A comprehensive characterization of PncA polymorphisms that confer resistance to pyrazinamide

Adam N Yadon,James C Sacchettini,Kashmeel Maharaj,Thomas R Ioerger,Alexander S Pym,Yi-Pin Lai,Eric J Rubin,John H Adamson

doi:10.1038/s41467-017-00721-2

Adam N Yadon, James C Sacchettini + Show 6 more

Open Access

https://doi.org/10.1038/s41467-017-00721-2

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Abstract

Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance. Resistance is mediated through mutations in the bacterial gene pncA. Methods for testing pyrazinamide susceptibility are difficult and rarely performed, and this means that the full spectrum of pncA alleles that confer clinical resistance to pyrazinamide is unknown. Here, we performed in vitro saturating mutagenesis of pncA to generate a comprehensive library of PncA polymorphisms resultant from a single-nucleotide polymorphism. We then screened it for pyrazinamide resistance both in vitro and in an infected animal model. We identify over 300 resistance-conferring substitutions. Strikingly, these mutations map throughout the PncA structure and result in either loss of enzymatic activity and/or decrease in protein abundance. Our comprehensive mutational and screening approach should stand as a paradigm for determining resistance mutations and their mechanisms of action.

Highlights

Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance
MTBDRplus (Hain Lifescience, Nehren, Germany) rapidly detect mutations that most frequently cause resistance to rifampin and INH23–30. Such assays rely on small genomic “hot-spot” regions where highly penetrant mutations directly correlate with phenotypic drug resistance
We found that 100% of clones that were enriched either in vitro or during infection in mice had an elevated minimum inhibitory concentrations (MICs) (>4-fold increase compared to the complemented with a WT pncA gene (Comp) control)

Summary

Introduction

Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance. We performed in vitro saturating mutagenesis of pncA to generate a comprehensive library of PncA polymorphisms resultant from a singlenucleotide polymorphism We screened it for pyrazinamide resistance both in vitro and in an infected animal model. MTBDRplus (Hain Lifescience, Nehren, Germany) rapidly detect mutations that most frequently cause resistance to rifampin and INH23–30. Such assays rely on small genomic “hot-spot” regions where highly penetrant mutations directly correlate with phenotypic drug resistance. Such an assay for PZA would be extremely beneficial

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