Teng Fang, Jacques-Palaz Karen D, Weinstock George M, Murray Barbara E
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 77030, USA.
Infect Immun. 2002 Apr;70(4):2010-5. doi: 10.1128/IAI.70.4.2010-2015.2002.
In previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis (epa) from Enterococcus faecalis strain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in Escherichia coli. Disruption of two open reading frames in the epa gene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with E. faecalis endocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecular-weight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF (P < or = 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF (P < or = 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the epa gene cluster of OG1RF (which is also present in the sequenced E. faecalis strain V583) was detected in 12 of 12 other clonally distinct E. faecalis strains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an E. faecalis endocarditis patient serum, and sera from four other patients with E. faecalis endocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the epa gene cluster is widespread among E. faecalis and confers some protection against human host defenses.
在先前的研究中，我们从粪肠球菌菌株OG1RF中克隆了一组参与多糖生物合成的基因（epa），并表明该基因簇介导了大肠杆菌中多糖的合成。破坏OG1RF的epa基因簇中的两个开放阅读框产生了两个突变体TX5179和TX5180，它们在小鼠腹膜炎模型中毒力减弱。在本研究中，用一名粪肠球菌心内膜炎患者的血清以及OG1RF和突变体TX5179和TX5180的多糖提取物进行了蛋白质印迹分析。OG1RF在高分子量区域显示出一条 smear 带，在低分子量区域显示出离散条带，而突变体中则没有这些条带；过碘酸盐处理和碳水化合物染色证实了该物质的多糖性质。在使用OG1RF吸附的正常人血清进行的中性粒细胞杀伤试验中，突变体TX5179和TX5180分别比野生型OG1RF对杀伤的敏感性高50倍和 2.4倍（P≤0.01）。通过荧光吞噬试验，中性粒细胞摄取的突变体比OG1RF多2.5至3倍（P≤0.001）。最后，在高严格条件下进行限制性消化和杂交，在测试的12株其他克隆不同的粪肠球菌菌株中的12株中检测到了OG1RF的epa基因簇（在已测序的粪肠球菌菌株V583中也存在）：使用粪肠球菌心内膜炎患者血清在蛋白质印迹上对这12株菌株检测到了相似的多糖模式，并且来自其他四名粪肠球菌心内膜炎患者的血清均与OG1RF的多糖提取物发生反应。这些结果表明，epa基因簇在粪肠球菌中广泛存在，并赋予了对人类宿主防御的一些保护作用。
