Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis

Anna Brzostek,Jaroslaw Dziadek,Zofia Sulowska,Marta Brzezinska,Magdalena Klink,Izabela Szulc,Martin G Marinus

doi:10.1371/journal.pone.0092799

Anna Brzostek, Jaroslaw Dziadek + Show 5 more

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https://doi.org/10.1371/journal.pone.0092799

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Abstract

The intracellular pathogen Mycobacterium tuberculosis (Mtb) is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs). These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR) and non-homologous ends joining (NHEJ), in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA), NHEJ [Δ(ku,ligD)], or both DSB repair systems [Δ(ku,ligD,recA)]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA) mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA) mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.

Highlights

Macrophages (MØs), which are derived from monocytes, are professional phagocytic cells specialized in ingesting and killing pathogens
The RecA, Ku, and LigD proteins are essential participants in homologous recombination (HR) and non-homologous ends joining (NHEJ) processes, both of which are involved in the repair of double-strand breaks (DSBs) in mycobacteria [5,6,7], [9], [26]
The repair of ultraviolet radiation (UV) radiation-induced DNA damage in Mycobacterium tuberculosis (Mtb) requires intact RecA It is well known that bacteria defective for RecA synthesis are hypersensitive to UV radiation [8], [27]

Summary

Introduction

Macrophages (MØs), which are derived from monocytes, are professional phagocytic cells specialized in ingesting and killing pathogens. The antimicrobial activity of MØs is due, in part, to the generation of large amounts of highly toxic molecules, including reactive oxygen species (ROS), such as superoxide anion (NO2-), hydrogen peroxide (H2O2), hydroxyl radicals (NOH) and hydroxyl anion (OH-), as well as reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite anion (ONOO-). These reactive species cause oxidative damage to a wide variety of targets, including DNA. Both HR and NHEJ systems have complementary roles in repairing DSBs, but act independently [12], [13]

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