Mycobacterium tuberculosis Survival in J774A.1 Cells Is Dependent on MenJ Moonlighting Activity, Not Its Enzymatic Activity
Mycobacterium tuberculosis Survival in J774A.1 Cells Is Dependent on MenJ Moonlighting Activity, Not Its Enzymatic Activity
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DOI：
10.1021/acsinfecdis.0c00312
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发表时间：
2020-10-09
影响因子：
5.3
通讯作者：
Crick, Dean C.
中科院分区：
文献类型：
--
作者：
Kumar, Santosh;Koehn, Jordan T.;Crick, Dean C.
MENJ是一种黄素蛋白氧化还原酶，负责分枝杆菌甲喹酮的β-异戊二烯单元的饱和，导致含有9个异戊二烯单元的甲喹酮(MK-9)转化为含有9个异戊二烯单元的甲喹酮，其中第二单元中的双键被还原[MK-9(II-H-2)]。MK-9的氢化提高了分枝杆菌电子传递系统的效率，而MenJ的缺失导致细菌在J774A.1巨噬细胞样细胞内的存活率下降，但不是培养中生长所必需的。因此，有人认为Menj可能代表结核分枝杆菌的上下文药物靶点，即仅在受感染的巨噬细胞背景下有效的药物靶点。然而，目前尚不清楚是MK-9向MK-9(II-H-2)的转换还是MenJ蛋白本身对细菌存活负责。为了解决这个问题，我们设计了一种表达折叠的、全长的、无活性的MenJ的质粒。初步序列分析表明，Menj与古细菌香叶基香叶还原酶具有保守的FAD结合、NADH结合、催化基序和C末端基序。在这些基序中任何一个缺失的Menj突变体都没有还原酶活性。因此，产生了保守基序中高度保守的氨基酸的点突变，并通过圆二色谱和氧化还原酶活性来监测重组蛋白的构象变化。突变分析表明，结核分枝杆菌Menj的色氨酸215(W215)和半胱氨酸46(C46)是MK-9氢化所必需的氨基酸，它们存在于已知的古香叶基香叶基还原酶和可能的菜喹酮饱和酶中。MANJ中C46突变为丝氨酸(C46S)或W215突变为亮氨酸(W215L)在体外完全丧失了催化活性，表达C46S或W2151突变蛋白的结核分枝杆菌MenJ基因敲除株不能将MK-9转化为MK-9(II-H-2)，但在J774A.1细胞内存活。因此，令人惊讶的是，结核分枝杆菌在J774A.1细胞中的生存依赖于Menj的表达，而不是它的氧化还原酶活性，即Mk9向MK-9(II-H-2)的转化。总体而言，目前的数据表明，Menj是一种兼职蛋白质。
MenJ, a flavoprotein oxidoreductase, is responsible for the saturation of the beta-isoprene unit of mycobacterial menaquinone, resulting in the conversion of menaquinone with nine isoprene units (MK-9) to menaquinone with nine isoprene units where the double bond in the second unit is reduced [MK-9(II-H-2)]. The hydrogenation of MK-9 increases the efficiency of the mycobacterial electron transport system, whereas the deletion of MenJ results in decreased survival of the bacteria inside J774A.1 macrophage-like cells but is not required for growth in culture. Thus, it was suggested that MenJ may represent a contextual drug target in M. tuberculosis, that is, a drug target that is valid only in the context of an infected macrophage. However, it was unclear if the conversion of MK-9 to MK-9(II-H-2) or the MenJ protein itself was responsible for bacterial survival. In order to resolve this issue, a plasmid expressing folded, full-length, inactive MenJ was engineered. Primary sequence analysis data revealed that MenJ shares conserved FAD binding, NADH binding, and catalytic and C-terminal motifs with archaeal geranylgeranyl reductases. A MenJ mutant deficient in any one of these motifs is devoid of reductase activity. Therefore, point mutations of highly conserved amino acids in the conserved motifs were generated and the recombinant proteins were monitored for conformational changes by circular dichroism and oxidoreductase activity. The mutational analysis indicates that amino acids tryptophan 215 (W215) and cysteine 46 (C46) of M. tuberculosis MenJ, conserved in known archaeal geranylgeranyl reductases and putative menaquinone saturases, are essential to the hydrogenation of MK-9. The mutation of either C46 to serine (C46S) or W215 to leucine (W215L) in MenJ completely abolishes the catalytic activity in vitro, and menJ knockout strains of M. tuberculosis expressing either the C46S or W2151, mutant protein are unable to convert MK-9 to MK-9(II-H-2) but survive inside the J774A.1 cells. Thus, surprisingly, the survival of M. tuberculosis in J774A.1 cells is dependent on the expression of MenJ rather than its oxidoreductase activity, the conversion of MK9 to MK-9(II-H-2) as previously hypothesized. Overall, the current data suggest that MenJ is a moonlighting protein.