Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis

Yee, Michelle; Gopal, Pooja; Dick, Thomas

doi:10.1128/aac.02342-16

Cited by 42 publications

(67 citation statements)

References 34 publications

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“…9,10 On the basis of our observation that POA resistance-conferring clpC1 mutations are attenuated in vivo, whereas panD mutations did not cause a growth defect in vivo, we hypothesized that panD mutations—if they do occur in vivo—may be found frequently in vivo. Targeted PCR sequencing of panD revealed that 23 (82%) of the 28 POA-resistant strains isolated after POA treatment of mice contained panD mutations observed in multiple selection experiments in vitro 9,11,12 (Supporting Information, Table S2).…”

Section: Resultsmentioning

confidence: 99%

“…10 M. tuberculosis ClpC1 works together with the ClpP1 and ClpP2 proteins of the caseinolytic protease complex and displays unfoldase and ATPase activities. 35,36 The Clp protease complex is crucial for viability of M. tuberculosis both in vitro and in vivo, 37,38 whereas the clpC1 gene on its own has been demonstrated to be essential for growth in vitro 31 and within macrophages.…”

Section: Discussion and Conclusionmentioning

confidence: 99%

“…Missense mutations in the aspartate decarboxylase panD (9,11,12) and in the unfoldase/ATPase clpC1 (10) were found to cause resistance to PZA and POA. Loss-of-function mutations of the phthiocerol dimycocerosate (PDIM) virulence factor producing polyketide synthases Mas and PpsA-E caused a lower level of resistance to PZA and POA.…”

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confidence: 99%

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In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1

et al. 2017

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Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.

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Section: Resultsmentioning

confidence: 99%

Section: Discussion and Conclusionmentioning

confidence: 99%

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In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1

et al. 2017

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“…confirms the earlier finding that PanD is indeed a target of PZA . In addition, recent studies also identified another possible target of PZA as ClpC1, part of a protease complex involved in protein degradation, which is presumably important for persister survival. Furthermore, a recent study identified novel mutations in LprG (rv1411c), rv0521, rv3630, rv0010c, ppsC and cyp128 associated with POA/PZA resistance in MTB , which sheds new light on mode of action and resistance of this intriguing persister drug.…”

Section: Mechanisms Of Drug Resistance In Mtbmentioning

confidence: 98%

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

et al. 2018

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Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is the deadliest infectious disease and the associated global threat has worsened with the emergence of drug resistance, in particular multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). Although the World Health Organization (WHO) End-TB Strategy advocates for universal access to antimicrobial susceptibility testing, this is not widely available and/or it is still underused. The majority of drug resistance in clinical MTB strains is attributed to chromosomal mutations. Resistance-related mutations could also exert certain fitness cost to the drug-resistant MTB strains and growth fitness could be restored by the presence of compensatory mutations. Understanding these underlying mechanisms could provide an important insight into TB pathogenesis and predict the future trend of MDR-TB global pandemic. This review covers the mechanisms of resistance in MTB and provides a comprehensive overview of current phenotypic and molecular approaches for drug susceptibility testing, with particular attention to the methods endorsed and recommended by the WHO.

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“…Mutations in pncA leading to the loss of PZase activity are the major mechanism of PZA resistance (4,10,11). PZA is known to interfere with multiple functions in Mycobacterium tuberculosis, including cytoplasmic acidification (12), disruption of membrane energy and transport function (12,13), inhibition of the protein degradation pathway via RpsA involved in trans-translation (14) and also ClpC1 protease (15,16), and energy production via PanD (17)(18)(19). Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1, clinical strains without these mutations such as 9739 (20) are known to exist.…”

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confidence: 99%

Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

Zhang

Cui

et al. 2017

Antimicrob Agents Chemother

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Pyrazinamide (PZA) is a critical drug used for the treatment of tuberculosis (TB). PZA is a prodrug that requires conversion to the active component pyrazinoic acid (POA) by pyrazinamidase (PZase) encoded by the pncA gene. Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1 mutations, clinical strains without these mutations are known to exist. While efflux of POA was demonstrated in Mycobacterium tuberculosis previously, the efflux proteins involved have not been identified. Here we performed POA binding studies with an M. tuberculosis proteome microarray and identified four efflux proteins (Rv0191, Rv3756c, Rv3008, and Rv1667c) that bind POA. Overexpression of the four efflux pump genes in M. tuberculosis caused low-level resistance to PZA and POA but not to other drugs. Furthermore, addition of efflux pump inhibitors such as reserpine, piperine, and verapamil caused increased susceptibility to PZA in M. tuberculosis strains overexpressing the efflux proteins Rv0191, Rv3756c, Rv3008, and Rv1667c. Our studies indicate that these four efflux proteins may be responsible for PZA/POA efflux and cause PZA resistance in M. tuberculosis. Future studies are needed to assess their roles in PZA resistance in clinical strains.

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Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis

Cited by 42 publications

References 34 publications

In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1

In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

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