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

Abstract

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.

Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD

“…In addition, these independent studies showed that mutations of H21 within the N-terminal region of the aspartate decarboxylase domain are also associated with POA resistance. 20,22,24,30 …”

“…Sequencing of POA-resistant M. tuberculosis strains isolated from lungs of mice treated with POA revealed that missense mutations in panD constitute the predominant mechanism of resistance to POA in vivo . 24 Probing of the panD -related resistance mechanism in vitro revealed that treatment of M. bovis BCG with POA results in a depletion of intracellular coenzyme A levels. 20,25 Our observation that resistance mutations in panD prevent POA-mediated depletion of intracellular coenzyme A levels 20 and that this PanD-mediated resistance phenotype can be mimicked by supplementation of downstream metabolites of PanD such as β -alanine and pantothenate 20,22,26 suggests a role of POA in disrupting the coenzyme A biosynthetic pathway.…”

Impact of the host environment on the antitubercular action of pyrazinamide

“…Recently, numerous studies have shown that a component of the coenzyme A (CoA) biosynthetic pathway may be a molecular target of POA ( Fig. 1 ) [40][41][42][43][44][45] . Analysis of spontaneous PZA and POA resistant isolates with functional PncA revealed missense mutations in panD that encodes l -aspartate-α-decarboxylase, a rate-limiting step in the CoA biosynthetic pathway [42,44] .…”

“…The C-terminal extension appears to be a hot spot for POA resistance mutations [45] . In addition to isolation of panD mutant strains in vitro, Gopal et al recovered POA resistant panD mutant strains (82% of POA resistant strains) from the lungs of mice that were infected with M. tuberculosis and treated with POA [41] . Mice infected with a panD missense mutant strain had bacterial lung burdens comparable to those infected with wild-type M. tuberculosis [41] , indicating that panD mutant strains maintain fitness in vivo.…”

The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD

“…What is certain is that PZA is a prodrug that is converted into pyrazinoic acid (POA) by the enzyme pyrazinamidase, PncA (Rv2043c) 8 , and that most of the PZA clinical resistance arises from loss of function mutations in pncA. However, multiple mechanisms of action and targets have been reported for PZA [9][10][11][12][13][14][15][16][17][18] . Recently, Zhang et al reported several PZA resistant mutations that mapped to the Mtb panD gene 19,20 , suggesting it was a target of PZA.…”