Mycobacterium tuberculosis H37Ra (Mtb-Ra) ORF MRA_1916 is annotated as a D-amino acid oxidase (DAO). These enzymes perform conversion of d-amino acids to corresponding imino acids followed by conversion into α-keto-acids. In the present study Mtb-Ra recombinants with DAO knockout (KO) and knockout complemented with DAO over-expressing plasmid (KOC) were constructed. The growth studies showed loss of growth of KO in medium containing glycerol as a primary carbon source. Substituting glycerol with acetate or with FBS addition, restored the growth. Growth was also restored in complemented strain (KOC). KO showed increased permeability to hydrophilic dye EtBr and reduced biofilm formation. Also, its survival in macrophages was low. Phagosome maturation studies suggested enhanced colocalization of KO, compared to WT, with lysosomal marker cathepsin D. Also, an increased intensity of Rab5 and iNOS was observed in macrophages infected with KO, compared to WT and KOC. The in vivo survival studies showed no increase in CFU of KO. This is the first study to show functional relevance of DAO encoded by MRA_1916 for Mtb-Ra growth on glycerol, its permeability and biofilm formation. Also, this study clearly demonstrates that DAO deletion leads to Mtb-Ra failing to grow in macrophages and in mice.

结核分枝杆菌H37Ra ( Mtb -Ra) ORF MRA_1916 被注释为 D-氨基酸氧化酶 (DAO)。这些酶将d-氨基酸转化为相应的亚氨基酸，然后转化为α-酮酸。在本研究中结核杆菌构建了具有DAO敲除(KO)和用DAO过表达质粒(KOC)补充的敲除的-Ra重组体。生长研究表明，在含有甘油作为主要碳源的培养基中，KO 的生长损失。用醋酸盐或添加 FBS 代替甘油，恢复了生长。补充菌株 (KOC) 也恢复了生长。KO 显示出对亲水性染料 EtBr 的渗透性增加并减少了生物膜的形成。此外，它在巨噬细胞中的存活率很低。吞噬体成熟研究表明，与 WT 相比，KO 与溶酶体标记组织蛋白酶 D 的共定位增强。此外，与 WT 和 KOC 相比，在感染 KO 的巨噬细胞中观察到 Rab5 和 iNOS 的强度增加。在体内生存研究显示 KO 的 CFU 没有增加。这是第一项显示 MRA_1916 编码的 DAO与甘油上Mtb -Ra 生长、其渗透性和生物膜形成的功能相关性的研究。此外，这项研究清楚地表明，DAO 缺失导致Mtb -Ra 无法在巨噬细胞和小鼠中生长。