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Abstract：
Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab\'s ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR\'s contribution to pathogenesis.
摘要：
脓肿分枝杆菌（Mab）是一种机会性病原体，困扰着患有潜在肺部疾病的个体，例如囊性纤维化（CF）或免疫缺陷。目前针对Mab感染的治疗策略受限于其固有的抗生素抗性和在其体内生态位中获得Mab的有限药物,导致30-50%的差的治愈率。Mab在巨噬细胞内存活的能力，肉芽肿和CF肺充满粘液的气道需要通过转录重塑来适应，以抵消缺氧等应激，硝酸盐含量增加，亚硝酸盐,和反应性氮中间体。已知结核分枝杆菌（Mtb）通过硝酸还原酶narGHJI诱导呼吸道硝酸盐同化来协调低氧适应。Mab,另一方面,不编码呼吸硝酸还原酶。此外,我们最近对Mab对缺氧的转录反应的研究揭示了含有推定的硝酸盐同化基因的基因座的明显下调，包括孤儿反应调节剂nnaR(硝酸盐/亚硝酸盐同化调节剂)。这些推定的硝酸盐同化基因，narK3（硝酸盐/亚硝酸盐转运蛋白），nirBD（亚硝酸还原酶），nnaR,和sirB（铁螯合酶）连续排列，而nasN（在这项工作中鉴定出的同化硝酸还原酶）在不同的基因座中编码。Mab中缺乏呼吸性硝酸还原酶和低氧中氮代谢基因的下调表明，低氧适应与硝酸盐同化之间的相互作用与Mtb中先前记录的不同。Mab在胁迫（例如缺氧）的背景下微调氮代谢的转录调节的机制，特别是NnaR的作用，仍然知之甚少。为了评估NnaR在硝酸盐代谢中的作用，我们构建了MabnnnaR敲除菌株（MabΔnnnaR）和补体（MabΔnnnaRC）来研究转录调控和表型。qRT-PCR显示NnaR对于调节硝酸盐和亚硝酸盐还原酶以及推定的硝酸盐转运蛋白是必需的。NnaR的损失损害了Mab吸收硝酸盐或亚硝酸盐作为唯一氮源的能力，这凸显了其必要性。这项工作为MabNnaR的作用提供了第一个见解，为未来研究NnaR对发病机理的贡献奠定了基础。
