Potential pathogenic factors produced by a clinical nontoxigenicVibrio cholerae O1

A clinical isolate of nontoxigenicVibrio cholerae O1 that caused intestinal fluid accumulation (FA) in adult mice produced proteolytic, hemolytic, and cytotoxic activities in in vitro assays. The linkage of these secreted factors to the FA activity was studied by transposon (TnphoA) mutagenesis. Ten of the 12 TnphoA insertion mutants that were defective for proteolytic activity produced FA, hemolytic and cytotoxic activities; the remaining two mutants lost these latter three activities. These results indicate that FA activity is independent of proteolytic activity but closely associated with cytotoxic and hemolytic activities. Our results with the adult mouse model and a nontoxigenicV. cholerae O1 are in general agreement with previous studies that demonstrated linkage of cytotoxin and hemolysin of toxigenicV. cholerae O1 and non-O1 with FA activity in rabbit ileal loops.     

Acid production and proteolytic activity in milk by selectedLactobacillus γ-radiation induced mutants

Selected γ radiation-induced mutants and parent cultures ofLactobacillus andL. casei were estimated for titratable and volatile acid production of biacetyl and acetoin and for proteolytic activity as compared to the parents.     

Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis

Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin-activated form involves a major conformational change of the N-terminal Domain I, which is remodelled into a tetrameric coiled-coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C-t Domains IV and V to sustain the conformational change necessary for toxicity.     

Correlation between delta-endotoxin and proteolytic activities produced by Bacillus thuringiensis var. kurstaki growing in an economic production medium

Abstract

Bacillus thuringiensis is a Gram positive bacterium that produces an insecticidal crystalline protein making it one of the most important biocontrol agents for pest management. Bioinsecticides based on B. thuringiensis were produced by fermentation processes in liquid media. Cultural conditions controlling proteolytic activities in different culture media were investigated to study the possible correlations between B. thuringiensis production of proteases and delta-endotoxins in a low-cost complex medium. Aeration appeared to play an important role in delta-endotoxin production. The correlation between proteolytic activity and aeration does not seem to be reliable. A negative correlation (correlation coefficient =? 0.774) was established between protease activity and delta-endotoxin production. In order to prove this correlation, protease hypo-producing and overproducing mutants were isolated through random mutagenesis of two wild strains, BUPM13 and BUPM5, by using nitrous acid. Interestingly, delta-endotoxin production of BUPM13-1, BUPM13-2 and BUPM13-3 was markedly improved when compared to the wild strain BUPM 13, reaching 2.1-fold, 3.69-fold and 8.13-fold, respectively. Maximal protease activity (540-2468 UI) obtained by BUPM5-1 and BUPM5-2 was 2.34-fold and 10.7-fold, respectively, more than that obtained by the wild strain BUPM5 with a drastic decrease of their delta-endotoxin production. Study of delta-endotoxin production by the selected mutants confirmed that insecticidal crystal protein stability in the culture strongly depends on the level of endogenous protease activity. This was also confirmed by bioassays measuring the LC₅₀ using larvae of Ephestia kuehniella. Determining protease activity in fermentation culture could be useful in indirectly predicting the potency of B. thuringiensis strains with high insecticidal activities. This would allow low-cost selection of overproducing wild isolates or mutants in the screening programmes for the reduction of production cost, which is important from a practical point of view.     

CUP-5, the C. elegans ortholog of the mammalian lysosomal channel protein MLN1/TRPML1, is required for proteolytic degradation in autolysosomes

The process of macroautophagy (herein referred to as autophagy) involves the formation of a closed double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes to form an autolysosome. Lysosomes are regenerated from autolysosomes after degradation of the sequestrated materials. In this study, we showed that mutations in cup-5, encoding the C. elegans Mucolipin 1 homolog, cause defects in the autophagy pathway. In cup-5 mutants, a variety of autophagy substrates accumulate in enlarged vacuoles that display characteristics of late endosomes and lysosomes, indicating defective proteolytic degradation in autolysosomes. We further revealed that lysosomes in coelomocytes (scavenger cells located in the body cavity) are smaller in size and more numerous in mutants with loss of autophagy activity. Furthermore, the enlarged vacuole accumulation abnormality and embryonic lethality of cup-5 mutants are partially suppressed by reduced autophagy activity. Our results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.     

Campylobacter jejuni outer membrane vesicle‐associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E‐cadherin and occludin

Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram‐negative bacteria. Campylobacter jejuni produces OMVs that trigger IL‐8, IL‐6, hBD‐3 and TNF‐α responses from T84 intestinal epithelial cells and are cytotoxic to Caco‐2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E‐cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co‐incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E‐cadherin and occludin. The addition of 11168H OMVs to the co‐culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time‐dependent and dose‐dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells.     

Proteolytic Activities of the Starch-Fermenting Ruminal Bacterium, Streptococcus bovis

The objective of this study was to characterize the extracellular proteolytic activity of Streptococcus bovis. Strains KEG, JB1, NCFB 2476, and K11.21.09.6C produced very similar large molecular weight (160–200 kDa) extracellular proteases that were specifically inhibited by PMSF, a serine protease inhibitor. Further experiments with S. bovis KEG indicated that cultures grown with casein as the sole added N source produced the greatest level of proteolytic activity, and the level of proteolytic activity was independent of growth rate. Clarified ruminal fluid (CRF) decreased proteolytic activity by 54% compared with cultures grown with casein alone, and addition of exogenous peptides and carbohydrates (CHO) to the CRF further reduced the level of proteolytic activity by 44% and 52%, respectively. These results suggested that the proteolytic activity of S. bovis KEG was modulated by available N source and that the proteolytic activity was present for reasons other than providing N for growth. The role of S. bovis in ruminal proteolysis requires further definition, but phenotypic similarity among some ruminal strains would suggest a common niche in ruminal proteolysis. The uniformity of proteolytic activities could make S. bovis a prime candidate for manipulation in ruminal proteolysis control strategies. Received: 12 January 1999 / Accepted: 19 May 1999     

The mitochondrial IMP peptidase of yeast: functional analysis of domains and identification of Gut2 as a new natural substrate

The mitochondrial inner membrane peptidase IMP of Saccharomyces cerevisiae is required for proteolytic processing of certain mitochondrially and nucleus-encoded proteins during their export from the matrix into the inner membrane or the intermembrane space. The membrane-associated signal peptidase complex is composed of the two catalytic subunits, Imp1 and Imp2, and the Som1 protein. The IMP subunits are thought to function in membrane association, interaction and stabilisation of subunits, substrate specificity, and proteolysis. We have analysed inner membrane peptidase mutants and substrates to gain more insight into the functions of various domains and investigate the basis of substrate recognition. The results suggest that certain conserved glycine residues in the second and third conserved regions of Imp1 and Imp2 are important for stabilisation of the Imp complex and for the proteolytic activity of the subunits, respectively. The non-conserved C-terminal parts of the Imp subunits are important for their proteolytic activities. The C-terminal region of Imp2, comprising a predicted second transmembrane segment, is dispensable for the stability of Imp2 and Imp1, and cannot functionally substitute for the C-terminal segment of Imp1. Alteration of the Imp2 cleavage site in cytochrome c ₁ (from AM to ND) reveals the specificity of the Imp2 peptidase. In addition, we have identified Gut2, the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase, as a new substrate for Imp1. Failure to cleave the Gut2 precursor may contribute to the pet phenotype of certain imp mutants. Gut2 is associated with the inner membrane, and is essential for growth on glycerol-containing medium. Suggested functions of the analysed residues and domains of the IMP subunits, characteristics of the cleavage sites of substrates and implications for the phenotypes of imp mutants are discussed.Communicated by C. P. Hollenberg     

Enzyme Activity of Lectins from the Nitrogen-Fixing Soil Bacterium Bacillus polymyxa

Lectins LI and LII, localized on the surface of the nitrogen-fixing soil bacterium Bacillus polymyxa 1460, were shown to possess proteolytic activity. A relationship was found between the proteolytic and hemagglutinating activities of the lectins. Blocking of hemagglutinating activity with specific carbohydrate haptens led to significant changes in the enzyme activity of both lectins. When lectin activity was blocked with glucuronic acid and fructose-1,6-diphosphate, the proteolytic activity of both LI and LII declined, whereas incubation with d-galactosamine and d-glucosamine promoted increases in the proteolytic activity of LII. This study proposes that the molecules of the B. polymyxa lectins may have two centers on their surfaces: one responsible for lectin activity and the other for proteolytic activity. Received: 27 March 2000 / Accepted: 26 April 2000     

Proteolytic Activity of Lectins from the Nitrogen-Fixing Bacterium Bacillus polymyxa

Two lectins (LI and LII) stripped from the surface of Bacillus polymyxa1460 cells were found to possess proteolytic activity, which was associated with their hemagglutinating activity. The inhibition of the hemagglutinating activity of lectins by specific carbohydrate haptens changed their enzyme activities. The inhibition of hemagglutinating activity by glucuronic acid or fructose 1,6-diphosphate decreased the proteolytic activities of both lectins, whereas the blocking of this activity with D-glucosamine or D-galactosamine increased the proteolytic activity of LII. The molecules of the B. polymyxalectins are suggested to contain two active centers responsible for hemagglutinating and proteolytic activities.     

Propeptide-Mediated Specific Inhibition of a Recombinant Serine Protease from Indian Malaria Vector, Anopheles culicifacies

Serine proteases are a class of proteolytic enzymes that are synthesized as enzymically inactive zymogens and when required in the cell, they are activated by the removal of proregion. The role of proregions as potent and specific inhibitors of their associated protease has been established. Here, we investigated the inhibition of a recombinantly expressed and refolded Anopheles c ulicifacies serine protease (ACSP) that was isolated from the body tissue of an Indian malaria vector, A. culicifacies by its own N-terminally located 19 amino acid residue propeptide. The synthetic peptide identical to the propeptide, its three deletion mutants and leupeptin (a general serine protease inhibitor) were tested in vitro for their inhibitory activity towards recombinant ACSP. Amongst the five peptides tested, leupeptin displayed maximum inhibition closely followed by native propeptide. The reduction or loss of inhibitory potential of deletion mutants of propeptide revealed the importance of charged residues present in the propeptide for inhibition of the cognate enzyme.     

Degradation and replenishment of the labile protein fraction in non-growing cells of the asporogenic mutant ofBacillus megaterium

Kinetics of degradation of labelled proteins was followed in two asporogenic mutants ofBacillus megaterium during incubation in a sporulation medium. Both the mutant producing exocellular protease (KM 1prn ⁺) and the mutant not producing the enzyme (KM 12prn) were found to contain a labile protein fraction, whose proportion decreases with prolonged time of labelling and whose half-life is about 1 h. Most proteins were relatively stable and were degraded at a rate of 1 %/h and 2 %/h in strains KM 1 and KM 12, respectively (half life 70–80 h and 35–40 h in strains KM 1 and KM 12, respectively). The intracellular proteolytic activity of the KM 12 mutant remains practically the same during incubation in the sporulation medium or slowly increases. The labile protein fraction practically disappears from the cells after a 3.5-h incubation. When such a culture is then subjected to a shift-up and transferred again to the sporulation medium, the rate of protein turnover temporarily increases. The temporary increase of the turnover rate is caused by a partial replenishment of the labile protein fraction rather than by an accelerated degradation of the relatively stable fraction. The intracellular proteolytic activity does not increase under these conditions. The wild sporogenic strain ofB. megaterium also contains the labile protein fraction. Its half protein life is 1 h or less. However, the second protein fraction is degraded much more rapidly than in the asporogenic mutants and its half life is 6–7 h.     

Increased ATP-dependent proteolytic activity in lon-deficient Escherichia coli strains lacking the DnaK protein.

Extracts made from Escherichia coli null dnaK strains contained elevated levels of ATP-dependent proteolytic activity compared with levels in extracts made from dnaK+ strains. This ATP-dependent proteolytic activity was not due to Lon, Clp, or Alp-associated protease. Comparison of the levels of ATP-dependent proteolytic activity present in lon rpoH dnaK mutants and in lon rpoH dnaK+ mutants showed that the level of ATP-dependent proteolytic activity was elevated in the lon rpoH dnaK mutant strain. These findings suggest that DnaK negatively regulates a new ATP-dependent proteolytic activity, independently of sigma 32. Other results indicate that an ATP-dependent proteolytic activity was increased in a lon alp strain after heat shock. It is not yet known whether the same protease is associated with the increased ATP-dependent proteolytic activity in the dnaK mutants and in the heat-shocked lon alph strain.     

Proteolytic Activity in Commercial Triacylglycerol Hydrolase Preparations

The proteolytic activity of 34 commercial lipase preparations (CLP) was determined using a labeled casein substrate. Only three CLP were free from proteolytic activity. Porcine pancreatic lipases exhibited levels of proteolytic activity comparable to or greater than that of a reference porcine trypsin. Bacterial lipases contained up to 10% of the proteolytic activity of commercial trypsin. Proteolytic activities in lipases from fungal species were present at low levels (<1% of the activity in trypsin). Among preparations of fungal origin, lipases from Aspergillus niger and Mucor javanicus were highest in proteolytic activity; Aspergillus oryzae and Pseudomonas cepacia lipases were lowest. Proteins in CLP were separated by non-denaturing PAGE; between 4 and 17 protein bands in the range &#104 6.5- &#83 200 kDa were observed. With the exception of a single pair of Rhizomucor miehei lipases, the distribution of apparent molecular weights (AMW) was unique to each preparation. Bands of caseinolytic activity in commercial lipases were visualized by applying a zymographic technique. CLP contained between 0 (P. cepacia lipases) and 6 (porcine pancreas lipase and Rhizopus oryzae lipase) discrete proteolytic bands. Common themes of proteolytic AMW emerged, including 21-23 kDa and 30-35 kDa bands.     

Spore control (Sco) mutations in Bacillus subtilis

Summary Spore control (Sco) mutants were isolated after nitrosoguanidine-induced mutagenesis of germinated spores. They were recognized as colonies showing high proteolytic activity on protein-agar (generally elastin-agar) test plates. Fourteen such mutants were isolated. The Sco mutations were transferred by transformation into an isogenic collection of genetically marked strains. Most of them appeared to be single mutations. Transduction experiments permitted the localisation of six Sco mutants in three loci, all in the argC-metC region. ScoA is located between argC and metC, ScoB is to the right of metC and ScoC is to the left of argC. ScoC and the previously described catA mutation are probably placed in the same gene.Two ScoC strains also appear to carry a second mutation, ScoD, probably localised in the same locus as ScoB or in a locus close to it. Eight other Sco mutations, apparently unlinked to the argC-metC region, were not localised. The results indicate complex regulation of sporulation-associated products such as the proteases, dependent on several genes.     

Stucture of the complex between <Emphasis Type="Italic">Mucor pusillus</Emphasis> pepsin and the key domain of κ-casein for site-directed mutagenesis: a combined molecular modeling and docking approach

In the structural-based mutagenesis of Mucor pusillus pepsin (MPP), understanding how κ-casein interacts with MPP is a great challenge for us to explore. Chymosin-sensitive peptide is the key domain of κ-casein that regulates milk clotting through the specific proteolytic cleavage of its peptide bond (Phe¹⁰⁵-Met¹⁰⁶) by MPP to produce insoluble para-κ-casein. Here, we built the model of this large peptide using molecular modeling technique. Docking study showed that MPP can accommodate the designed model with a favorable binding energy and the docked complex has proven to locally resemble the inhibitor-chymosin complex. The catalytic mechanism for the peptide model binding with MPP was explored in terms of substrate-enzyme interaction and property of contacting surface. Some critical amino acid residues in the substrate binding cleft have been identified as an important guide for further site-directed mutagenesis. Glu13 and Leu11 in the S3 region of MPP, predicted as the special mutation sites, were confirmed to retain clotting activity and decrease the proteolytic activity. These novel mutants may provide a promising application for improving cheese flavor.     

A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae

Peptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3. PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N-end-rule pathway of the ubiquitin-dependent proteolytic system. Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into ceils. Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N-end-rule pathway. Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S. cerevisiae. These results establish a physiological role for a protein previously known to be required for the degradation of N-end-rule substrates. Our findings show that peptide transport and the ubiquitin pathway—two dynamic phenomena universal to eukaryotic cells—share a common component, namely UBR1/PTR1.     

Degradation of insect cuticle by Paecilomyces farinosus proteases

The entomopathogenic fungus Paecilomyces farinosus showed proteolytic activity in both solid and semi-liquid culture with gelatin as sole N and C source. Semi-liquid cultures were used to characterise proteases. Zymography of crude culture filtrates showed several bands of gelatin degradation in electrophoresis gels. Gel filtration chromatography of these filtrates revealed two peaks of proteolytic activity. Ion-exchange absorption eliminated gelatin from culture filtrates while retaining activity and was used to semipurify P. farinosus proteases. Semipurified culture filtrates had basic pH (8.5 approx.) optimum for proteolytic activity. Treatment of these filtrates with effectors revealed that P. farinosus proteases are serine proteases containing sulphydryl groups. Isoelectrofocusing combined with zymography revealed the presence of several active basic isoforms. Larvae of the lepidopteran Galleria mellonella showed cuticle damage and protein release 1h after incubation with semipurified extracts of P. farinosus. These results indicate that proteolytic enzymes could be involved in insect host penetration by P. farinosus.