Guerrini Valentina, Subbian Selvakumar, Santucci Pierre, Canaan Stéphane, Gennaro Maria Laura, Pozzi Gianni
Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
Laboratory of Molecular Microbiology and Biotechnology, Department of Medical Biotechnologies, University of Siena, Siena, Italy.
PLoS One. 2016 Dec 13;11(12):e0167989. doi: 10.1371/journal.pone.0167989. eCollection 2016.
Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.
从临床样本中分离出的人类病原体结核分枝杆菌菌株表现出遗传异质性。这种变异可能源于病原体在巨噬细胞内遇到的应激环境，巨噬细胞是结核杆菌寄生的宿主细胞。为了研究结核分枝杆菌基因组在巨噬细胞内生长过程中的进化，我们建立了一种细胞内培养模型，其中细菌在类似巨噬细胞的THP-1细胞中连续传代约80代细菌。对感染周期前后分离的单个细菌菌落进行基因组测序，结果显示结核分枝杆菌的突变率约为5.7×10-9 / bp/代，这与体内感染期间计算的突变率一致。对突变体在巨噬细胞和小鼠体内生长的分析表明，循环感染后鉴定出的突变相对于野生型对突变体没有优势。此外，对携带其中一种突变的重组蛋白进行活性测试表明，该突变的存在不影响酶活性。本研究中开发的用于研究结核分枝杆菌基因组微进化的连续感染方案可应用于暴露于应激源，以确定它们对巨噬细胞内生长过程中基因组重塑的影响。
