Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis
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.
References
[1]
Cadet J, Ravanat JL, TavernaPorro M, Menoni H, Angelov D (2012) Oxidatively generated complex DNA damage: tandem and clustered lesions. Cancer Lett 327: 5–15. doi: 10.1016/j.canlet.2012.04.005
[2]
Bogdan C, R？llinghoff M, Diefenbach A (2000) Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 12: 64–76. doi: 10.1016/s0952-7915(99)00052-7
[3]
Kowalczykowski SC (1991) Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins. Biochimie 73: 289–304. doi: 10.1016/0300-9084(91)90216-n
[4]
Kowalczykowski SC (1994) In vitro reconstitution of homologous recombination reactions. Experientia 50: 204–215. doi: 10.1007/bf01924003
[5]
Della M, Palmbos PL, Tseng HM, Tonkin LM, Daley JM, et al. (2004) Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine. Science 306: 683–685. doi: 10.1126/science.1099824
[6]
Gong C, Bongiorno P, Martins A, Stephanou NC, Zhu H, et al. (2005) Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C. Nat Struct Mol Biol. 12: 304–312. doi: 10.1038/nsmb915
[7]
Korycka-Machala M, Brzostek A, Rozalska S, Rumijowska-Galewicz A, Dziedzic R, et al. (2006) Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis. FEMS Microbiol Lett 258: 83–91. doi: 10.1111/j.1574-6968.2006.00199.x
[8]
Stephanou NC, Gao F, Bongiorno P, Ehrt S, Schnappinger D, et al. (2007) Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks. J Bacteriol 189: 5237–5246. doi: 10.1128/jb.00332-07
[9]
Pitcher RS, Green AJ, Brzostek A, Korycka-Machala M, Dziadek J, et al. (2007) NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation. DNA Repair (Amst) 6: 1271–1276. doi: 10.1016/j.dnarep.2007.02.009
[10]
Gupta R, Barkan D, Redelman-Sidi G, Shuman S, Glickman MS (2011) Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways. Mol Microbiol 79: 316–330. doi: 10.1111/j.1365-2958.2010.07463.x
[11]
Aniukwu J, Glickman MS, Shuman S (2008) The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends. Genes Dev 22: 512–527. doi: 10.1101/gad.1631908
[12]
Bowater R, Doherty AJ (2006) Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining. PLoS Genet 2(2): e8. doi: 10.1371/journal.pgen.0020008
[13]
Takata M, Sasaki MS, Sonoda E, Morrison C, Hashimoto M, et al. (1998) Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J 17: 5497–5508. doi: 10.1093/emboj/17.18.5497
[14]
Gorna AE, Bowater RP, Dziadek J (2010) DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection. Clin Sci 119: 187–202. doi: 10.1042/cs20100041
[15]
Pitcher RS, Brissett NC, Doherty AJ (2007) Nonhomologous end-joining in bacteria: a microbial perspective. Annu Rev Microbiol 61: 259–282. doi: 10.1146/annurev.micro.61.080706.093354
[16]
Cabiscol E, Tamarit J, Ros J (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol 3: 3–8.
[17]
Kurthkoti K, Varshney U (2012) Distinct mechanisms of DNA repair in mycobacteria and their implications in attenuation of the pathogen growth. Mech Ageing Dev 133: 138–146. doi: 10.1016/j.mad.2011.09.003
[18]
Warner DF, Mizrahi V (2006) Tuberculosis chemotherapy: the influence of bacillary stress and damage response pathways on drug efficacy. Clin Microbiol Rev 19: 558–570. doi: 10.1128/cmr.00060-05
[19]
Wojcik EA, Brzostek A, Bacolla A, Mackiewicz P, Vasquez KM, et al. (2012) Direct and inverted repeats elicit genetic instability by both exploiting and eluding DNA double-strand break repair systems in mycobacteria. PLoS One 7: e51064 doi:10.1371/journal.pone.0051064.
[20]
Parish T, Stoker NG (2000) Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146: 1969–1975.
[21]
Dziadek J, Rajagopalan M, Parish T, Kurepina N, Greendyke R, et al. (2002) Mutations in the CCGTTCACA DnaA box of Mycobacterium tuberculosis oriC that abolish replication of oriC plasmids are tolerated on the chromosome. J Bacteriol 184: 3848–3855. doi: 10.1128/jb.184.14.3848-3855.2002
[22]
Brzostek A, Dziadek B, Rumijowska-Galewicz A, Pawelczyk J, Dziadek J (2007) Cholesterol oxidase is required for virulence of Mycobacterium tuberculosis. FEMS Microbiol Lett 275: 106–112. doi: 10.1111/j.1574-6968.2007.00865.x
[23]
Brzostek A, Pawelczyk J, Rumijowska-Galewicz A, Dziadek B, Dziadek J (2009) Mycobacterium tuberculosis is able to accumulate and utilize cholesterol. J Bacteriol 191: 6584–6591. doi: 10.1128/jb.00488-09
[24]
Brzostek A, Rumijowska-Galewicz A, Dziadek B, Wojcik EA, Dziadek J (2013) ChoD and HsdD can be dispensable for cholesterol degradation in mycobacteria. J Steroid Biochem Mol Biol 134: 1–7. doi: 10.1016/j.jsbmb.2012.09.028
[25]
Brzezinska M, Szulc I, Brzostek A, Klink M, Kielbik M, et al. (2013) The role of 3-ketosteroid 1(2)-dehydrogenase in the pathogenicity of Mycobacterium tuberculosis. BMC Microbiol 13: 43. doi: 10.1186/1471-2180-13-43
[26]
Pitcher RS, Tonkin LM, Green AJ, Doherty AJ (2005) Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis. J Mol Biol 351: 531–544. doi: 10.1016/j.jmb.2005.06.038
[27]
Sander P, Papavinasasundaram KG, Dick T, Stavropoulos E, Ellrott K, et al. (2001) Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model. Infect Immun 69: 3562–3568. doi: 10.1128/iai.69.6.3562-3568.2001
[28]
Richardson AR, Soliven KC, Castor ME, Barnes PD, Libby SJ, et al. (2009) The Base Excision Repair system of Salmonella enterica serovar typhimurium counteracts DNA damage by host nitric oxide. PLoS Pathog 5(5): e1000451 doi:10.1371/journal.ppat.1000451.
[29]
Carpenter EP, Corbett A, Thomson H, Adacha J, Jensen K, et al. (2007) AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis. EMBO J 26: 1363–1372. doi: 10.1038/sj.emboj.7601593
[30]
Mertens K, Lantsheer L, Ennis DG, Samuel JE (2008) Constitutive SOS expression and damage-inducible AddAB-mediated recombinational repair systems for Coxiella burnetii as potential adaptations for survival within macrophages. Mol Microbiol 69: 1411–1426. doi: 10.1111/j.1365-2958.2008.06373.x
[31]
Davies BW, Bogard RW, Dupes NM, Gerstenfeld TA, Simmons LA, et al. (2011) DNA damage and reactive nitrogen species are barriers to Vibrio cholerae colonization of the infant mouse intestine. PLoS Pathog 7(2): e1001295 doi:10.1371/journal.ppat.1001295.
[32]
Boshoff HI, Reed MB, Barry CE 3rd, Mizrahi V (2003) DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113: 183–193. doi: 10.1016/s0092-8674(03)00270-8
[33]
Sassetti CM, Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A 100: 12989–12994. doi: 10.1073/pnas.2134250100
[34]
Darwin KH, Nathan CF (2005) Role for nucleotide excision repair in virulence of Mycobacterium tuberculosis. Infect Immun 73: 4581–4587. doi: 10.1128/iai.73.8.4581-4587.2005
[35]
Houghton J, Townsend C, Williams AR, Rodgers A, Rand L, et al. (2012) Important role for Mycobacterium tuberculosis UvrD1 in pathogenesis and persistence apart from its function in nucleotide excision repair. J Bacteriol 194: 2916–2923. doi: 10.1128/jb.06654-11
[36]
Dutta NK, Mehra S, Didier PJ, Roy CJ, Doyle LA, et al. (2010) Genetic requirements for the survival of tubercle bacilli in primates. J Infect Dis 201: 1743–1752. doi: 10.1086/652497
[37]
Cuccui J, Easton A, Chu KK, Bancroft GJ, Oyston PC, et al. (2007) Development of signature-tagged mutagenesis in Burkholderia pseudomallei to identify genes important in survival and pathogenesis. Infect Immun 75: 1186–1195. doi: 10.1128/iai.01240-06
[38]
Yeager CM, Bottomley PJ, Arp DJ (2001) Requirement of DNA repair mechanisms for survival of Burkholderia cepacia G4 upon degradation of trichloroethylene. Appl Environ Microbiol 67: 5384–5391. doi: 10.1128/aem.67.12.5384-5391.2001
[39]
Buchmeier NA, Lipps CJ, So MYH, Heffron F (1993) Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages. Mol Microbiol 7: 933–936. doi: 10.1111/j.1365-2958.1993.tb01184.x
[40]
Aranda J, Bardina C, Beceiro A, Rumbo S, Cabral MP, et al. (2011) Acinetobacter baumannii RecA protein in repair of DNA damage, antimicrobial resistance, general stress response, and virulence. J Bacteriol 193: 3740–3747. doi: 10.1128/jb.00389-11
[41]
Fletcher HM, Morgan RM, Macrina FL (1997) Nucleotide sequence of the Porphyromonas gingivalis W83 recA homolog and construction of a recA-deficient mutant. Infect Immun 65: 4592–4597.
[42]
Rengarajan J, Bloom BR, Rubin EJ (2005) Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages. Proc Natl Acad Sci U S A 102: 8327–8332. doi: 10.1073/pnas.0503272102
[43]
Fu LM, Shinnick TM (2007) Genome-wide analysis of intergenic regions of Mycobacterium tuberculosis H37Rv using Affymetrix GeneChips. EURASIP J Bioinform Syst Biol doi: 10.1155/2007/23054.
[44]
Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA, et al. (2003) Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment. J Exp Med 198: 693–704. doi: 10.1084/jem.20030846
[45]
Dos Vultos T, Mestre O, Tonjum T, Gicquel B (2009) DNA repair in Mycobacterium tuberculosis revisited. FEMS Microbiol Rev 33: 471–487. doi: 10.1111/j.1574-6976.2009.00170.x
[46]
Raja A (2004) Immunology of tuberculosis. Indian J Med Res 120: 213–232.
[47]
Yang CS, Yuk JM, Jo EK (2009) The role of nitric oxide in mycobacterial infections. Immune Netw 9: 46–52. doi: 10.4110/in.2009.9.2.46
[48]
Szabó C (2003) Multiple pathways of peroxynitrite cytotoxicity. Toxicol Lett 140–141: 105–112. doi: 10.1016/s0378-4274(02)00507-6
[49]
Fang FC (1997) Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity. J Clin Invest 99: 2818–2825. doi: 10.1172/jci119473
