Extracellular Collagenase Research Articles

A Dual-Enzyme Amplification Loop for the Sensitive Biosensing of Endopeptidases.

A rapid and sensitive approach for the detection of endopeptidases via a new analyte-triggered mutual emancipation of linker-immobilized enzymes (AMELIE) mechanism has been developed and demonstrated using a matrix metallopeptidase, a collagenase, as the model endopeptidase analyte. AMELIE involves an autocatalytic loop created by a pair of selected enzymes immobilized on solid substrates via linkers with specific sites that can be proteolyzed by one another. These bound enzymes are spatially separated so that they cannot act upon their corresponding substrates until the introduction of the target endopeptidase analyte that can also cleave one of the linkers. This triggers the self-sustained loop of enzymatic activities to emancipate all the immobilized enzymes. In this proof of concept, signal transduction was achieved by a colorimetric horseradish peroxidase-tetramethylbenzidine (HRP-TMB-H2O2) reaction with HRP that are also being immobilized by one of the linkers. The pair of immobilized enzymes were collagenase and alginate lyase, and they were immobilized by an alginate linker and a short peptide chain containing the amino acid sequence of Leu-Gly-Pro-Ala for collagenase. A detection limit of 2.5 pg collagenase mL-1 with a wide linear range up to 4 orders of magnitude was achieved. The AMELIE biosensor can detect extracellular collagenase in the supernatant of various bacteria cultures, with a sensitivity as low as 103 cfu mL-1 of E. coli. AMELIE can readily be adapted to provide the sensitive detection of other endopeptidases.

Extracellular collagenase isolated from Streptomyces antibioticus UFPEDA 3421: purification and biochemical characterization

Collagenases are proteases able to degrade native and denatured collagen, with broad applications such as leather, food, and pharmaceutical industries. The aim of this research was to purify and characterize a collagenase from Streptomyces antibioticus. In the present work, the coffee ground substrate provided conditions to obtaining high collagenase activity (377.5 U/mL) using anion-exchange DEAE-Sephadex G50 chromatographic protocol. SDS-PAGE revealed the metallo-collagenase with a single band of 41.28 kDa and was able to hydrolyzed type I and type V collagen producing bioactive peptides that delayed the coagulation time. The enzyme activity showed stability across a range of pH (6.0–11) and temperature (30–55 °C) with optima at pH 7.0 and 60 °C, respectively. Activators include Mg+2, Ca+2, Na+, K+, while full inhibition was given by other tested metalloproteinase inhibitors. Kinetic parameters (Km of 27.14 mg/mol, Vmax of 714.29 mg/mol/min, Kcat of 79.9 s−1 and Kcat/Km of 2.95 mL/mg/s) and thermodynamic parameters (Ea of 65.224 kJ/mol, ΔH of 62.75 kJ/mol, ΔS of 1.96 J/mol, ΔG of 62.16 kJ/mol, ΔGE-S of 8.18 kJ/mol and ΔGE-T of −2.64 kJ/mol) were also defined. Coffee grounds showed to be an interesting source to obtaining a collagenase able to produce bioactive peptides with anticoagulant activity.

Antimicrobial efficacy of grape seed extract in terminating the ramifications of plaque microorganisms: a randomized control study.

BackgroundGrape seed extract contains Proanthocyanin, which reduces collagen degradation by inhibiting interstitial and extracellular collagenase, thus having the potential to reduce the progression of periodontitis. Here we compare and evaluate the aerobic and anaerobic microorganism’s CFUs (colony forming units) in plaque samples of Group A, Group B, Group C and severity of periodontal disease on day 0 and 7.MethodsForty-five subjects in age range 18–30 years were selected among undergraduate students and randomly divided into Group A: 15, 2% grape seed extract mouthwash (GSE), Group B: 15, 0.2% chlorhexidine mouthwash (CHX) and Group C: 15, distilled water (control). The supragingival plaque was collected into transport media. Kruskal Wallis test followed by Mann Whitney test was used to compare the mean CFUs (×103) of microorganisms and severity of periodontal disease was compared, by clinical parameters among all groups on day 0 and 7.ResultsThere was a significant difference concerning mean scores of all clinical parameters (P<0.001) and mean CFUs of microorganisms between 3 study groups (P=0.005) at 7 days post-intervention period. Intragroup comparison, mean scores were significantly reduced on day 7 as compared to day 0 at (P<0.001) in Group A and B, but no significant difference was noted with Group C.ConclusionIntervention with GSE mouthwash showed a positive effect on reducing CFUs in the plaque when compared with the control group. GSE group also showed similar results in reducing CFUs in plaque when compared to CHX group, thereby demonstrating the agent’s antimicrobial efficacy, therapeutic effect and its potential usefulness in controlling plaque and periodontal diseases.

Protein Truncating Variants of colA in Clostridium perfringens Type G Strains.

Extracellular matrix (ECM) degrading enzymes produced by Clostridium perfringens may play an important role during the initial phases of avian necrotic enteritis by facilitating toxin entry in the intestinal mucosa and destruction of the tissue. C. perfringens is known to produce several ECM-degrading proteases, such as kappa toxin, an extracellular collagenase that is encoded by the colA gene. In this study, the colA gene sequence of a collection of 48 C. perfringens strains, including pathogenic (i.e. toxinotype G) and commensal (i.e. toxinotype A) chicken derived strains and strains originating from other host species, was analyzed. Although the colA gene showed a high level of conservation (>96% nucleotide sequence identity), several gene variants carrying different nonsense mutations in the colA gene were identified, leading to the definition of four truncated collagenase variant types (I-IV). Collagenase variant types I, III and IV have a (nearly) complete collagenase unit but lack parts of the C-terminal recruitment domains, whereas collagenase variant types II misses the N-terminal part of collagenase unit. Gene fragments encoding a truncated collagenase were mainly linked with necrotic enteritis associated C. perfringens type G strains with collagenase variant types I and II being the most prevalent types. Gelatin zymography revealed that both recombinant full-length and variant type I collagenase have active auto-cleavage products. Moreover, both recombinant fragments were capable of degrading type I as well as type IV collagen, although variant type I collagenase showed a higher relative activity against collagen type IV as compared to full-length collagenase. Consequently, these smaller truncated collagenases might be able to break down collagen type IV in the epithelial basement membrane of the intestinal villi and so contribute to the initiation of the pathological process leading to necrotic enteritis.

Taxonomic and Enzymatic Characterization of Flocculibacter collagenilyticus gen. nov., sp. nov., a Novel Gammaproteobacterium With High Collagenase Production.

Collagens from marine animals are an important component of marine organic nitrogen. Collagenase-producing bacteria and their collagenases play important roles in collagen degradation and organic nitrogen recycling in the ocean. However, only a few collagenase-producing marine bacteria have been so far discovered. Here, we reported the isolation and characterization of a collagenase-secreting bacterium, designated strain SM1988T, isolated from a green alga Codium fragile sample. Strain SM1988T is a Gram-negative, aerobic, oxidase-, and catalase-positive, unipolar flagellated, and rod-shaped bacterium capable of hydrolyzing casein, gelatin and collagens. Phylogenetic analysis revealed that strain SM1988T formed a distinct phylogenetic lineage along with known genera within the family Pseudoalteromonadaceae, with 16S rRNA gene sequence similarity being less than 93.3% to all known species in the family. Based on the phylogenetic, genomic, chemotaxonomic and phenotypic data, strain SM1988T was considered to represent a novel species in a novel genus in the family Pseudoalteromonadaceae, for which the name Flocculibacter collagenilyticus gen. nov., sp. nov. is proposed, with the type strain being SM1988T (= MCCC 1K04279T = KCTC 72761T). Strain SM1988T showed a high production of extracellular collagenases, which had high activity against both bovine collagen and codfish collagen. Biochemical tests combined with genome and secretome analyses indicated that the collagenases secreted by strain SM1988T are serine proteases from the MEROPS S8 family. These data suggest that strain SM1988T acts as an important player in marine collagen degradation and recycling and may have a promising potential in collagen resource utilization.

Binding Mode Characterization and Early in Vivo Evaluation of Fragment-Like Thiols as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa.

The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).

Early reversal cells in adult human bone remodeling: osteoblastic nature, catabolic functions and interactions with osteoclasts.

The mechanism coupling bone resorption and formation is a burning question that remains incompletely answered through the current investigations on osteoclasts and osteoblasts. An attractive hypothesis is that the reversal cells are likely mediators of this coupling. Their nature is a big matter of debate. The present study performed on human cancellous bone is the first one combining in situ hybridization and immunohistochemistry to demonstrate their osteoblastic nature. It shows that the Runx2 and CD56 immunoreactive reversal cells appear to take up TRAcP released by neighboring osteoclasts. Earlier preclinical studies indicate that reversal cells degrade the organic matrix left behind by the osteoclasts and that this degradation is crucial for the initiation of the subsequent bone formation. To our knowledge, this study is the first addressing these catabolic activities in adult human bone through electron microscopy and analysis of molecular markers. Periosteoclastic reversal cells show direct contacts with the osteoclasts and with the demineralized resorption debris. These early reversal cells show (1) ¾-collagen fragments typically generated by extracellular collagenases of the MMP family, (2) MMP-13 (collagenase-3) and (3) the endocytic collagen receptor uPARAP/Endo180. The prevalence of these markers was lower in the later reversal cells, which are located near the osteoid surfaces and morphologically resemble mature bone-forming osteoblasts. In conclusion, this study demonstrates that reversal cells colonizing bone surfaces right after resorption are osteoblast-lineage cells, and extends to adult human bone remodeling their role in rendering eroded surfaces osteogenic.

A metalloprotease secreted by the type II secretion system links Vibrio cholerae with collagen.

Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program.

Collagen: Basis of Life

Collagen is a group of naturally occurring proteins found in animals, especially in the flesh and connective tissues of mammals, making up about 25% of the whole-body protein content. Collagens represent a large family of proteins and at least 28 different collagen types have been described so far which are genetically, chemically and immunologically distinct. All collagens are composed of three polypeptide alpha chains coiled around each other to form the triple-helix configuration, which are stabilized by numerous hydrogen bonds. The primary structural motif of mature collagen is (Gly-X-Pro/Hyp) n . Every third residue is glycine, which is preceded by either a prolyl or a hydroxyprolyl residue. Collagen biosynthesis takes place both intra and extracellularly. It is synthesized by fibroblasts, chondroblasts, osteoblasts and other cells. Degradation of collagen also involves two pathways, the major intracellular collagenase independent and the extracellular collagenase mediated. Collagen related diseases such as osteogenesis imperfecta, ehler danlos syndrome, scurvy, epidermolysis bullosa, collagenopathy most commonly arise from mutations in the collagen genes or the genes encoding some of the enzymes involved in the post-translational modifications in normal collagen production. Through this is a review article, we aim to discuss the various aspects of collagen and its clinical implications in periodontal health and disease.

Differential Actions of the Endocytic Collagen Receptor uPARAP/Endo180 and the Collagenase MMP-2 in Bone Homeostasis

A well-coordinated remodeling of uncalcified collagen matrices is a pre-requisite for bone development and homeostasis. Collagen turnover proceeds through different pathways, either involving extracellular reactions exclusively, or being dependent on endocytic processes. Extracellular collagen degradation requires the action of secreted or membrane attached collagenolytic proteases, whereas the alternative collagen degradation pathway proceeds intracellularly after receptor-mediated uptake and delivery to the lysosomes. In this study we have examined the functional interplay between the extracellular collagenase, MMP-2, and the endocytic collagen receptor, uPARAP, by generating mice with combined deficiency of both components. In both uPARAP-deficient and MMP-2-deficient adult mice the length of the tibia and femur was decreased, along with a reduced bone mineral density and trabecular bone quality. An additional decrease in bone length was observed when combining the two deficiencies, pointing to both components being important for the remodeling processes in long bone growth. In agreement with results found by others, a different effect of MMP-2 deficiency was observed in the distinct bone structures of the calvaria. These membranous bones were found to be thickened in MMP-2-deficient mice, an effect likely to be related to an accompanying defect in the canalicular system. Surprisingly, both of the latter defects in MMP-2-deficient mice were counteracted by concurrent uPARAP deficiency, demonstrating that the collagen receptor does not support the same matrix remodeling processes as the MMP in the growth of the skull. We conclude that both uPARAP and MMP-2 take part in matrix turnover processes important for bone growth. However, in some physiological situations, these two components do not support the same step in the growth process.

Purification and characterization of collagenase from Bacillus licheniformis F11.4

The extracellular collagenase, produced by Bacillus licheniformis F11.4, was purified by ammonium sulfate precipitation followed by DEAE Sephadex A-50. The purified collagenase showed a 26.3-fold increase in specific activity being 1.0 U/mg and 2.6% recovery. The collagenase has an apparent molecular weight of 124 and 26 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymography. The optimal temperature and pH were 50°C and pH 7.0, respectively. The collagenase activity was inhibited by Fe2+ (1 mM), Mg2+ (1 mM), Mn2+ (1 mM), Co2+ (1 mM), EDTA (1 mM), and β-mercaptoetanol (1 mM). However, Ca2+ (1 mM) and Cu2+ (1 mM) increased its activity. The collagenase from B. licheniformis F11.4 was capable of hydrolyzing other protein substrates such as casein, gelatin, and fibrin. The Km and Vmax of the enzyme for collagen were 0.26 mg/ml and 0.27 U, respectively. Key words: Collagenase, Bacillus licheniformis F11.4, purification, characterization.

Production and characterization of a collagenolytic serine proteinase by Penicillium aurantiogriseum URM 4622: A factorial study

A 24 full factorial design was used to identify the main effects and interactions of the initial medium pH, soybean flour concentration, temperature and orbital agitation speed on extracellular collagenase production by Penicillium aurantiogriseum URM4622. The most significant variables for collagenase production were soybean flour concentration and initial medium pH that had positive main effects, and temperature that had a negative one. Protein concentration in soybean flour revealed to be a significant factor for the production of a collagenase serine proteinase. The most favorable production conditions were found to be 0.75% soybean flour, pH 8.0, 200 rpm, and 28°C, which led to a collagenase activity of 164 U. The enzyme showed an optimum activity at 37°C and pH 9.0, was stable over wide ranges of pH and temperature (6.0 ∼ 10.0 and 25 ∼ 45°C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonylfluoride. The firstorder rate constants for collagenase inactivation in the crude extract, calculated from semi-log plots of the residual activity versus time, were used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E*d = 107.4 kJ/mol and ΔH*d = 104.7 kJ/mol). The enzyme is probably an extracellular neutral serine collagenase effective on azocoll, gelatin and collagen decomposition.

Repetitive mechanical stretch increases extracellular collagenase activity in vaginal fibroblasts.

OBJECTIVES: The objectives were 1) to determine whether human vaginal fibroblasts are mechanosensitive and 2) to study the impact of mechanical stretch on these cells in the presence and absence of hormones. METHODS: Fibroblasts obtained from biopsies of full thickness vagina of 3 women were cyclically biaxially stretched at a magnitude of 8 and 16% for 72 hours with or without 17-β-estradiol plus progesterone. Culture media was collected and total collagenase activity was measured in duplicate using a fluorogenic substrate degradation assay. Data were analyzed at the 0.05 level of significance using Student t-test. RESULTS: Cells remained 90% viable throughout the experiments. Relative to the controls, hormonal treatment alone decreased collagenase activity (P=0.008). In the presence of mechanical stretch and in the absence of hormones, collagenase activity was increased (8% elongation, P=0.04; 16% elongation, P=0.001, respectively). The increase in collagenase activity was linearly correlated with magnitude (P<0.001). In the presence of hormones, the increase in enzyme activity by mechanical stretch was suppressed to baseline control levels (P=0.46). There was no difference in suppression by hormones by magnitude (P=0.48). CONCLUSIONS: Vaginal connective tissue fibroblasts are mechanosensitive with increased collagenase activity in the presence of stretch. This degradative behavior is inhibited in the presence of hormones. The data provide a mechanism by which events that induce vaginal stretch may lead to progression of pelvic organ prolapse, particularly, in the absence of hormones. Further studies are needed to determine whether these events lead to tissue with inferior mechanical properties.

Production of a collagenase from Candida albicans URM3622

Abstract Culture conditions (pH, time, temperature, inoculum size, orbital agitation speed and substrate concentration) for an extracellular collagenase produced by Candida albicans URM3622 were studied using three experimental designs (one 2 6−2 fractionary factorial and two 2 3 full factorial). The analysis of the 2 6−2 fractionary design data indicated that agitation speed and substrate concentration had the most significant effect on collagenase production. Based on these results, two successive 2 3 full factorial design experiments were run in which the effects of substrate concentration, orbital agitation speed and pH were further studied. These two sets of experiments showed that all variables chosen were significant for the enzyme production, with the maximum collagenolytic activity of 6.8 ± 0.4 U achieved at pH 7.0 with an orbital agitation speed of 160 rpm and 2% substrate concentration. Maximum collagenolytic activity was observed at pH 8.2 and 45 °C. The collagenase was stable within a pH range of 7.2–8.2 and over a temperature range of 28–45 °C. These results clearly indicate that C. albicans URM3622 is a potential resource for collagenase production and could be of interest for pharmaceutical, cosmetic and food industry.

Differential gene expression and extracellular secretion of the collagenolytic enzymes by the pathogen Vibrio parahaemolyticus

Vibrio parahaemolyticus, a causative agent of wound infections as well as food poisoning, harbors two collagenase genes: vppC and prtV. When cultivated at 26 degrees C in gelatin broth supplemented with 3.0% NaCl, significant collagenolytic activity was detected in the culture supernatant at the early stationary phase. Native polyacrylamide gel electrophoresis analysis revealed a 90-kDa protein, and N-terminal amino acid sequencing showed that this protein was VppC, generated through truncation of 72 N-terminal amino acid residues. Additionally, significant expression of only vppC was observed by reverse transcriptase PCR. By contrast, a vppC-negative mutant constructed through single crossover homologous recombination secreted a 50-kDa-collagenolytic enzyme; however, this enzyme was a serine protease that was reported previously. These results suggest that VppC is a primary extracellular collagenase produced by V. parahaemolyticus.

Extracellular Collagenases and the Endocytic Receptor, Urokinase Plasminogen Activator Receptor-associated Protein/Endo180, Cooperate in Fibroblast-mediated Collagen Degradation

The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis of large collagen fragments. First, we show that collagen that has been pre-cleaved by a mammalian collagenase is taken up much more efficiently than intact, native collagen by uPARAP/Endo180-positive cells. Second, we demonstrate that this preference is governed by the acquisition of a gelatin-like structure by the collagen, occurring upon collagenase-mediated cleavage under native conditions. Third, we demonstrate that the growth of uPARAP/Endo180-deficient fibroblasts on a native collagen matrix leads to substantial extracellular accumulation of well defined collagen fragments, whereas, wild-type fibroblasts possess the ability to direct an organized and complete degradation sequence comprising both the initial cleavage, the endocytic uptake, and the intracellular breakdown of collagen.

Enhanced prolidase activity and decreased collagen content in breast cancer tissue

Adherent interactions between integrins and extracellular matrix (ECM) proteins play an important role in tumorigenicity and invasiveness. The major component of ECM is collagen that plays a central role in the interaction with integrins. The expression of certain collagenases (gelatinases) by tumour cells is one of the characteristic features of the so-called metastatic phenotype, presumably by breaking down ECM barriers as well by altering the ECM-cell interaction. Although extracellular collagenases initiate the breakdown of collagen, the final step of collagen degradation is catalysed by intracellular prolidase. Collagen deposition, gelatinolytic and prolidase activities, expression of beta(1)-integrin receptor and their possible relationships were studied in seven operable breast cancer cases. In breast cancer tissue, we have found significant decrease in the amount of collagen. The decrease in collagen deposition in breast cancer tissue was accompanied by increase in the tissue gelatinolytic and prolidase activities. Simultaneously, a slight decrease in the expression of beta(1)-integrin receptor in breast cancer tissue was observed. These results suggest that alteration in collagen metabolism in breast cancer tissue may reflect tissue remodelling, characteristic for invasive phenotype of cancer cells. Increased gelatinolytic and prolidase activities in breast cancer tissue may enhance stromal matrix degradation and thus may promote metastatic dissemination. On the basis of the data, it seems that compounds endowed with gelatinolytic and prolidase inhibitory activities may be considered as a potential drug candidates for breast cancer therapy.

Role of collagenase in colonic anastomoses: a reappraisal.

Increased collagenolysis, with reduction in collagen concentration, has been incriminated in the breakdown of colonic anastomoses but previous studies have measured only collagen levels and non-specific collagenolytic activity. Collagenase, the initiating enzyme in collagen degradation, is synthesized on demand and controlled by tissue inhibitor of metalloproteinases (TIMP). Antibodies to collagenase and TIMP were applied to colonic anastomoses in rabbits to investigate the role of the enzyme during healing. Within 12 h of operation, secreting cells and extracellular collagenase were identified at the everted edges of the bowel wall. After 24 h, collagenase activity was accompanied by TIMP secretion in the same localized regions, and by the third postoperative day very few cells were still synthesizing enzyme in these areas, although extracellular activity remained visible. TIMP-secreting cells, however, were seen in a layer of connective tissue sealing the serosal surface of the anastomosis. At 7 days, both enzyme and inhibitor were found only in small aggregates of secreting cells in the deeper layers. The localization and extent of collagenase and TIMP activity accorded well with a normal healing response as, at all times, the enzyme was confined to the immediate vicinity of the suture line.

Role of exopolysaccharide, the rugose phenotype and VpsR in the pathogenesis of epidemic Vibrio cholerae

Vibrio cholerae, the causative agent of cholera can produce an exopolysaccharide (EPS). Some strains can also phenotypically switch from a smooth to a ‘rugose’ phenotype characterized by small wrinkled colonies, overproduction of EPS, increased biofilm formation in vitro and increased resistance to various stressful conditions. High frequency switching to the rugose phenotype is more common in epidemic strains than in non-pathogenic strains, suggesting EPS production and the rugose phenotype are important in cholera epidemiology. VpsR up-regulates Vibrio polysaccharide (VPS) genes and the synthesis of extracellular EPS (VPS). However, the function of VPS, the rugose phenotype and VpsR in pathogenesis is not well understood. We report that rugose strains of both classical and El Tor biotypes of epidemic V. cholerae are defective in the in vitro production of extracellular collagenase activity. In vivo studies in rabbit ileal loops suggest that VpsR mutants are attenuated in reactogenicity. Intestinal colonization studies in infant mice suggest that VPS production, the rugose phenotype and VpsR have a role in pathogenesis. Our results indicate that regulated VPS production is important for promoting in vivo biofilm formation and pathogenesis. Additionally, VpsR might regulate genes with roles in virulence. Rugose strains appear to be a subpopulation of cells that might act as a ‘helper’ phenotype promoting the pathogenesis of certain strains. Our studies provide new insight into the potential role of VPS, the rugose phenotype and VpsR in the pathogenesis of epidemic V. cholerae.

Construction and virulence testing of a collagenase mutant of Clostridium perfringens

Clostridium perfringens produces several extracellular toxins and enzymes, including an extracellular collagenase or κ toxin that is encoded by the colA gene. To determine if the ability to produce collagenase was a significant virulence factor in cases of gas gangrene or clostridial myonecrosis that are caused by C. perfringens, a chromosomal colA mutant was constructed by homologous recombination and subsequently virulence tested in the mouse myonecrosis model. The results clearly indicate that loss of the ability to produce collagenase does not alter the ability of the mutant to establish a virulent infection. By contrast, infection with a mutant unable to produce α-toxin led to a marked decrease in virulence. These results indicate that collagenase is not a major determinant of virulence in C. perfringens -mediated clostridial myonecrosis.