McCoy Andrea J, Sandlin Robin C, Maurelli Anthony T
Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
J Bacteriol. 2003 Feb;185(4):1218-28. doi: 10.1128/JB.185.4.1218-1228.2003.
Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.
衣原体属的微生物是专性细胞内寄生的革兰氏阴性细菌，具有双态发育周期，整个过程在称为包涵体的膜结合液泡内进行。衣原体异常是指这样一个事实：细胞壁活性抗生素会抑制衣原体生长，且基因组中存在肽聚糖（PG）合成基因，但却没有PG合成的生化证据。在这项研究中，我们采用基于遗传学的方法，通过对MurA进行表征来重新评估衣原体异常，MurA是一种UDP-N-乙酰葡糖胺烯醇丙酮酸转移酶，催化PG合成的第一步关键反应。沙眼衣原体血清型L2的murA基因被克隆，并置于阿拉伯糖诱导、葡萄糖抑制的ara启动子控制下，然后转化到大肠杆菌中。将致死性的ΔmurA突变导入该菌株后，大肠杆菌菌株的活力变得依赖于沙眼衣原体murA的表达。对沙眼衣原体murA的DNA序列分析预测，MurA活性位点内的一个关键残基发生了半胱氨酸到天冬氨酸的变化。在大肠杆菌中，此前已证明相同的突变会导致对磷霉素产生抗性，磷霉素是一种专门靶向MurA的强效抗生素。衣原体MurA的体外活性对高水平的磷霉素具有抗性。沙眼衣原体的生长也对磷霉素具有抗性。此外，表达衣原体murA的大肠杆菌菌株也获得了磷霉素抗性。沙眼衣原体原体向网状体的转化以及细胞分裂与murA mRNA的表达相关。在沙眼衣原体感染期间也检测到了PG途径中第二个酶促反应的murB的mRNA。我们的研究结果以及其他团队的研究表明，衣原体属中存在功能性的PG途径。我们提出，衣原体PG对于发育周期的进程以及细胞分裂至关重要。阐明衣原体属中PG的存在对于开发针对衣原体细胞壁的新型抗生素具有重要意义。
