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Article

Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses

1
Institute of Medical Biology of the Polish Academy of Sciences, Lodowa 106, 93-232 Łódź, Poland
2
Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland
3
Department of Molecular Microbiology, Faculty of Biology and Environmental Protection, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland
4
Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pomorska 139, 90-235 Łódź, Poland
*
Authors to whom correspondence should be addressed.
These authors equally contributed to this work.
Academic Editor: Francesco Dieli
Cells 2021, 10(5), 1168; https://doi.org/10.3390/cells10051168
Received: 26 March 2021 / Revised: 6 May 2021 / Accepted: 6 May 2021 / Published: 11 May 2021
Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level. View Full-Text
Keywords: DNA damage repair; SOS response; tuberculosis DNA damage repair; SOS response; tuberculosis
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MDPI and ACS Style

Brzostek, A.; Płociński, P.; Minias, A.; Ciszewska, A.; Gąsior, F.; Pawełczyk, J.; Dziadek, B.; Słomka, M.; Dziadek, J. Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses. Cells 2021, 10, 1168. https://doi.org/10.3390/cells10051168

AMA Style

Brzostek A, Płociński P, Minias A, Ciszewska A, Gąsior F, Pawełczyk J, Dziadek B, Słomka M, Dziadek J. Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses. Cells. 2021; 10(5):1168. https://doi.org/10.3390/cells10051168

Chicago/Turabian Style

Brzostek, Anna, Przemysław Płociński, Alina Minias, Aneta Ciszewska, Filip Gąsior, Jakub Pawełczyk, Bożena Dziadek, Marcin Słomka, and Jarosław Dziadek. 2021. "Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses" Cells 10, no. 5: 1168. https://doi.org/10.3390/cells10051168

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