Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579

Abstract

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.

Biochemical characterisation of a collagenase from Bacillus cereus strain Q1

“…In fact, ColQ1 is much more active against triple-helical collagen than ColG, the clostridial collagenase with the highest reported collagenolytic activity 12 . This is consistent with our recent report on the activity of ColA from B. cereus type strain ATCC 14579 28 and indicates that, although historically less studied and therefore much less well described, bacillial collagenases may in general be much more efficient collagenolytic enzymes than clostridial collagenases. By determination of the kinetics towards collagen and its thermally denatured variant gelatin we showed that this difference can be explained by a higher helicase activity of ColQ1.…”

“…While there are no reports that implicate bacillial collagenases as virulence factors in B. cereus -associated foodborne diseases, in case of B. cereus mediated endophthalmitis 32 and periodontal diseases, however, collagenases have been found in the isolates of vitreous humor and dental plaques 32 , 33 , 51 , 52 . Considering the high collagenolytic efficiency of bacillial collagenases, as shown here for ColQ1 and for B. cereus ATCC 14579 ColA 28 , we propose that these enzymes facilitate the infiltration of the target tissues by degradation of the protective surrounding extracellular matrix, which then allows the diffusion of other virulence factors into the tissue. Beecher et al 32 , for example, observed a progressive degradation of the collagenous lens capsule prior to colonization of the lens cortex by B. cereus .…”

“…In contrast to clostridial collagenases, there is only little known about collagenolytic enzymes from bacilli. It has been shown that, similarly to clostridia, bacilli can secrete collagenases into the culture medium 28 – 30 and these can play a role in pathogenesis. Bacillus thuringiensis , a widely used bacterial insecticide, utilizes ColB for host invasion 31 and it has been observed that collagenases play a role in B. cereus mediated endophthalmitis 32 and periodontal disease 33 .…”

Complete Genome Sequence of Brevibacillus laterosporus Bl-zj, an Algicidal Bacterium Isolated from Soil

“…For a better understanding of the algicidal activity of B. laterosporus Bl-zj as reflected in the genome, the virulence genes, secretory proteins, transporters, and pathogen-host interaction factors were further predicted and analyzed. As expected, epsilon toxin, mosquitocidal toxin, alveolysin, lethal factor, chitinase, microbial collagenase, and various carbohydrate hydrolases and peptidases were identified, which are important virulence characteristics of bacteria in invasion and infection, as well as algicidal responses (14–18).…”

The Food Poisoning Toxins of Bacillus cereus

“…It has been shown recently that the most important effector for this is the mature NprA protein, which is cleaved by InhA1 [ 477 ]. Additionally, the phosphatidylinositol- and phosphocholine-specific phospholipase C (PI-PLC and PC-PLC) as well as the collagenase ColA are considered as pathogenicity factors [ 105 , 110 , 478 , 479 , 480 , 481 ]. The bifunctional protein CalY, formerly known as camelysin, has been shown to play an important role in cell surface adhesion and virulence, as well as in biofilms [ 482 , 483 , 484 , 485 ].…”