Role of an extracellular neutral protease in infection against nematodes by <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis> strain G4

Proteases have been proposed as virulence factors in microbial pathogenicity against nematodes. However, what kinds of extracellular proteases from these pathogens and how they contribute to the pathogenesis of infections against nematode in vivo remain largely unknown. A previous analysis using a strain with a deletion in an extracellular alkaline protease BLG4 gene from Brevibacillus laterosporus demonstrated that BLG4 was responsible for the majority of nematicidal activity by destroying host’s cuticle. In recent studies, a neutral protease NPE-4, purified from the mutant BLG4–6, was found to be responsible for the majority of the remaining EDTA-inhibited protease activity. However, the purified NPE-4 and recombinant NPE-4 in a related species Bacillus subtilis showed little nematicidal activity in vitro and were unable to degrade the intact cuticle of the host. It is interesting to note that the addition of NPE-4 improved the pathogenicity of crude enzyme extract from wild-type B. laterosporus but had no effect on the BLG4-deficient mutant. This result suggests that NPE-4 functions in the presence of protease BLG4. Moreover, NPE-4 could degrade proteins from the inner layer of purified cuticles from nematode Panagrellus redivivus in vitro. These results indicated that the two different bacterial extracellular proteases might play differential roles at different stages of infection or a synthetic role in penetration of nematode cuticle in B. laterosporus. This is among the first reports to systematically evaluate and define the roles of different bacterial extracellular proteases in infection against nematodes.     

Isolation and Characterization of a Serine Protease from the Nematophagous Fungus, <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis>, Displaying Nematicidal Activity

Lecanicillium psalliotae produced an extracellular protease (Ver112) which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 32 kDa. The optimum activity of Ver112 was at pH 10 and 70 °C (over 5 min). The purified protease degraded a broad range of substrates including casein, gelatin, and nematode cuticle with 81% of a nematode (Panagrellus redivivus) being degraded after treating with Ver112 for 12 h. The protease was highly sensitive to PMSF (1 mM) indicating it to be a serine protease. The N-terminal amino acid residues of Ver112 shared a high degree of similarity with other cuticle-degrading proteases from nematophagous fungi which suggests a role in nematode infection.     

Development and industrialisation of enzymatic shrink-resist process based on modified proteases for wool machine washability

There is currently considerable interest in the use of enzymes to achieve a variety of finishing effects on wool, but it is apparent that the extent of fibre degradation by enzymes is of major concern during their commercial application. Proteolytic enzymes are known to penetrate and degrade the internal wool structure during processing, causing fibre damage, rather than limiting the degradation to the cuticle cells. The ability to be able to control the exact location of proteolytic attack on wool protein structures will lead to the successful development of enzymatic treatments for achieving a variety of finishing effects for wool-containing products. This present work describes the modification of proteases so that enzymatic modification of wool fibres is restricted to the cuticle scales of the fibres.

Bulk trials have demonstrated that novel modifications of the enzyme enable the reaction of the enzyme with wool to be controlled, so that less degradation of the wool occurs than in similar treatments with the native protease. An anti-felting effect has been achieved without any significant weight loss being caused by the modified protease during the treatment. This novel enzymatic process leads to environmentally friendly production of machine washable wool.     

Purification and cloning of a novel serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Dactylellina varietas</Emphasis> and its potential roles in infection against nematodes

From the culture filtrate of the fungus Dactylellina varietas (syn. Dactylella varietas), an extracellular protease (designed Dv1) was purified by cation exchange and hydrophobic interaction chromatography. The purified protease showed a molecular mass of approximately 30 kDa and displayed an optimal activity at pH 8 and 60.5°C (more than 20 min). This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. However, its proteolytic activity was highly sensitive to the serine protease inhibitor Phenylmethylphonylfuoride (1 mM), indicating that it belongs to the serine-type peptidase group. This protease could immobilize the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans and hydrolyze the purified cuticle of P. redivivus, suggesting it may play a role in infection against nematodes. The encoding gene of Dv1 and its promoter sequence were cloned using degenerate primers and the DNA walking technology. Its open-reading frame contains 1,224 base pairs and without any intron. The deduced amino-acid sequence shared low identity to serine proteases from other nematode-trapping fungi. Our report identified a novel pathogenic protease from the nematode-trapping fungus D. varietas, and the three-dimensional structure of this protease was predicted using the Swiss-Prot method. Jinkui Yang and Lianming Liang contributed equally to this work.     

Purification and characterization of the cuticle-degrading protease produced by the entomopathogenic fungus,Beauveria bassiana in the presence of Sunn pest,Eurygaster integriceps (Hemiptera: Scutelleridae) cuticle

The extracellular protease from the entomopathogenic fungus, Beauveria bassiana in the presence of Eurygaster integriceps cuticle was isolated, purified and characterized. Isolate B1 of B. bassiana that shows high virulence against E. integriceps was examined for the production of the cuticle-degrading proteases. Results showed that both subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases were produced and the enzyme kinetic properties showed better activity of Pr1 in comparison with Pr2. The proteases were purified using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 5.09-fold increase in specific activity and 21.86% recovery. The enzyme molecular weight was estimated to be 47 kDa and the optimal pH and temperature were 8 and 45°C, respectively. The purified protease was activated by divalent cations, Ca^{2 +} and Mg^{2 +}, and inhibited by NaCl, KCl and determined as a serine protease by inhibition of its activity due to using PMSF, EDTA, mercaptoethanol and SDS. Studies on the timing of the protease secretion in the presence of cuticular substrates could provide information about the role of the accumulated hydrolytic enzymes during pathogenesis to better understand these processes.     

Molecular epidemiology ofCladosporium carrionii

The effects of cuticle from larvae ofTrichoplusia ni andHelicoverpa (=Heliothis)zea on expression of proteases and chitinases by germinating conidia ofNomuraea rileyi in submerged cultures were studied. Increasing the concentration ofT. ni orH. zea cuticle resulted in a 13- and 15-fold increase in protease activity, respectively. Endochitinase and N-acetylglucosaminidase activity on theT. ni andH. zea substrates increased as cuticular concentrations increased to 2.5%, then stabilized or decreased thereafter. The simultaneous expression of both proteases and chitinases suggests that they are controlled by a multiple-regulatory system.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by US Department of Agriculture.     

Production and Regulation of Cuticle-Degrading Proteases from <Emphasis Type="Italic">Beauveria bassiana</Emphasis> in the Presence of <Emphasis Type="Italic">Rhammatocerus schistocercoides</Emphasis> Cuticle

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.     

Restricting detergent protease action to surface of protein fibres by chemical modification

Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1′-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (ΔE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres.     

A neutral protease from Bacillus nematocida, another potential virulence factor in the infection against nematodes

A neutral protease (npr) (designated Bae16) toxic to nematodes was purified to homogeneity from the strain Bacillus nematocida. The purified protease showed a molecular mass of approximately 40 kDa and displayed optimal activity at 55°C, pH 6.5. Bioassay experiments demonstrated that this purified protease could destroy the nematode cuticle and its hydrolytic substrates included gelatin and collagen. The gene encoding Bae16 was cloned, and the deduced amino acid sequence showed 94% sequence identity with npr gene from B. amyloliquefaciens, but had low similarity (13–43%) with the previously reported virulence serine proteases from fungi or bacteria, which reflected their differences. Recombinant mature Bae16 (rm-Bae16) was expressed in Escherichia coli BL21 using pET30 vector system, and its nematicidal activity confirmed that Bae16 could be involved in the infection process. Our present study revealed that the npr besides the known alkaline serine protease could serve as a potential virulence factor in the infection against nematodes, furthermore, the two proteases with different characteristics produced by the same strain co-ordinated efforts to kill nematodes. These data helped to understand the interaction between this bacterial pathogen and its host.Qiuhong Niu, Xiaowei Huang have contributed equally to this work.     

Purification and characterization of a neutral serine protease with nematicidal activity from <Emphasis Type="Italic">Hirsutella rhossiliensis</Emphasis>

Serine protease plays an important role in fungal infection to invertebrate hosts. An extracellular protease (Hnsp) was detected in liquid culture of Hirsutella rhossiliensis OWVT-1 with nematodes (Panagrellus redivivus) as the unique nitrogen source and purified to homogeneity by ammonium sulphate precipitation, anion exchange chromatography and gel filtration. Its molecular mass was about 32 kDa, and the optimal reaction pH value and temperature were pH 7 and 40°C, respectively. The Hnsp activity was stable at pH 6–8 and decreased radically at 50°C for 10 min. Hnsp was highly sensitive to inhibitor of PMSF and well decomposed the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, suggesting that it belonged to the chymotrypsin/subtilisin of serine proteases. The N-terminal amino acid sequence of Hnsp was SVTDQQGADCGLARISHRE, which showed high homology with other serine proteases from nematophagous fungi. Ability to kill nematode and degrade its cuticle in vitro indicated that Hnsp could be involved in the infection of nematode.     

Combined use of mild oxidation and cutinase/lipase pretreatments for enzymatic processing of wool fabrics

A cutinase from Thermobifida fusca WSH04 and two lipases, L3126 and Lipex 100L, were applied to the enzymatic pretreatment of wool fabrics followed by protease treatment, aiming at hydrolyzing the outmost bound lipids on the wool surface. A mild oxidation with 2 g/L hydrogen peroxide (30%) was selectively carried out before the enzymatic treatments. The cooperative actions of mild oxidation, cutinase and lipase pretreatments during wool processing were investigated. The results showed that lipase pretreatment alone had less impact on the wettability and anti‐felting ability of wool fabrics than cutinase treatment. Combined use of cutinase and lipase pretreatments did not evidently improve the properties of the wool fabric compared with the individual cutinase pretreatment. By contrast, mild oxidation slightly enhanced the activity of cutinase toward the wool surface and promoted the subsequent proteolytic reactions. The wetting time and contact angle of the protease‐treated fabric deceased to 1.2 min and 55°, respectively; the area shrinkage decreased to 3.1%, with an acceptable strength loss from 489 to 418 N. The changes in the cuticle scales of the wool fibers, confirmed by scanning electron microscopy, further proved the cooperative actions of mild oxidation and cutinase pretreatment during enzymatic wool processing.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, <Emphasis Type="Italic">Lecanicillium</Emphasis> spp: implications for host specificity

The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data).     

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae     

Varroa treatment with bromfenvinphos markedly suppresses honeybee biochemical defence levels

We examined the influence of bromfenvinphos, a commonly used acaricide, on activities of many metabolic enzymes affecting the biochemical defences/physiology of the western honeybee, Apis mellifera L. (Hymenoptera: Apidae), as well as on some metabolic compound concentrations, percentage of global DNA methylation, and Nosema spp. infection levels. Bromfenvinphos‐treated workers had decreased haemolymph volumes and higher protein concentrations on their cuticle but lower protein concentrations in the haemolymph. They had higher global DNA methylation levels independent of the age‐related variants. Bromfenvinphos decreased the activities of antioxidant enzymes (SOD, GPx, CAT, GST), acidic, neutral, and alkaline protease inhibitors and enzymatic physiological markers (AST, ALT, ALP), and concentrations of urea, uric acid, creatinine, cholesterol, glucose, Mg²⁺, and Ca²⁺ in worker haemolymph, depending on the age of the bees. Protease activities were higher only in the haemolymph of young bromfenvinphos‐treated bees in comparison with untreated bees. This compound decreased the activities of alkaline proteases and neutral protease inhibitors on the cuticle. Unexpectedly, in the treated bees, the activities of acidic and neutral proteases, and acidic and alkaline protease inhibitors, were higher in the young bees and lower in the older workers in comparison to the untreated group. The bromfenvinphos‐treated workers were more heavily infested with Nosema spp. Thus, bromfenvinphos not only supressed many levels of biochemical defences, and therefore stress‐resistance‐related biochemical pathways but also visibly increased the Nosema spp. infection levels.     

Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus

The gene encoding the extracellular neutral metalloprotease ShpI from Staphylococcus hyicus subsp. hyicus was cloned. DNA sequencing revealed an ORF of 1317 nucleotides encoding a 438 amino acid protein with Mr of 49698. When the cloned gene was expressed in Staphylococcus carnosus, a 42 kDa protease was found in the culture medium. The protease was purified from both S. carnosus (pCAshp1) and S. hyicus subsp. hyicus. The N-terminal amino acid sequences of the two proteases revealed that ShpI is organized as a pre-pro-enzyme with a proposed 26 amino acid signal peptide, a 75 amino acid hydrophilic pro-region, and a 337 amino acid extracellular mature form with a calculated Mr of 38394. The N-termini showed microheterogeneity in both host strains. ShpI had a maximum proteolytic activity at 55°C and pH 7.4–8.5. The protease, which had a low substrate specificity, could be inhibited by metal- and zinc-specific inhibitors, such as EDTA and 1,10-phenanthroline. Insensitivity to phosphoramidon separates ShpI from the thermolysin-like family. The conserved Zn²⁺ binding motif, the only homology to other proteases, and the reactivation of the apoenzyme by Zn²⁺, indicated that Zn²⁺ is the catalytic ion. Ca²⁺ very probably acts as a stabilizer. We also demonstrated the presence of a second extracellular protease in S. hyicus subsp. hyicus.     

Effect of aeration and agitation on the protease production by S<Emphasis Type="Italic">taphylococcus aureus</Emphasis> mutant RC128 in a stirred tank bioreactor

The modified rotating simplex method has been successfully used to determine the best combination of agitation rate and aeration rate for maximum production of extracellular proteases by Staphylococcus aureus mutant RC128, in a stirred tank bioreactor operated in a discontinuous way. This mutant has shown altered exoprotein production, specially enhanced protease production. Maximum production of proteases (15.28 UP/ml), measured using azocasein as a substrate, was obtained at exponential growth phase when the bioreactor was operated at 300 rpm and at 2 vvm with a volumetric oxygen transfer coefficient (K _L a) of 175.75 h⁻¹. These conditions were found to be more suitable for protease production.     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.