DOI: 10.4103/ijmy.ijmy_8_26 ISSN: 2212-5531
Gene-specific Functional Roles of MSMEG_5046, MSMEG_0241, and MSMEG_0232 in Pyrazinoic Acid Efflux Identified through Clustered Regularly Interspaced Short Palindromic Repeat Interference
Kevin R. Obando Ballardo, Kiara Aricoche-Del Campo, B Carlos Alonso Flores, Robert H. Gilman, Mirko Zimic, Patricia Sheen
Background:
Efflux-mediated export of pyrazinoic acid (POA) has been associated with pyrazinamide (PZA) resistance, yet the specific transport components remain incompletely defined.
Mycobacterium smegmatis
, which exhibits intrinsically high POA efflux, provides a quantitative model to study PZA/POA transport mechanisms.
Methods:
Using clustered regularly interspaced short palindromic repeats interference, we silenced three efflux pump orthologs of
Mycobacterium tuberculosis
in
M. smegmatis
:
MSMEG_5046
(
Rv1250c
),
MSMEG_0241
(
Rv0202c
/MmpL11), and
MSMEG_0232
(
Rv0191c
). Gene knockdown was validated by quantitative reverse transcription polymerase chain reaction, achieving 45.1-, 14.6-, and 4.18-fold repression, respectively. POA export kinetics were assessed after PZA loading (final concentration 6.5 mM; 800 µg/mL) using a colorimetric assay over 0–60 min. Efflux rates were calculated from slope values, normalized to intracellular protein content, and compared across biological replicates using analysis of covariance.
Results:
All silenced strains showed significant differences in efflux slope compared with controls:
MSMEG_5046
(
P
= 0.0184),
MSMEG_0241
(
P
= 0.0497), and
MSMEG_0232
(
P
< 0.0001). At 60 min, normalized POA export changed by +32% (0.0048 mM POA/protein) for
MSMEG_5046
, −89.33% (0.0134 mM POA/protein) for
MSMEG_0241
, and −39.33% (0.0059 mM POA/protein) for
MSMEG_0232
relative to wild type.
MSMEG_0241
and
MSMEG_0232
knockdowns reduced both efflux slope and total export, supporting a direct role in POA transport, whereas
MSMEG_5046
repression increased efflux, suggesting compensatory activity.
Conclusion:
This protein-normalized, slope-based POA export assay resolves gene-specific contributions within a networked efflux system and prioritizes targeted validation in
M. tuberculosis.