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

采用阳离子交换色谱法、超滤法和高效液相色谱法从表皮葡萄球菌（GluSE）中纯化了一种新的谷氨酰基内肽酶。蛋白酶的表观分子质量为27 kDa，蛋白酶水解活性的最佳pH为8.0。该蛋白酶特异性催化了合成肽Z-LLE-MCA的谷氨酸羰基末端的水解。考虑到纯化蛋白的n端氨基酸序列和金黄色葡萄球菌谷氨酰内肽酶相对保守区域的DNA序列，利用反聚合酶链反应从表皮葡萄球菌ATCC 14990基因组DNA中克隆出编码GluSE （gse）的基因。预测的gse基因产物由282个氨基酸组成，分子质量为30,809，前序有66个氨基酸残基。细胞在琼脂培养基上的透析膜上优先产生GluSE，而在培养基上不优先产生GluSE，其产生与生物膜的形成明显相关。因此，我们推测GluSE在细菌通过生物膜附着在塑料装置表面的机制中发挥了一定的作用。南方杂交分析表明，GluSE基因以单拷贝形式存在于染色体DNA上。用PCR方法检测了GluSE基因在凝固酶阴性葡萄球菌临床分离株中的分布。该基因在所有表皮葡萄球菌分离株中扩增（65/65），但在金黄色葡萄球菌或其他CNS分离株中未扩增，包括S. capitis，S. CNS。溶血性链球菌、人型链球菌和沃纳利链球菌。此外，72.3%（47/65）的表皮葡萄球菌分离株可产生GluSE。这些结果表明，GluSE基因在表皮葡萄球菌中是普遍存在的，并且是专门保存的。

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.