Wladyka Benedykt, Bista Michal, Sabat Artur J, Bonar Emilia, Grzeszczuk Sabina, Hryniewicz Waleria, Dubin Adam
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
Acta Biochim Pol. 2008;55(3):525-36. Epub 2008 Sep 4.
Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No proteolytic activity was detected when the bacteria were grown in regular liquid media. Unexpectedly, supplementation of the medium with calcium ions resulted in expression of a metalloprotease and profound changes in the profile of extracellular proteins. On the basis of homology to other staphylococcal metalloproteases, the nucleotide sequence of the gene encoding this protease (Pst) and its flanking regions was determined. The full-length pst codes for a protein with an open reading frame of 505 amino acids. The internal region contains the HEXXH catalytic domain that is conserved in members of the thermolysin family. Regardless of the presence of calcium in the medium, the expression of the protease gene was of the same intensity. This suggests that regulation of the metalloprotease production by calcium ions is at a post-transcriptional level. Isolates of S. pseudintermedius exhibit a proteolytic phenotype due to the metalloprotease expression, however only in presence of calcium ions, which most probably stabilize the structure of the protease.
嗜热菌蛋白酶构成了一类分泌型细菌金属蛋白酶家族，多种病原体均可表达此类酶。从患病犬只中分离出的中间型葡萄球菌菌株，经脱脂乳琼脂平板培养判定为蛋白酶阳性，并对其蛋白酶含量进行了研究。当细菌在常规液体培养基中生长时，未检测到蛋白水解活性。出乎意料的是，向培养基中添加钙离子会导致金属蛋白酶的表达以及细胞外蛋白质谱的显著变化。基于与其他葡萄球菌金属蛋白酶的同源性，确定了编码该蛋白酶（Pst）的基因及其侧翼区域的核苷酸序列。全长pst编码一个具有505个氨基酸开放阅读框的蛋白质。内部区域包含嗜热菌蛋白酶家族成员中保守的HEXXH催化结构域。无论培养基中是否存在钙离子，蛋白酶基因的表达强度相同。这表明钙离子对金属蛋白酶产生的调控处于转录后水平。中间型葡萄球菌分离株由于金属蛋白酶的表达而呈现蛋白水解表型，但仅在存在钙离子的情况下，钙离子很可能稳定了蛋白酶的结构。
