Feng Zhengyu, Barletta Raúl G
Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583-0905, USA.
Antimicrob Agents Chemother. 2003 Jan;47(1):283-91. doi: 10.1128/AAC.47.1.283-291.2003.
D-Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes D-alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of D-alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of L- and D-alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of L- over D-alanine. In all strains, DCS treatment led to a significant accumulation of L-alanine and a concomitant decease of D-alanine, with approximately a 20-fold excess of L-alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the D-alanine branch of mycobacterial peptidoglycan biosynthesis.
D-环丝氨酸（DCS）作用于肽聚糖生物合成酶D-丙氨酸消旋酶（Alr）和D-丙氨酸：D-丙氨酸连接酶（Ddl）。此前，我们证明耻垢分枝杆菌中Alr的过量表达决定了DCS耐药表型。在本研究中，我们调查了Alr和Ddl在耻垢分枝杆菌中DCS作用机制和耐药性中的作用。我们发现，在耻垢分枝杆菌中过表达耻垢分枝杆菌或结核分枝杆菌的ddl基因均可赋予对DCS的耐药性，但耐药水平低于alr基因的过表达。此外，同时过表达alr和ddl基因的菌株显示出高八倍的耐药水平。为了验证DCS抑制Alr会减少细胞内D-丙氨酸池这一假说，我们测定了经或未经DCS处理的耻垢分枝杆菌野生型和重组菌株中的丙氨酸池。未接触DCS的Alr过表达菌株GPM14细胞在稳态下显示L-丙氨酸和D-丙氨酸的量几乎等摩尔。野生型菌株和Ddl过表达菌株中L-丙氨酸比D-丙氨酸多两倍。在所有菌株中，DCS处理导致L-丙氨酸显著积累，同时D-丙氨酸减少，在Ddl过表达菌株中L-丙氨酸过量约20倍。这些数据表明，在抑制Alr的浓度下，DCS对Ddl没有显著抑制作用。本研究对于确定DCS的致死靶点以及开发靶向分枝杆菌肽聚糖生物合成D-丙氨酸分支的新型药物具有重要意义。
