<i>Mycobacterium abscessus</i> virulence traits unraveled by transcriptomic profiling in amoeba and macrophages

Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival.自由生活阿米巴被认为是一个环境生态位，耐阿米巴细菌可在此环境中逐步演化出致病性。为深入解析参与胞内生存适应的相关调控因子，我们对新兴致病菌脓肿分枝杆菌（Mycobacterium abscessus）在阿米巴与小鼠巨噬细胞（Mϕ）中的转录组活性进行了系统表征，并将其与亲缘关系密切但致病性较弱的龟分枝杆菌（Mycobacterium chelonae）的阿米巴内转录组进行了对比分析。针对阿米巴中上调基因的分析结果显示，脓肿分枝杆菌可通过相关蛋白抵御环境应激、诱导宿主防御机制，并实现碳源代谢从碳水化合物向脂肪酸的转换。我们针对阿米巴及/或巨噬细胞中11个上调幅度最高的基因，分别构建了脓肿分枝杆菌的单基因敲除突变菌株；经后续毒力实验分析发现，其中10株突变株在阿米巴及/或巨噬细胞内的增殖能力显著衰减。此外，将其中2个基因转入龟分枝杆菌基因组后，重组菌株在巨噬细胞内的存活能力得到显著提升。其中一株敲除了编码Eis N-乙酰基转移酶蛋白（Eis N-acetyl transferase protein，MAB_4532c）的基因的突变株，在巨噬细胞内的增殖能力出现了尤为显著的衰减。综上，脓肿分枝杆菌的阿米巴内与巨噬细胞内转录组数据揭示了其适应胞内生存模式的能力，而阿米巴在很大程度上促进了脓肿分枝杆菌在巨噬细胞内的存活能力提升。