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

Background: The Mycobacterium tuberculosis gene, Rv2745c, leads to induction of downstream genes that is condition-dependent. Results: Rv2745c is induced during hypoxia and reaeration conditions. An isogenic mutant leads to differential transcriptional profiles. Conclusion: Rv2745c plays a role during hypoxia and reaeration. Significance: Rv2745c is important for survival during reaeration, implicating that Rv2745c is important for growth during reactivation in vivo. Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease worldwide and is the causative agent of tuberculosis (Chao, M. C., and Rubin, E. J. (2010) Annu. Rev. Microbiol. 64, 293–311). Throughout infection, Mtb encounters a variety of host pressures. Thus, responding to these host stresses via the induction of multiple regulatory networks is needed for survival within the host. The Clp protease gene regulator, Rv2745c (clgR), is induced in response to environmental stress conditions, implicating its potential role in Mtb pathogenesis. Transcriptional activation of genes downstream of Rv2745c occurs in a condition-dependent manner. Our isogenic Mtb:ΔRv2745c mutant expresses a significantly different phenotype upon reaeration conditions. Transcriptional analysis revealed differential gene expression profiles relative to wild-type Mtb. Rv2745c is strongly induced in response to hypoxic and reaeration conditions, implicating a role of Rv2745c in vivo during both establishment of infection and reactivation. We found dysregulation of downstream genes within both the σH/σE regulon as well as the dosR regulon in the isogenic mutant, Mtb:ΔRv2745c. Upon hypoxic and reaeration conditions, Clp protease induction occurred within wild-type Mtb, indicating that activation of clgR, which subsequently leads to Clp protease induction, is crucial for degradation of misfolded proteins and ultimately survival of Mtb upon specific stress conditions. Our data indicate the diverse response of Rv2745c, σH and σE in response to a variety of stress conditions. Activation of Rv2745c in response to various stress conditions leads to differential activation of downstream genes, indicating the diverse role of Rv2745c and its importance for Mtb survival in vivo.

[1]  Tige R. Rustad,et al.  The Enduring Hypoxic Response of Mycobacterium tuberculosis , 2008, PloS one.

[2]  J. Ernst,et al.  Tuberculosis pathogenesis and immunity. , 2012, Annual review of pathology.

[3]  D. Russell.  The evolutionary pressures that have molded Mycobacterium tuberculosis into an infectious adjuvant. , 2013, Current opinion in microbiology.

[4]  A. Goldberg,et al.  Mycobacterium tuberculosis ClpP1 and ClpP2 Function Together in Protein Degradation and Are Required for Viability in vitro and During Infection , 2012, PLoS pathogens.

[5]  S. Chauhan,et al.  Powerful Induction of Divergent tgs1-Rv3131 Genes in Mycobacterium tuberculosis Is Mediated by DevR Interaction with a High-Affinity Site and an Adjacent Cryptic Low-Affinity Site , 2009, Journal of bacteriology.

[6]  T. Niu,et al.  Role of TNF in the Altered Interaction of Dormant Mycobacterium tuberculosis with Host Macrophages , 2014, PloS one.

[7]  Tanya Parish,et al.  Validation of the Essential ClpP Protease in Mycobacterium tuberculosis as a Novel Drug Target , 2011, Journal of bacteriology.

[8]  Kyle J. Minch,et al.  Hypoxia: a window into Mycobacterium tuberculosis latency , 2009, Cellular microbiology.

[9]  G. Schoolnik,et al.  Role of the extracytoplasmic‐function σ Factor σH in Mycobacterium tuberculosis global gene expression , 2002 .

[10]  A. Tyagi,et al.  The sigma factors of Mycobacterium tuberculosis: regulation of the regulators , 2010, The FEBS journal.

[11]  Alfred Goldberg,et al.  Lassomycin, a ribosomally synthesized cyclic peptide, kills mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2. , 2014, Chemistry & biology.

[12]  Smriti Mehra,et al.  A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine , 2010, PloS one.

[13]  Richard D. Smith,et al.  Activated ClpP kills persisters and eradicates a chronic biofilm infection , 2013, Nature.

[14]  Daniel J. Bretl,et al.  Adaptation to Environmental Stimuli within the Host: Two-Component Signal Transduction Systems of Mycobacterium tuberculosis , 2011, Microbiology and Molecular Reviews.

[15]  R. Fleischmann,et al.  Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative σ factor, SigH , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  V. Nizet,et al.  Pharmacological Inhibition of the ClpXP Protease Increases Bacterial Susceptibility to Host Cathelicidin Antimicrobial Peptides and Cell Envelope-Active Antibiotics , 2012, Antimicrobial Agents and Chemotherapy.

[17]  Yves Van de Peer,et al.  The Mycobacterium tuberculosis regulatory network and hypoxia , 2013, Nature.

[18]  G. Dougan,et al.  Sequence-Based Analysis Uncovers an Abundance of Non-Coding RNA in the Total Transcriptome of Mycobacterium tuberculosis , 2011, PLoS pathogens.

[19]  J. Pape,et al.  Correlation between Genotypic and Phenotypic Testing for Resistance to Rifampin in Mycobacterium tuberculosis Clinical Isolates in Haiti: Investigation of Cases with Discrepant Susceptibility Results , 2014, PloS one.

[20]  S. Gygi,et al.  Post-Translational Regulation via Clp Protease Is Critical for Survival of Mycobacterium tuberculosis , 2014, PLoS pathogens.

[21]  E. Rubin,et al.  Letting sleeping dos lie: does dormancy play a role in tuberculosis? , 2010, Annual review of microbiology.

[22]  T. Parish,et al.  Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities , 2013, PloS one.

[23]  James A. Raleigh,et al.  Tuberculous Granulomas Are Hypoxic in Guinea Pigs, Rabbits, and Nonhuman Primates , 2008, Infection and Immunity.

[24]  D. Kaushal,et al.  Functional Genomics Reveals Extended Roles of the Mycobacterium tuberculosis Stress Response Factor σH , 2009, Journal of bacteriology.

[25]  W. Bishai,et al.  Role of the dosR-dosS Two-Component Regulatory System in Mycobacterium tuberculosis Virulence in Three Animal Models , 2008, Infection and Immunity.

[26]  Sadhna Sharma,et al.  Immunogenic potential of latency associated antigens against Mycobacterium tuberculosis. , 2014, Vaccine.

[27]  Sébastien Rodrigue,et al.  σ Factors and Global Gene Regulation in Mycobacterium tuberculosis , 2004, Journal of bacteriology.

[28]  Ashley M. Sherrid,et al.  Characterization of a Clp Protease Gene Regulator and the Reaeration Response in Mycobacterium tuberculosis , 2010, PloS one.

[29]  Y Av-Gay,et al.  The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. , 2000, Trends in microbiology.

[30]  TAC from Mycobacterium tuberculosis: a paradigm for stress-responsive toxin–antitoxin systems controlled by SecB-like chaperones , 2012, Cell Stress and Chaperones.

[31]  D. Saini,et al.  Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis. , 2004, FEMS microbiology letters.

[32]  David G. Russell,et al.  Who puts the tubercle in tuberculosis? , 2007, Nature Reviews Microbiology.

[33]  Pawan Gupta,et al.  The mechanism of redox sensing in Mycobacterium tuberculosis. , 2012, Free radical biology & medicine.

[34]  J. Basu,et al.  RseA, the SigE specific anti‐sigma factor of Mycobacterium tuberculosis, is inactivated by phosphorylation‐dependent ClpC1P2 proteolysis , 2010, Molecular microbiology.

[35]  W. Yam,et al.  High-Resolution Melting Analysis for the Rapid Detection of Fluoroquinolone and Streptomycin Resistance in Mycobacterium tuberculosis , 2012, PloS one.

[36]  P. Genevaux,et al.  Multiple Toxin-Antitoxin Systems in Mycobacterium tuberculosis , 2014, Toxins.

[37]  W. Bishai,et al.  Expression of a Subset of Heat Stress Induced Genes of Mycobacterium tuberculosis Is Regulated by 3',5'-Cyclic AMP , 2014, PloS one.

[38]  Eric J. Rubin,et al.  Bacterial proteolytic complexes as therapeutic targets , 2012, Nature Reviews Drug Discovery.

[39]  Smriti Mehra,et al.  The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress , 2014, PloS one.

[40]  Denise E. Kirschner,et al.  Multi-Scale Modeling Predicts a Balance of Tumor Necrosis Factor-α and Interleukin-10 Controls the Granuloma Environment during Mycobacterium tuberculosis Infection , 2013, PloS one.

[41]  F. Falciani,et al.  Global transcriptional response to vancomycin in Mycobacterium tuberculosis. , 2009, Microbiology.

[42]  R. Hunter,et al.  The ΔfbpA mutant derived from Mycobacterium tuberculosis H37Rv has an enhanced susceptibility to intracellular antimicrobial oxidative mechanisms, undergoes limited phagosome maturation and activates macrophages and dendritic cells , 2008, Cellular microbiology.

[43]  G. Schoolnik,et al.  Role of the extracytoplasmic-function sigma factor sigma(H) in Mycobacterium tuberculosis global gene expression. , 2002, Molecular microbiology.

[44]  Jianping Xie,et al.  The Mycobacterium DosR regulon structure and diversity revealed by comparative genomic analysis , 2013, Journal of cellular biochemistry.

[45]  Smriti Mehra,et al.  Mycobacterium tuberculosis MT2816 encodes a key stress-response regulator. , 2010, The Journal of infectious diseases.