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Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Speaker Abstracts
28
Visualizing the Mycobacterial Mutasome
Michael A. Reiche
1
, Dirk Lang
2
, Valerie Mizrahi
1,3
, Digby F. Warner
1,3
.
1
University of Cape Town, Cape Town, Western Cape, South Africa,
2
University of Cape Town,
Cape Town, Western Cape, South Africa,
3
University of Cape Town, Cape Town, Western Cape,
South Africa.
Previous work in our laboratory identified a DNA damage-inducible mutagenic DNA repair
system that is required for adaptive mutagenesis, including the development of drug resistance,
in
Mycobacterium tuberculosis
(
Mtb
), the causative agent of tuberculosis (TB). Moreover,
genetic studies established that this so-called “mycobacterial mutasome” comprises a minimum
of three essential components: a
dnaE2
-encoded PolIIIα subunit, and
imuB
- and
imuA
’- encoded
accessory factors. Current work aims to investigate the recruitment dynamics and sub-cellular
localization of the mutasome components following exposure of bacilli to genotoxic stress. To
this end, we constructed a panel of
M
.
smegmatis
(
Msm
) reporter mutants encoding fluorescently
tagged mutasome proteins. The DNA damage survival and induced mutagenesis functions of the
recombinant proteins were assessed, and fluorescence visualized and quantified in a series of
DNA damage assays. In addition, population-wide expression characteristics were assessed by
flow cytometry. Results indicate that expression of enhanced green fluorescent protein (EGFP)
from the DNA damage-responsive imuA’ promoter initiates 60 minutes post exposure to
mitomycin C (MMC), a known genotoxin, with maximal EGFP expression achieved 360 minutes
post treatment. These results indicate that
Msm
responds rapidly to DNA damage, with dynamics
and level of expression correlating with the amount of damage incurred. Moreover, cellular
localization of mutasome components indicates differential recruitment and localization of
ImuA’ and ImuB in MMC-exposed cells. In combination, our results establish the utility of
combining fluorescence imaging with functional genetics to elucidate the mechanisms regulating
expression and activity of a major error-prone damage tolerance pathway in
Mtb
.