Abfalter Carmen M, Schönauer Esther, Ponnuraj Karthe, Huemer Markus, Gadermaier Gabriele, Regl Christof, Briza Peter, Ferreira Fatima, Huber Christian G, Brandstetter Hans, Posselt Gernot, Wessler Silja
Department of Molecular Biology, Division of Microbiology, Paris-Lodron University of Salzburg, Salzburg, Austria.
Department of Molecular Biology, Division of Structural Biology, Paris-Lodron University of Salzburg, Salzburg, Austria.
PLoS One. 2016 Sep 2;11(9):e0162433. doi: 10.1371/journal.pone.0162433. eCollection 2016.
Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.
细菌胶原酶在结构和功能上有很大差异。溶组织梭菌的胶原酶ColH和ColG以及产气荚膜梭菌表达的ColA是已得到充分表征的能切割三螺旋胶原的胶原酶，因此被称为“真正的”胶原酶。蜡样芽孢杆菌的ColA也被归入真正胶原酶之列。然而，蜡样芽孢杆菌ColA的分子特征尚不太清楚。在本研究中，我们鉴定出蜡样芽孢杆菌ATCC 14579分泌的真正胶原酶ColA，其转录受调节子磷脂酶C调节因子（PlcR）控制。克隆蜡样芽孢杆菌ATCC 14579的ColA以表达重组野生型ColA（ColAwt），并将其突变为蛋白水解无活性的版本（ColAE501A）。检测重组ColAwt的明胶分解和胶原分解活性，并用ColAE501A制备多克隆抗ColA抗体。将ColAwt活性与蜡样芽孢杆菌狭义菌株（蜡样芽孢杆菌狭义）的同源蛋白酶以及相关梭菌胶原酶进行比较，结果表明蜡样芽孢杆菌ATCC 14579 ColA是一种高活性的肽分解和胶原分解蛋白酶。这些发现可能有助于更深入地了解用于医学和生物技术应用的细菌胶原酶的功能和机制。
