Impairment of D-alanine biosynthesis in Mycobacterium smegmatis determines decreased intracellular survival in human macrophages
Impairment of D-alanine biosynthesis in Mycobacterium smegmatis determines decreased intracellular survival in human macrophages
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DOI：
10.1099/mic.0.024901-0
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发表时间：
2009-05-01
期刊：
影响因子：
2.8
通讯作者：
Barletta, Raul G.
中科院分区：
文献类型：
--
作者：
Chacon, Ofelia;Bermudez, Luiz E.;Barletta, Raul G.
D-丙氨酸是分枝杆菌肽聚糖的结构组分。在真细菌中D-丙氨酸生物合成的主要途径是由L-丙氨酸转化为对映体，该反应由D-丙氨酸消旋酶（Alr）催化。耻垢分枝杆菌插入突变体的生长不依赖于D-丙氨酸，并显示出与D-丙氨酸生物合成的替代途径一致的代谢模式。在这项研究中，我们证明了M。丝霉酸插入突变株TAM 23合成D-丙氨酸的水平低于亲本菌株。alr基因的插入失活也降低了突变株在原代人单核细胞衍生的巨噬细胞内的细胞内存活。通过互补研究，我们证实alr基因功能的损伤是导致生存率下降的原因。抑制超氧阴离子和一氧化氮的形成，抑制巨噬细胞的差异生存。相反，对于在肉汤中生长的细菌，两种菌株对过氧化氢、酸化亚硝酸钠、低pH和多粘菌素B的敏感性大致相同。相反，TAM 23表现出对溶菌酶的抗性增加。D-丙氨酸补充显著增加了TAM 23在营养缺乏培养基中和巨噬细胞内的活力。这些结果表明，吞噬细胞中的营养剥夺结合活性中间体介导的杀伤是alr突变体存活率降低的基础。这些知识可能对构建分枝杆菌营养缺陷型候选疫苗有价值。
D-Alanine is a structural component of mycobacterial peptidoglycan. The primary route of D-alanine biosynthesis in eubacteria is the enantiomeric conversion from L-alanine, a reaction catalysed by D-alanine racemase (Alr). Mycobacterium smegmatis alr insertion mutants are not dependent on D-alanine for growth and display a metabolic pattern consistent with an alternative pathway for D-alanine biosynthesis. In this study, we demonstrate that the M. smegmatis alr insertion mutant TAM23 can synthesize D-alanine at lower levels than the parental strain. The insertional inactivation of the alr gene also decreases the intracellular survival of mutant strains within primary human monocyte-derived macrophages. By complementation studies, we confirmed that the impairment of alr gene function is responsible for this reduced survival. Inhibition of superoxide anion and nitric oxide formation in macrophages suppresses the differential survival. In contrast, for bacteria grown in broth, both strains had approximately the same susceptibility to hydrogen peroxide, acidified sodium nitrite, low pH and polymyxin B. In contrast, TAM23 exhibited increased resistance to lysozyme. D-Alanine supplementation considerably increased TAM23 viability in nutritionally deficient media and within macrophages. These results suggest that nutrient deprivation in phagocytic cells combined with killing mediated by reactive intermediates underlies the decreased survival of alr mutants. This knowledge may be valuable in the construction of mycobacterial auxotrophic vaccine candidates.