Szulc-Kielbik Izabela, Brzezinska Marta, Kielbik Michal, Brzostek Anna, Dziadek Jaroslaw, Kania Katarzyna, Sulowska Zofia, Krupa Agnieszka, Klink Magdalena
Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.
FEBS J. 2015 Apr;282(7):1289-306. doi: 10.1111/febs.13219. Epub 2015 Feb 13.
Our knowledge about the mechanisms utilized by Mycobacterium tuberculosis to survive inside macrophages is still incomplete. One of the mechanism that protects M. tuberculosis from the host's microbicidal products and allows bacteria to survive involves DNA repair systems such as the homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways. It is accepted that any pathway that contributes to genome maintenance should be considered as potentially important virulence factor. In these studies, we investigated reactive oxygen species, nitric oxide and tumor necrosis factor-α production by macrophages infected with wild-type M. tuberculosis, with an HR-defective mutant (∆recA), with an NHEJ-defective mutant [∆(ku,ligD)], with a mutant defective for both HR and NHEJ [∆(ku,ligD,recA)], or with appropriate complemented strains. We also assessed the involvement of extracellular signal-regulated kinases (ERKs) 1 and 2 in the response of macrophages to infection with the above-mentioned strains, and ERK1/2 phosphorylation in M. tuberculosis-infected macrophages. We found that mutants lacking RecA induced a greater bactericidal response by macrophages than did the wild-type strain or an NHEJ-defective mutant, and activated ERK1/2 was involved only in the response of macrophages to recA deletion mutants [∆(ku,ligD,recA) and ∆recA]. We also demonstrated that only the triple mutant induced ERK1/2 phosphorylation in phorbol-12-myristate-13-acetate-stimulated macrophages. Moreover, HR-defective mutants induced lower amounts of tumor necrosis factor-α secretion than did the wild-type or ∆(ku,ligD). Our results indicate that RecA contributes to M. tuberculosis virulence, and also suggest that diminished ERK1/2 activation in macrophages infected with M. tuberculosis possessing recA may be an important mechanism by which wild-type mycobacteria escape intracellular killing.
我们对结核分枝杆菌在巨噬细胞内存活所利用机制的了解仍不完整。保护结核分枝杆菌免受宿主杀菌产物影响并使其得以存活的机制之一涉及DNA修复系统，如同源重组（HR）和非同源末端连接（NHEJ）途径。人们认为，任何有助于基因组维持的途径都应被视为潜在的重要毒力因子。在这些研究中，我们调查了野生型结核分枝杆菌、HR缺陷型突变体（∆recA）、NHEJ缺陷型突变体[∆(ku,ligD)]、HR和NHEJ均缺陷的突变体[∆(ku,ligD,recA)]或相应互补菌株感染巨噬细胞后活性氧、一氧化氮和肿瘤坏死因子-α的产生情况。我们还评估了细胞外信号调节激酶（ERK）1和2在巨噬细胞对上述菌株感染的反应中的作用，以及结核分枝杆菌感染的巨噬细胞中ERK1/2的磷酸化情况。我们发现，缺乏RecA的突变体比野生型菌株或NHEJ缺陷型突变体诱导巨噬细胞产生更强的杀菌反应，并且活化的ERK1/2仅参与巨噬细胞对recA缺失突变体[∆(ku,ligD,recA)和∆recA]的反应。我们还证明，只有三重突变体在佛波醇-12-肉豆蔻酸酯-13-乙酸酯刺激的巨噬细胞中诱导ERK1/2磷酸化。此外，HR缺陷型突变体诱导的肿瘤坏死因子-α分泌量低于野生型或∆(ku,ligD)。我们的结果表明，RecA有助于结核分枝杆菌的毒力，并且还表明，在感染含有recA的结核分枝杆菌的巨噬细胞中ERK1/2活化减弱可能是野生型分枝杆菌逃避细胞内杀伤的重要机制。
