Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin

We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.     

Production and properties of heat-stable extracellular hemolysin from Pseudomonas aeruginosa.

Of 12 strains of Pseudomonas aeruginosa, 10 were found to produce heat-stable extracellular hemolysin in highly aerated peptone broth supplemented with glycerol, fructose, or mannitol. Glucose supported good hemolysin production only in medium that was highly buffered. The yield of both cells and hemolysin was lower with organic acids as supplement. Growth-limiting phosphate concentrations produced maximum hemolysin levels. Purified hemolysin preparations contained two hemolytic glycolipids. The kinetics of hemolysis at various levels of purified lysin and the effects of variation in lysin and erythrocyte concentration are described.     

The origin and properties of extracellular DNA: from PAMP to DAMP

DNA is a polymeric macromolecule whose biological activities depend on location as well as binding to associated molecules. Inside the cell, DNA is the source of genetic information and binds histones to form nucleosomes. DNA can exit the cell, however, to enter the extracellular space primarily during cell death, either apoptosis or necrosis, as well as NETosis. While bacterial DNA is a potent immune stimulant by virtue of its CpG motifs, mammalian DNA, which is ordinarily inactive, can acquire activity by associating with nuclear, cytoplasmic and serum proteins which promote its uptake into cells to stimulate internal DNA sensors, including Toll-like receptor 9. Among these proteins, anti-DNA autoantibodies can form immune complexes with DNA to stimulate plasmacytoid dendritic cells to produce type 1 interferon. Together, these findings suggest that the immune properties of DNA are mutable and diverse, reflecting its context and the array of attached molecules.     

Some biological and immunological properties of extracellular slime from Pseudomonas aeruginosa.

Extracellular slime from 8 different Pseudomonas aeruginosa strains was extracted and purified. All slime-preparations exhibited immunogenic properties in rabbits vaccinated with detoxified or not detoxified slime. The antisera of both groups of immunized animals possessed strong hemagglutination activity against homologous slime. Immune hemagglutinins were present in the IgM and IgG fractions of serum globulins. The high value of these antibodies was found in rabbit's sera short after injection of slime-extract. The hemagglutinins quickly reached the peak value and maintained in serum over 60-70 days. Biological properties of lyophilized slime-preparations were defined in rabbit-skin test, local Shwartzman test, pyrogenic reaction and measured as LD50 for mouse. Intravenous injection of slime elicited marked changes in the number of leukocytes in the peripheral blood of rabbit. The animals responded to slime either with leukopenia passing into leukocytosis or with leukocytosis without leukopenia.     

Production and properties of an extracellular bacteriocin from Streptococcus mutans bacteriocidal for group A and other streptococci.

An extracellular bacteriocidal substance is produced by a serotype c strain of Streptococcus mutans in liquid meduim during the stationary phase of growth. The lethal effect of the substance was demonstrated by the decrease in viable counts of a standardized suspension of group A streptococci in broth. No lysis of affected cells was observed and no changes in appearance of these cells was seen in electron micrographs. The material was effective against certain strains of immmunological groups A, C, D, G, H, L, and O streptococci. It was inactive against strains of S. mutans belonging to the a, b, c, and d serotypes, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The factor was purified 273-fold from the culture fluid by column chromatography. It was sensitive to trysin and Pronase and resistant to catalase. It possessed a molecular weight of more than 20,000 and was not dialyzable. The properties of this substance indicate that it is a bacteriocin. Group A streptococci, which had been treated with antiserum specific for the cell wall group and type antigens, were susceptible to the bacteriocin. Streptococcal strains resistant to the lethal action of the bacteriocin adsorbed the bacteriocin from the solutions, as did the sensitive cells. The bacteriocin was not adsorbed at 0 C.     

A novel extracellular proteinase from Bacillus pumilus

Bacillus pumilus NCTC AII6/73 produces a novel extracellular alkaline proteinase whose optimum is pH 9.5. The purified product from ion-exchange chromatography (DEAE-cellulose) was resolved into two bands by polyacrylamide-sodium dodecyl sulphate (SDS) gel electrophoresis. These had approximate molecular weights of 15.6 × 10³ and 11.1 × 10³ daltons, respectively, and a high content of glycine and proline, comprising roughly 50% of the total amino acid residues.     

Prothrombin activation by a metalloprotease from Staphylococcus aureus.

Formation of thrombin during incubation of purified bovine prothrombin with purified staphylococcal metalloprotease has been investigated. Thrombin activity was estimated by examination of clotting time and by digestion of a synthetic substrate, Chromozym TH. The metalloprotease caused direct activation of prothrombin which was inhibited by the addition of ethylenediaminetetraacetic acid. Metalloprotease produced by some strains of Staphylococcus aureus may simulate staphylocoagulase activity.     

Migration through the extracellular matrix by the parasitic protozoan Leishmania is enhanced by surface metalloprotease gp63

Leishmania species engineered to express high levels of the surface metalloprotease gp63 have enhanced capacity of migration through extracellular matrix in vitro. This correlates with gp63 degradation of extracellular matrix components, such as collagen type IV and fibronectin, and suggests an important role for gp63 in the pathogenesis of leishmaniasis.     

Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection.     

Identification and characterization of a metalloprotease activity from Helicobacter pylori.

Helicobacter pylori produces a metalloprotease with a native molecular size of approximately 200 kDa, as determined by size-exclusion chromatography. Subcellular distribution studies demonstrated that the activity was associated with the outer membrane fraction of the bacterium. In addition, the protease was secreted by the bacterium when grown in liquid culture. The enzyme activity was measured by hydrolysis of azocasein and biotinylated casein and exhibited optimal caseinolytic activity at pH 8.0 (37 degrees C). The activity was inhibited by EDTA, 1,10-phenanthroline, phosphoramidon, pyridine-2,6-dicarboxylic acid, and 8-hydroxyquinoline-5-sulfonic acid (HQSA). Inhibition by HQSA was reversed by zinc, whereas inhibition due to EDTA was reversed by excess calcium, thus indicating that the enzyme was a zinc-dependent, calcium-stabilized endoproteinase. Furthermore, titration with Zn2+ of a desalted, active-site zinc-chelated preparation of the protease demonstrated that Zn2+ was essential for activity. Leupeptin, phenylmethylsulfonyl fluoride, E-64, pepstatin A, dithiothreitol, and 2-mercaptoethanol had no effect on enzymatic activity. Addition of Ca2+ or Mg2+ to the incubation medium resulted in approximately a twofold stimulation of the azocaseinolytic activity of the enzyme. The protease was stably expressed since it was active even after repeated subculture of the bacterium. Bovine serum albumin, hide powder azure, and elastin-Congo red remained intact even after prolonged exposure to the enzyme. The surface expression of this metalloprotease activity raises the possibility that this enzyme may be involved in the proteolysis of a variety of host proteins in vivo and thereby contributes to gastric pathology.     

A cold-active heat-labile t-RNA modification GTPase from a psychrophilic bacterium Pseudomonas syringae (Lz4W)

A cold-active heat-labile t-RNA modification GTPase (TrmE) from psychrophilic bacterium Pseudomonas syringae (Lz4W) has been purified and characterized. The purified TrmE is a 53 kDa protein, has GTPase activity and hydrolyses only the oxy and deoxy forms of GTP but not the other nucleotide triphosphates. The enzyme exhibits optimal activity at 12–18 °C and retains 65% of its optimal activity at 4 °C, indicating that it is a cold-active enzyme. The enzyme is also heat-labile and loses 60% of its activity at 30 °C. This is the first report on the purification and characterization of a TrmE from a psychrophilic bacterium.     

Induction of an extracellular esterase from Candida albicans and some of its properties.

An extracellular esterase from Candida albicans A-714 was found to be induced in a medium containing 0.7% yeast nitrogen base and 2.5% Tween 80 (polyoxyethylenesorbitan compounds). Enzyme activity, which exists predominantly in the extracellular space, was measured by a colorimetric method using alpha-naphthyl palmitate as a substrate. The induction level of the esterase activity was found to be well correlated with fungal growth and was dependent on the Tween 80 concentration. Such esterase activity was observed only in medium containing Tween 80 or other Tweens as the sole carbon source and therefore was not observed in either peptone-glucose medium or peptone-glucose medium supplemented with Tween 80. The induced esterase was heat labile and had maximum activity at pH 5.5. Enzyme activity was stimulated by the addition of sodium taurocholate, an activator of lipase. Thin-layer chromatography revealed that this enzyme does not hydrolyze triolein and L-alpha-lecithin, suggesting that it is a monoester hydrolase (not a lipase in the strict sense of the word). Esterase activity was examined in 85 clinical isolates of Candida species; C. albicans, C. tropicalis, and C. parapsilosis tended to have higher enzyme activities than C. kefyr, C. krusei, C. glabrata, and C. guilliermondii. Although the physiological properties of this esterase are not clear at present, it was found to be crucial for fungal growth under specific conditions.     

A hydrogel derived from decellularized dermal extracellular matrix

The ECM of mammalian tissues has been used as a scaffold to facilitate the repair and reconstruction of numerous tissues. Such scaffolds are prepared in many forms including sheets, powders, and hydrogels. ECM hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the inherent bioactivity of native matrix. However, material properties of ECM hydrogels and the effect of these properties upon cell behavior are neither well understood nor controlled. The objective of this study was to prepare and determine the structure, mechanics, and the cell response in?vitro and in?vivo of ECM hydrogels prepared from decellularized porcine dermis and urinary bladder tissues. Dermal ECM hydrogels were characterized by a more dense fiber architecture and greater mechanical integrity than urinary bladder ECM hydrogels, and showed a dose dependent increase in mechanical properties with ECM concentration. In?vitro, dermal ECM hydrogels supported greater C2C12 myoblast fusion, and less fibroblast infiltration and less fibroblast mediated hydrogel contraction than urinary bladder ECM hydrogels. Both hydrogels were rapidly infiltrated by host cells, primarily macrophages, when implanted in a rat abdominal wall defect. Both ECM hydrogels degraded by 35 days in?vivo, but UBM hydrogels degraded more quickly, and with greater amounts of myogenesis than dermal ECM. These results show that ECM hydrogel properties can be varied and partially controlled by the scaffold tissue source, and that these properties can markedly affect cell behavior.     

Mitogenic properties of major extracellular proteins

The major plasma and extracellular matrix proteins are multifunctional molecules. Some, such as fibrinogen or C3, have one domain that binds adhesion receptors and another that specifically binds and activates a separate, mitogenic receptor. In this review, Jean-Pierre Lévesque, Antoinette Hatzfeld and Jacques Hatzfeld describe adhesion and mitogenic receptors that bind to distinct domains of the same extracellular matrix protein and discuss the possibility of common ancestral genes for cell adhesion molecules, extracellular matrix proteins, integrins, immunoglobulins, growth factors and their receptors.     

A fourth metalloprotease gene in Erwinia chrysanthemi

Erwinia chrysanthemi, a Gram-negative phytopathogenic bacterium, was previously shown to secrete 3 related extracellular metalloproteases, A, B and C via a specific signal-peptide-independent pathway. A new gene (prtG) encoding a fourth, 52-kDa metalloprotease was identified on the same recombinant cosmid (pEW1) that carries the genes for the previously described proteases (prtA, prtB and prtC), for the specific secretion factors (prtD, prtE and prtF) and for a protease inhibitor (inh) cloned from E. chrysanthemi B374. The predicted sequence of PrtG was similar to those of PrtA, PrtB and PrtC, its secretion required PrtD, PrtE and PrtF; its secretion signal was located at the C terminus but its proteolytic activity was distinct from that of the 3 other proteases. Results presented here suggest that prtG could be the first gene of an operon that includes inh, prtD, prtE and prtF.     

Procaspase-3 activation by a metalloprotease secreted from Vibrio vulnificus

Vibrio vulnificus is a marine bacterium and a human pathogen capable of causing wound infection and septicemia. We previously showed that the metalloprotease vEP secreted by V. vulnificus activates prothrombin in vitro. To further investigate the ability of vEP to activate other zymogens, we used a mutant form of procaspase-3 which lacks the native cleavage sites as a zymogen. The mutant zymogen was activated by vEP to yield a mature enzyme with a maximum increase in caspase-3 activity of approximately 14-fold in a time-dependent manner. However, the increase started to decline with prolonged incubation and with higher protease concentration as a result of further cleavage of the mature enzyme. Western blot analysis revealed a band of approximately 17 kDa for the cleavage product, which corresponded with the change in caspase-3 activity. The activated procaspase-3 by vEP was also able to cleave poly(ADP-ribose) polymerase in a cell-free system, and was inhibited by Ac-DEVD-CHO, a potent caspase-3 inhibitor. The results presented are the first to demonstrate the in vitro activation of one of the crucial enzymes involved in cell death by a bacterial extracellular metalloprotease.     

Purification and characterization of a 43.5 kDa keratinolytic metalloprotease from Microsporum canis.

A keratinolytic protease secreted by a feline clinical isolate of Microsporum canis cultivated in a broth containing feline keratin as the sole nitrogen source was purified from the culture filtrate by affinity chromatography on bacitracin-agarose and by hydrophobic chromatography on octyl-agarose. The enzyme had an apparent molecular mass of 43.5 kDa and the pI was 7.7. It had a significant activity against keratin azure, elastin-Congo red and denatured type I collagen (azocoll). Using the latter substrate, the optimum pH was around 8 and the apparent optimum temperature around 50 degrees C. The protease was strongly inhibited by 1,10-phenanthroline, phosphoramidon and EDTA. The first 13 N-terminal amino acid sequence showed a 61% homology with that of the extracellular metalloprotease of Aspergillus fumigatus and with the neutral protease I of A. oryzae, confirming that this 43.5 kDa keratinase is a metalloprotease. This keratinolytic metalloprotease could be a virulence-related factor involved in pathophysiological mechanisms of M. canis dermatophytosis.     

High-level production of a cold-active B-mannanase from Bacillus subtilis Bs5 and its molecular cloning and expression

Mannanases can be useful in the food, feed, pulp and paper industries. In this research a Bacillus subtilis strain (named Bs5) which produced hign-level β-mannanase was isolated. Maximum level of β-mannanase (1231.41 U/mL) was reached when B. subtilis Bs5 was grown on konjac powder as the carbon source for nine hours at 32°C. The β-mannanase was a typical cold-active enzyme and its optimal temperature of 35°C was the lowest among those of the known mannanases from bacteria. In addition, the optimal pH was 5.0 and much wide pH range from 3.0–8.0 was also observed in the β-mannanase. These properties make the β-mannanase more attractive for biotechnological applications. The DNA sequence coding the β-mannanase was cloned and the open reading frame consisted of 1089 bp encoding 362 amino acids. A phylogenetic tree of the β-mannanase based on the similarity of amino acid sequences revealed that the β-mannanase formed a cluster with the β-mannanases of B. subtilis, which was separated from the mannanases of fungi and other bacteria The β-mannanase gene could be expressed in Escherichia coli and the recombinant β-mannanase was characterized by western blot. This study provided a new source of carbohydrate hydrolysis enzyme with novel characteristics from B. subtilis.