the mutant (black).