Analysis of Physiological role of glutamic acid-specific protease produced by Gram-positive bacteria in the oral cavity

我们已经纯化了一种新的谷氨酰内肽酶从表皮葡萄球菌（GluSE）的阳离子交换色谱，超滤，和尺寸排阻HPLC。该蛋白酶的表观分子量为27 kDa，最适pH为8.0。该蛋白酶特异性地催化合成肽Z-LLE-MCA的谷氨酸羰基末端的水解。利用反向聚合酶链反应（PCR）技术，从S.表皮葡萄球菌ATCC 14990的纯化蛋白的N-末端氨基酸序列和金黄色葡萄球菌谷氨酰内肽酶的相对保守区域的DNA序列。预测的gse基因产物由282个氨基酸组成，分子量为30，809，具有66个氨基酸残基的前体序列。当细胞在琼脂培养基上的透析膜上培养而不是在培养基中培养时，优先证明GluSE的产生，并且这种产生显然与生物膜的形成相关。因此，推测GluSE在细菌通过生物膜附着于塑料器械表面的机制中发挥了一定作用。Southern杂交分析表明GluSE基因在染色体DNA上以单拷贝存在。GluSE基因的分布也通过PCR在凝固酶阴性葡萄球菌的临床分离株进行了检查。该基因在所有的S.表皮葡萄球菌分离株（65/65），而S.金黄色葡萄球菌或其他CNS分离株，包括S. capitis、S. haemolyticus、S. hominis和S.瓦内里。此外，72.3%（47/65）的S.表皮分离物。这些结果表明，GluSE基因是普遍存在的，并专门保存在S。表皮

We have purified a novel glutamyl endopeptidase from Staphylococcus epidermidis (GluSE) by cation-exchange chromatography, ultrafiltration, and size-exclusion HPLC. An apparent molecular mass of the protease was 27 kDa and the optimal pH of the proteolytic activity was 8.0. The protease specifically catalyzed the hydrolysis of a carbonyl end of glutamic acid of a synthetic peptide, Z-LLE-MCA. The gene encoding GluSE (gse) was cloned by the inverse polymerase chain reaction from genome DNA of S. epidermidis ATCC 14990 in consideration of the N-terminal amino acid sequence of the purified protein and DNA sequence of a relatively conserved region of glutamyl endopeptidases of Staphylococcus aureus. The predicted gse gene-product consists of 282 amino acids, a molecular mass of 30,809, with 66 amino acid residues of a preprosequence. The production of GluSE was preferentially demonstrated when the cells were cultured on the dialysis membrane sheeted on agar medium, but not in culture medium, and this production apparently correlated with formation of biofilms. Therefore, it was speculated that GluSE played some roles in the attachment mechanism of the bacteria by biofilms to the surface of plastic devices. Southern hybridization analysis indicated that the GluSE gene was present as a single copy on the chromosomal DNA. The distribution of the GluSE gene was also examined by PCR in clinical isolates of coagulase-negative staphylococci. The gene was amplified in all the S. epiderinidis isolates (65/65), but not in S. aureus or in the other CNS isolates including S. capitis,S. haemolyticus, S. hominis, and S. warneri. Further, the production of GluSE was demonstrated in 72.3% (47/65) of S. epidermidis isolates. These results suggest that the GluSE gene is ubiquitous and exclusively preserved in S. epidermidis.