Non- pncA Gene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

Abstract

Pyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDR) tuberculosis (TB). gene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDR TB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-type gene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy: or mutations identified by whole-genome sequencing (WGS) ( = 7), heteroresistance ( = 8), and mixed populations with ( = 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDR clinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymous mutation (Thr87Met), whereas a nonsynonymous mutation was found in 17 isolates. None of these isolates carried a nonsynonymous mutation, while all 15 of the MDR isolates harbored a nonsynonymous mutation. Our findings indicate that it is necessary to consider the occurrence of mutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymous mutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role of in PZA resistance.

Pyrazinoic Acid Inhibits the Bifunctional Enzyme (Rv2783) in Mycobacterium tuberculosis by Competing with tmRNA

“…Further structural studies indicate that POA binds to RpsA through hydrophobic interactions by hydrogen bonds, which are mainly mediated by residues (Lys303, Phe307, Phe310, and Arg357) that are also crucial for tmRNA binding [7]. rpsA mutations seem to play a role in the PZA resistance of some other clinical isolates without pncA mutations [8,9]. However, rpsA polymorphisms are seemingly found in some PZA-susceptible clinical strains from closely related geographical regions.…”

Introducing RpsA Point Mutation Δ438A or D123A into the Chromosome of M. tuberculosis Confirms its Role in Causing Resistance to Pyrazinamide

“…That RpsA is a target of PZA (13) is supported by a number of observations including association of RpsA mutations and PZA resistance in clinical strains (3, 5, 9, 14, 18, 23), rpsA overexpression causing PZA resistance (13), as well as biochemical and structural studies showing the binding of the drug to the RpsA (4, 15, 25). Despite these supporting studies, and in view of the question about the role of RpsAΔ438A mutation in PZA resistance, point mutation construction into the chromosome, though challenging, is by far the most convincing method to prove if a specific mutation is the cause for drug resistance.…”

“…It is well established that mutations in the pncA gene encoding nicotinamidase/pyrazinamidase (PZase) involved in conversion of PZA to active form pyrazinoic acid (POA) are the major mechanism of PZA resistance (11, 32), accounting for85% of all PZA resistance (32). However, some PZA-resistant strains without pncA mutations may have mutations in potential targets of PZA, including ribosomal protein S1 (RpsA) involved in trans-translation (5, 9, 13, 17, 24), aspartate decarboxylase (PanD) (12, 27) involved in synthesis of β-alanine – a precursor for pantothenate and CoA biosynthesis, and ATP-dependent protease ClpC1(26, 28). We have previously identified a 3–base pair “GCC” deletion resulting in loss of an Alanine at amino acid 438 in RpsA in a low-level PZA-resistant clinical isolate DHM444 (MIC = 200 to 300 μg/ml PZA compared with 100 μg/ml in susceptible M. tuberculosis) without pncA mutation (13).…”

Pyrazinamide/Pyrazinoic Acid Resistance in Mycobacterium Tuberculosis: Recent Findings and Implications for Improving the Treatment of Tuberculosis

“…RpsA is a component of trans-translation, a rescue mechanism for stalled ribosomes. Although the role of rpsA mutations in PZA resistance is disputed, association studies of larger collections of clinical isolates does suggest some involvement [63]. RspA does not appear to be a target of PZA/POA, as recent work using laboratory mutants did not show any effect of PZA (POA) on trans-translation or the expression of RpsA [17], but RspA does seem to play a role in the susceptibility to PZA/POA.…”