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Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Speaker Abstracts
21
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.