Characterization of a Clp Protease Gene Regulator and the Reaeration Response in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (MTB) enters a non-replicating state when exposed to low oxygen tension, a condition the bacillus encounters in granulomas during infection. Determining how mycobacteria enter and maintain this state is a major focus of research. However, from a public health standpoint the importance of latent TB is its ability to reactivate. The mechanism by which mycobacteria return to a replicating state upon re-exposure to favorable conditions is not understood. In this study, we utilized reaeration from a defined hypoxia model to characterize the adaptive response of MTB following a return to favorable growth conditions. Global transcriptional analysis identified the ∼100 gene Reaeration Response, induced relative to both log-phase and hypoxic MTB. This response includes chaperones and proteases, as well as the transcription factor Rv2745c, which we characterize as a Clp protease gene regulator (ClgR) orthologue. During reaeration, genes repressed during hypoxia are also upregulated in a wave of transcription that includes genes crucial to transcription, translation and oxidative phosphorylation and culminates in bacterial replication. In sum, this study defines a new transcriptional response of MTB with potential relevance to disease, and implicates ClgR as a regulator involved in resumption of replication following hypoxia.

The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy

“…Hypoxia and reaeration treatment were followed based on Sherrid et al (18). Briefly, for hypoxia treatment, a modified Wayne model was used in which cultures were transferred to 50-ml conical tubes with a headspace of 0.…”

“…In Mtb, clpP1 and clpP2 are encoded on a single operon that is activated by Rv2745c (9). Rv2745c, clgR, is a Clp protease gene regulator that is induced under a variety of conditions, but ultimately induction of Rv2745c leads to differential activation of downstream genes in a condition-dependent manner (9,17,18).…”

“…Rv2745c expression is induced under multiple stress conditions, such as redox stress, SDS treatment, heat shock, and acid stress (19) as well as hypoxia (9,18) implicating that Rv2745c plays a role in response to various environmental pressures. Rv2745c up-regulation in response to redox stress or envelope damage by thioridazine (20) or vancomycin (21) is dependent upon H and/or E levels and does not lead to the induction of clp proteases (9,17,19).…”

“…Rv2745c up-regulation in response to redox stress or envelope damage by thioridazine (20) or vancomycin (21) is dependent upon H and/or E levels and does not lead to the induction of clp proteases (9,17,19). However, induction of Rv2745c during the late stages of hypoxia as well as during reaeration leads to clp protease induction (18). As to how the induction of Rv2745c by various stressors can lead to different downstream outcomes is, however, not well understood.…”

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

“…For example, Mycobacterium tuberculosis, a pathogenic actinobacterium, encodes cotranscribed clpP1 and clpP2 genes (24,25). The importance of Clp-family proteolysis in M. tuberculosis is highlighted by the facts that the clpP1, clpP2, clpX, and clpC1 genes are all essential and that mechanism-based ClpP inhibitors suppress growth (24,(26)(27)(28).…”