Mycobacterium tuberculosis Growth following Aerobic Expression of the DosR Regulon

The Mycobacterium tuberculosis regulator DosR is induced by multiple stimuli including hypoxia, nitric oxide and redox stress. Overlap of these stimuli with conditions thought to promote latency in infected patients fuels a model in which DosR regulon expression is correlated with bacteriostasis in vitro and a proxy for latency in vivo. Here, we find that inducing the DosR regulon to wildtype levels in aerobic, replicating M. tuberculosis does not alter bacterial growth kinetics. We conclude that DosR regulon expression alone is insufficient for bacterial latency, but rather is expressed during a range of growth states in a dynamic environment

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

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 Enduring Hypoxic Response of Mycobacterium tuberculosis

deletion mutant. mutant. for long-term bacteriostasis in the face of oxygen deprivation

Ectopic expression of DosR induces the DosR regulon.

<p>Scatterplot displaying transcript levels of all <i>M. tuberculosis</i> genes after 24 hours of treatment with either 10 ng/mL Atc (induced) or an equivalent volume of sterile DMSO (uninduced). Three biological replicates were RMA-normalized and the median pixel intensity data are plotted on a log₂ scale. Genes of the DosR regulon are represented as dark gray circles. Significantly induced genes (moderated t-test with Benjamini-Hochberg FDR correction, p<0.05) not part of the DosR regulon are presented as black diamonds, and the <i>dosR</i> transcript is indicated with a star.</p

Genes Required for Intrinsic Multidrug Resistance in Mycobacterium avium

Genes required for intrinsic multidrug resistance by Mycobacterium avium were identified by screening a library of transposon insertion mutants for the inability to grow in the presence of ciprofloxacin, clarithromycin, and penicillin at subinhibitory concentrations. Two genes, pks12 and Maa2520, were disrupted in multiple drug-susceptible mutants. The pks12 gene (Maa1979), which may be cotranscribed with a downstream gene (Maa1980), is widely conserved in the actinomycetes. Its ortholog in Mycobacterium tuberculosis is a polyketide synthase required for the synthesis of dimycocerosyl phthiocerol, a major cell wall lipid. Mutants of M. avium with insertions into pks12 exhibited altered colony morphology and were drug susceptible, but they grew as well as the wild type did in vitro and intracellularly within THP-1 cells. A pks12 mutant of M. tuberculosis was moderately more susceptible to clarithromycin than was its parent strain; however, susceptibility to ciprofloxacin and penicillin was not altered. M. avium complex (MAC) and M. tuberculosis appear to have different genetic mechanisms for resisting the effects of these antibiotics, with pks12 playing a relatively more significant role in MAC. The second genetic locus identified in this study, Maa2520, is a conserved hypothetical gene with orthologs in M. tuberculosis and Mycobacterium leprae. It is immediately upstream of Maa2521, which may code for an exported protein. Mutants with insertions at this locus were susceptible to multiple antibiotics and slow growing in vitro and were unable to survive intracellularly within THP-1 cells. Like pks12 mutants, they exhibited increased Congo red binding, an indirect indication of cell wall modifications. Maa2520 and pks12 are the first genes to be linked by mutation to intrinsic drug resistance in MAC