Biochemical characterisation of a collagenase from Bacillus cereus strain Q1.

Collagen is the most abundant protein in higher animals and as such it is a valuable source of amino acids and carbon for saprophytic bacteria. Due to its unique amino acid composition and triple-helical tertiary structure it can however only be cleaved by specialized proteases like the collagenases secreted by some bacteria. Among the best described bacterial collagenases are ColG and ColH from Clostridium histolyticum. Many Bacillus species contain homologues of clostridial collagenases, which play a role in some infections caused by B. cereus. Detailed biochemical and enzymatic characterizations of bacillial collagenases are however lacking at this time. In an effort to close this gap in knowledge we expressed ColQ1 from B. cereus strain Q1 recombinantly, investigated its metal dependency and performed peptide, gelatin and collagen degradation assays. Our results show that ColQ1 is a true collagenase, cleaving natively folded collagen six times more efficiently than ColG while at the same time being a similarly effective peptidase as ColH. In both ColQ1 and ColG the rate-limiting step in collagenolysis is the unwinding of the triple-helix. The data suggest an orchestrated multi-domain mechanism for efficient helicase activity.

Surgical outcomes after collagenase Clostridium histolyticum failure in patients with Peyronie's disease in a multicenter clinical study.

In the present study we aimed to investigate the surgical outcomes of patients with persistent penile curvature (PC) after Collagenase Clostridium histolyticum (CCH) intraplaque injections. Data from 90 patients with persistent PC after CCH in a multicentre study from 6 andrological centres were retrospectively reviewed. Three standardized surgical techniques were performed. Group 1: plaque incision grafting (PIG) with penile prosthesis implant (PPI); Group 2: PIG without PPI; Group 3: Nesbit technique. Hospital stay, operative time, postoperative complications and PC persistency/recurrence (> 20°) were evaluated. Overall satisfaction and functional outcomes were assessed through International Index of Erectile Function-Erectile Function (IIEF-EF), Peyronie's Disease Questionnaire (PDQ), Female Sexual Function Index (FSFI) administered pre and 3 months postoperatively. Of all, 25 (27.8%) patients received grafting procedure + PPI (Group 1), 18 (20.0%) patients belonged to Group 2, and 47 (52.2%) to Group 3. Bovine pericardium graft and collagen fleece have been used in in 22 (51.2%) and 21 (48.8%) patients, respectively. Median penile length after surgery was 13.0 cm (IQR 12.0-15.0). After surgery, Group 1 showed higher increase in penile length after surgery and better improvements in terms of PDQ-PS. In contrast, both IIEF-EF and FSFI scores did not differ among groups. Overall, 86 (95.6%) did not report any complication. 4 (4.4%) patients had PC recurrence; of those, 2 (8.0%), 1 (5.6%) and 1 (2.1%) cases were observed in Group 1, Group 2 and Group 3, respectively. In case of persistent PC after CCH, surgical correction by grafting with or without concomitant PPI or Nesbit technique emerged as a technically feasible, effective and safe procedure, with no significant postoperative complications.

Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation.

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

Cathepsin D regulates cathepsin B activation and disease severity predominantly in inflammatory cells during experimental pancreatitis.

Acute pancreatitis is a complex disorder involving both premature intracellular protease activation and inflammatory cell invasion. An initiating event is the intracellular activation of trypsinogen by cathepsin B (CTSB), which can be induced directly via G protein-coupled receptors on acinar cells or through inflammatory cells. Here, we studied CTSB regulation by another lysosomal hydrolase, cathepsin D (CTSD), using mice with a complete (CTSD-/-) or pancreas-specific conditional CTSD knockout (KO) (CTSDf/f/p48Cre/+). We induced acute pancreatitis by repeated caerulein injections and isolated acinar and bone marrow cells for ex vivo studies. Supramaximal caerulein stimulation induced subcellular redistribution of CTSD from the lysosomal to the zymogen-containing subcellular compartment of acinar cells and activation of CTSD, CTSB, and trypsinogen. Of note, the CTSD KO greatly reduced CTSB and trypsinogen activation in acinar cells, and CTSD directly activated CTSB but not trypsinogen in vitro During pancreatitis in pancreas-specific CTSDf/f/p48Cre/+ animals, markers of severity were reduced only at 1 h, whereas in the complete KO, this effect also included the late disease phase (8 h), indicating an important effect of extra-acinar CTSD on course of the disease. CTSD-/- leukocytes exhibited reduced cytokine release after lipopolysaccharide (LPS) stimulation, and CTSD KO also reduced caspase-3 activation and apoptosis in acinar cells stimulated with the intestinal hormone cholecystokinin. In summary, CTSD is expressed in pancreatic acinar and inflammatory cells, undergoes subcellular redistribution and activation during experimental pancreatitis, and regulates disease severity by potently activating CTSB. Its impact is only minimal and transient in the early, acinar cell-dependent phase of pancreatitis and much greater in the later, inflammatory cell-dependent phase of the disease.

A Sensitive, Rapid, and Specific Technique for the Detection of Collagenase Using Zymography.

In-gel zymography is a commonly employed tool to identify active enzymes in a quantitative and qualitative manner. In this work, apart from the incorporation of substrate which is traditionally employed in zymography, the identification of collagenase by incubation of the enzyme resolved on a polyacrylamide gel with substrate solution is described. The two methods are quite fast and result in specific detection of bacterial collagenase.

The effects of fibrin and fibrin-agarose on the extracellular matrix profile of bioengineered oral mucosa.

Several studies have developed efficient oral mucosa constructs using different types of scaffold. However, the changes in the morphology and gene and protein expression profile that could occur in these artificial constructs remain unknown. This study compared the histology and expression of several extracellular matrix molecules in human artificial oral mucosa developed using two different types of scaffolds: fibrin and fibrin-agarose. To that end, bioengineered oral mucosa stromas were constructed from biopsy samples of human oral mucosa and the substitute generated was analyzed at different periods of time in culture. Histological analysis was carried out by light and transmission electron microscopy and the expression of collagen types I, III, and VI, the proteoglycans decorin and biglycan, and the different chains of laminin, were assessed by immunoperoxidase technique. This study found that fibrin scaffolds accelerated fibroblast growth and remodeling of the scaffold, thus enhancing collagen fibrillogenesis. In the fibrin-agarose scaffold, the morphology and organization of the fibroblasts did not change during the culture period. All extracellular matrix proteins analyzed were expressed in both scaffolds. However, in fibrin scaffolds, these proteins were widely distributed and replaced the scaffold during the follow-up period. These results show that the substitutes generated showed histological and molecular similarities with native human oral mucosa stroma. In addition, it was observed that the nature of the biomaterial influenced the behaviour of the oral stromal fibroblasts, thereby modulating their growth, protein synthesis, and collagen fibrillogenesis.

In vitro fermentation of grape seed flavan-3-ol fractions by human faecal microbiota: changes in microbial groups and phenolic metabolites.

With the aim of investigating the potential of flavan-3-ols to influence the growth of intestinal bacterial groups, we have carried out the in vitro fermentation, with human faecal microbiota, of two purified fractions from grape seed extract (GSE): GSE-M (70% monomers and 28% procyanidins) and GSE-O (21% monomers and 78% procyanidins). Samples were collected at 0, 5, 10, 24, 30 and 48 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and for analysis of phenolic metabolites. Both GSE-M and GSE-O fractions promoted growth of Lactobacillus/Enterococcus and decrease in the Clostridium histolyticum group during fermentation, although the effects were only statistically significant with GSE-M for Lactobacillus/Enterococcus (at 5 and 10 h of fermentation) and GSE-O for C. histolyticum (at 10 h of fermentation). Main changes in polyphenol catabolism also occurred during the first 10 h of fermentation; however, no significant correlation coefficients (P > 0.05) were found between changes in microbial populations and precursor flavan-3-ols or microbial metabolites. Together, these data suggest that the flavan-3-ol profile of a particular food source could affect the microbiota composition and its catabolic activity, inducing changes that could in turn affect the bioavailability and potential bioactivity of these compounds.

Structural comparison of ColH and ColG collagen-binding domains from Clostridium histolyticum.

Clostridium histolyticum secretes collagenases, ColG and ColH, that cause extensive tissue destruction in myonecrosis. The C-terminal collagen-binding domain (CBD) of collagenase is required for insoluble collagen fibril binding and subsequent collagenolysis. The high-resolution crystal structures of ColG-CBD (s3b) and ColH-CBD (s3) are reported in this paper. The new X-ray structure of s3 was solved at 2.0-Å resolution (R = 17.4%; R(free) = 23.3%), while the resolution of the previously determined s3b was extended to 1.4 Å (R = 17.9%; R(free) = 21.0%). Despite sharing only 30% sequence identity, the molecules resemble one another closely (root mean square deviation [RMSD] C(α) = 1.5 Å). All but one residue, whose side chain chelates with Ca(2+), are conserved. The dual Ca(2+) binding site in s3 is completed by an unconserved aspartate. Differential scanning calorimetric measurements showed that s3 gains thermal stability, comparable to s3b, by binding to Ca(2+) (holo T(m) = 94.1°C; apo T(m) = 70.2°C). holo s3 is also stabilized against chemical denaturants urea and guanidine HCl. The three most critical residues for collagen interaction in s3b are conserved in s3. The general shape of the binding pocket is retained by altered loop structures and side chain positions. Small-angle X-ray scattering data revealed that s3 also binds asymmetrically to minicollagen. Besides the calcium-binding sites and the collagen-binding pocket, architecturally important hydrophobic residues and the hydrogen-bonding network around the cis-peptide bond are well conserved within the metallopeptidase subfamily M9B. CBDs were previously shown to bind to the extracellular matrix of various tissues. Compactness and extreme stability in physiological Ca(2+) concentration possibly make both CBDs suitable for targeted growth factor delivery.

Temporal and spatial distribution of Bacillus and Clostridium histolyticum in swine manure composting by fluorescent in situ hybridization (FISH).

The temporal and spatial distribution of the genus Bacillus and Clostridium histolyticum group in swine manure composting was determined by fluorescent in situ hybridization using fluorescently labeled 16S rRNA-targeted oligonucleotide probes LGC353b and Chis150, respectively. The temporal distribution of total bacteria, Bacillus and C. histolyticum, detected in each layer of the composting pile was noticeable in that the number of them detected at the high-temperature stage was higher than that of the cooling stage. The number detected at the cooling stage was higher than that of the temperature-rising stage. The number of the total bacteria distributed in three locations achieved balance at the stage of cooling. The spatial distribution of the genus Bacillus cells was that the number and the relative abundance of Bacillus cells detected in the middle layer of composting pile were the lowest at each stage of composting. However, the minimum value of the relative abundance exceeded 8%. Compared with Bacillus spp., the C. histolyticum group displayed higher relative abundance in the same layer at different stages of composting except in the top layer at the stage of high temperature. However, the characteristic of the spatial distribution was not noticeable. The detected limits of the genus Bacillus and C. histolyticum group were both found to be the high cell density of 10(6) cells g(-1) (wet weight). These results indicated that the genus Bacillus and C. histolyticum group were the predominant bacteria in the swine manure composting process and may play important role in this complex environment.

Characterization and functional assessment of Clostridium histolyticum class I (C1) collagenases and the synergistic degradation of native collagen in enzyme mixtures containing class II (C2) collagenase.

OBJECTIVES: Clostridium histolyticum expresses two classes of collagenases, C1 and C2. However, degradation of these enzymes by proteases during the fermentation or purification process may lead to numerous molecular forms that lead to inconsistent release of islets from human pancreata. This report defines the amino acid sequence of the truncated forms of C1 (C1b or C1c) that contain a single collagen-binding domain (CBD) and investigates the synergy between the different forms of C1 collagenase and C2 to degrade native collagen. METHODS: Highly purified collagenase isoforms were purified from C. histolyticum culture supernatants using established column chromatography techniques and analyzed using high-pressure liquid chromatograph (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The collagen-degrading activity (CDA) assay was used to investigate the synergy between different collagenase molecular forms. RESULTS: MS was used to confirm the sequence of full-length C2 and C1 from the reported gene sequence. These results were correlated with the molecular weights observed on the SDS- PAGE and elution after analytical anion-exchange HPLC. HPLC peaks designated as C1b and C1c were both confirmed to be C1 lacking the terminal CBD. The only difference being the cleavage site leading to a 12 amino acid difference between the two forms. A non-additive synergy in CDA relative to activity of individual collagenases was observed for C2 with each of the three C1 molecular forms. The C1 molecular forms did not display this synergy in the absence of C2. CONCLUSIONS: These observations support earlier reports that suggest the two collagenases bind to different portions of the collagen and have different specificities to cut native collagen. Although the implications of this are not yet understood, they are fundamental in advancing the understanding of how collagenases work together along with the neutral protease to breakdown the extracellular matrix for islet isolation.