Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Speaker Abstracts

Comprehensive Mutational Analysis of PncA SNPs Conferring in Vitro and in Vivo Pyrazinamide Resistance in M. Tuberculosis Adam N. Yadon 1,2 , Kashmeel Maharaj 2 , Thomas R. Ioerger 3 , Alex Pym 2 , Eric J. Rubin 1 . 1 Harvard TH Chan School of Public Health, Boston, MA, USA, 2 KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, KwaZulu-Natal, South Africa, 3 Texas A&M University, College Station, TX, USA. Pyrazinamide (PZA) is an integral component of chemotherapy for both drug-susceptible and drug-resistant tuberculosis. Unfortunately, the requirement of acidic media significantly complicates the reproducibility of phenotypic drug-susceptibility testing (DST), thus hindering its widespread use. A faster, molecular diagnostic for identifying PZA susceptibility is urgently required. The primary resistance mechanism to PZA is variants in PncA. This enzyme encodes the bacterial pyrazinamidase that is required for conversion of PZA to its active form, pyrazinoic acid (POA-). Unfortunately, single-nucleotide polymorphisms (SNPs) occur across the entire length of pncA in clinically resistant isolates. The phenotypic consequences of these mutations are unclear. To address this, we have developed an in vitro and in vivo screen to unbiasedly assay for phenotypic drug-susceptibility of all pncA SNPs. We constructed a library of pncA variants using random PCR mutagenesis to complement a ΔpncA strain of M. tuberculosis . The in vitro selection was performed using a range of PZA concentrations (4-500 μg ml -1 ) in a BD BACTEC MGIT 960 PZA Kit. A complementary in vivo screen was also performed by infecting mice by tail vein injection or aerosolization. Treatment with 150 mg ml -1 PZA or a saline control was then administered for up to 42 days. Resistant clones from both the lungs and spleens were evaluated. Illumina sequencing was performed to identify enriched SNPs following in vitro and in vivo selection. Our results have enabled us to identify SNPs conferring phenotypic resistance to PZA and has allowed us to classify these clones as high- or low-level resistance mutations. Importantly, structurally modeling these SNPs onto PncA has furthered our mechanistic understanding of PZA resistance. These results will enable the development of a comprehensive genetic based diagnostic for PZA susceptibility.

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