Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in M. tuberculosis strains with existing resistance to isoniazid and rifampicin (MDR) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, Pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotype pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of nineteen phenotypically PZA mono-resistant isolates collected in Sweden and compare their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDRPZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p-138) and N-terminal (ClpC1Val63Ala). Mutations have emerged in both these regions under POA selection in vitro and ClpC1N-terminal has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1Val63Ala mutants spanned 4 Indo-oceanic sublineages. Indo-oceanic isolates invariably harbored ClpC1Val63Ala and were starkly overrepresented (OR=22.2, p <0.00001) among PZA mono-resistant isolates (11/19) compared to MDRPZA-R isolates (5/80). The genetic basis of Indo-oceanic isolates’ overrepresentation in PZA mono-resistant TB remains undetermined, but substantial circumstantial evidence suggests ClpC1Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.

Pyrazinamide Susceptibility Is Driven by Activation of the SigE-Dependent Cell Envelope Stress Response in Mycobacterium tuberculosis

“…Of these isolates, 10 had unique insertions and 4 appeared to be siblings. Consistent with other recent studies ( 20 – 22 , 32 – 34 ), insertions were identified in the carboxy-terminal coding region of panD and in the promoter region of clpC1 ( Table S2 ). Eight additional unique insertions were identified within seven other genes ( Table S2 ), four of which we recently associated with PZA or POA resistance ( 35 ).…”

“…Considering that most clinical PZA resistance is mediated by pncA mutations and an intact SigE regulon is essential for tolerating other antitubercular therapeutics ( 46 ), we reasoned that any enrichment for sigE mutants would be exceedingly rare and appear more frequently—if not exclusively—in pncA WT PZA-monoresistant isolates. Accordingly, we contrasted the prevalence of sigE mutations in a large global set of clinical isolates ( n = 1,215) to a recently curated set of PZA-monoresistant isolates ( n = 18) in which pncA WT isolates were markedly overrepresented ( 34 ), including three that are both pncA WT and panD WT . Among all isolates, six nonsynonymous sigE mutations were observed.…”

Novel and Reported Compensatory Mutations in rpoABC Associate Specifically with Predominant Mycobacterium tuberculosis Rifampicin Resistance Marker rpoB:S450L

“…However, as rpoB :I1106T/X were only carried by isolates from this cluster, it is uncertain whether the mutation truly improved fitness, or was simply a neutral mutation carried by the common ancestor of the outbreak. Monophyletic mutations can still effect phenotype 30 , however as with any association, follow-up mutagenesis is needed to show causation.…”

“…Monophyletic mutations can still effect phenotype 30 , however as with any association, follow-up mutagenesis is needed to show causation.…”

Development of amidase-dependent pyrazinoic acid prodrugs with activity against pyrazinamide resistant Mycobacterium tuberculosis

“…The main driver of clinical resistance to PZA is through development of mutations in the PncA encoding gene, pncA (18)(19)(20)(21)(22). Resistant mutants raised on high concentrations of POA in vitro, as well as roughly 10% of PZA-resistant clinical Mtb isolates, can also develop mutations in panD (23)(24)(25). The panD gene encodes for an aspartate decarboxylase which carries out the penultimate step in pantothenate biosynthesis, an essential precursor of coenzyme A synthesis.…”