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The enduring hypoxic response of Mycobacterium tuberculosis.

Rustad TR, Harrell MI, Liao R, Sherman DR - PLoS ONE (2008)

Bottom Line: These results suggested that additional genes may be essential for entry into and maintenance 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.Our results suggest a reassessment of the role of DosR and the initial hypoxic response in MTB physiology.

View Article: PubMed Central - PubMed

Affiliation: Seattle Biomedical Research Institute, Seattle, Washington, USA.

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.

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Related in: MedlinePlus

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.
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pone-0001502-g001: 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.

Mentions: In the defined hypoxic model, a constant flow of low oxygen gas over the surface of a stirred, early log phase culture is used to deplete the oxygen in a rapid and highly reproducible way. This model was used initially to characterize the MTB transcriptional response to hypoxia; the DosR regulon is induced within two hours and bacteriostasis is evident within 24 hours, with less than a single doubling occurring after the initial exposure to low oxygen conditions [21]. In this system, the dosR mutant and wild-type strains showed no survival difference over a one-week period (Figure S1). Longer time points are not feasible in this system, due to complications from evaporation. To test survival following exposure to prolonged hypoxia, we employed a standing culture model. Wild-type and mutant bacilli were cultured in competition in small cryovials with no head space for up to 1 year. By 90 days, survival of the dosR mutant was about one log lower than that of wild-type (Figure 1a). This difference was still evident after one year in standing culture.


The enduring hypoxic response of Mycobacterium tuberculosis.

Rustad TR, Harrell MI, Liao R, Sherman DR - PLoS ONE (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198943&req=5

pone-0001502-g001: 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.
Mentions: In the defined hypoxic model, a constant flow of low oxygen gas over the surface of a stirred, early log phase culture is used to deplete the oxygen in a rapid and highly reproducible way. This model was used initially to characterize the MTB transcriptional response to hypoxia; the DosR regulon is induced within two hours and bacteriostasis is evident within 24 hours, with less than a single doubling occurring after the initial exposure to low oxygen conditions [21]. In this system, the dosR mutant and wild-type strains showed no survival difference over a one-week period (Figure S1). Longer time points are not feasible in this system, due to complications from evaporation. To test survival following exposure to prolonged hypoxia, we employed a standing culture model. Wild-type and mutant bacilli were cultured in competition in small cryovials with no head space for up to 1 year. By 90 days, survival of the dosR mutant was about one log lower than that of wild-type (Figure 1a). This difference was still evident after one year in standing culture.

Bottom Line: These results suggested that additional genes may be essential for entry into and maintenance 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.Our results suggest a reassessment of the role of DosR and the initial hypoxic response in MTB physiology.

View Article: PubMed Central - PubMed

Affiliation: Seattle Biomedical Research Institute, Seattle, Washington, USA.

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.

Show MeSH
Related in: MedlinePlus