Role of the protease in the permeability enhancement by Vibrio vulnificus

The protease produced by Vibrio vulnificus enhances vascular permeability through histamine release from mast cells and activation of the plasma kallikrein-kinin system which generates bradykinin when injected into the dorsal skin. V. vulnificus living cells also enhanced vascular permeability within a few hours after the injection into the dorsal skin. The permeability-enhancing activity of living cells was greatly reduced by addition of soybean trypsin inhibitor, a specific inhibitor for plasma kallikrein-kinin system, or anti-protease IgG. Two protease-deficient mutants induced by nitrosoguanidine treatment had only one-tenth permeability-enhancing activity of a wild-type strain. These results indicate that V. vulnificus elaborates the protease in vivo and that the protease elaborated enhances vascular permeability through release of chemical mediators such as histamine and bradykinin and forms edema.     

Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization

Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transfer-based HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 microm and blocked HIV-1 replication with IC(50) values of 0.0002-0.48 microm. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1.     

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.     

Identification of a myofibril-bound serine protease and its endogenous inhibitor in mouse skeletal muscle

Myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. A proteolytic activity associated to myofibrils was found in mouse skeletal muscle, as show electrophoretic patterns, and denominated by us, as protease M. During incubation of whole myofibrils at 37 degrees C, myosin heavy chain, alpha actinin, actin and troponin T suffered degradation. These effects were inhibited selectively by serine protease inhibitors (soybean trypsin inhibitor, di-isopropyl phosphofluoridate, phenylmethanesulfonyl fluoride). Using myofibrils as protease M source, azocaseinolytic activity was also detected. Endogenous inhibitor and various compounds effects on protease M activity were also quantified by trichloroacetic acid soluble products formation, using radiolabeled myofibrils. An endogenous trypsin inhibitor isolated from the muscle cytoplasmic fraction could inhibit protease M activity on myofibrillar proteins and on azocasein. While K(+) increased protease M activity, the presence of Ca(2+) did not show any effect. Data presented in this study suggest that reported protease M may be implicated in myofibrillar degradation in vivo and isolated endogenous inhibitor may provide a mechanism to control its action in mouse skeletal muscle.     

Kinetic studies on the inhibitions of mast cell chymase by natural serine protease inhibitors: indications for potential biological functions of these inhibitors

We have found that degranulation from mast cells is specifically inhibited by the inhibitors of chymase (10). Among the natural serine protease inhibitors tested, Bowman-Birk soybean protease inhibitor, Eglin C, and human alpha 1-antichymotrypsin inhibited chymase more strongly than did chymostatin, Kunitz soybean protease inhibitor, and phosphatidylserine. Of the inhibitors tested, Bowman-Birk soybean protease inhibitor was the strongest inhibitor of chymase, its Ki value being 13.2 X 10(-9) M. Kinetic studies showed that these inhibitors were all noncompetitive inhibitors of chymase. Bowman-Birk and Kunitz soybean protease inhibitors inhibited both chymotrypsin-type and trypsin-type serine proteases but Eglin C specifically inhibited chymotrypsin-type proteases.     

Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity

We report here the continued characterization of a 41-kDa protease expressed in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin-cleavage activity and an echinoderm-specific collagen-cleavage activity. In the experiments reported here, we have explored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10-phenanthroline (a metalloprotease inhibitor), and E-64 (a thiol protease inhibitor) were without effect on the gelatin-cleavage activity of the 41-kDa species. Using a gelatin substrate gel zymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamide appeared to partially inhibit gelatin-cleavage activity. This result was confirmed in a quantitative gelatin-cleavage assay using the water soluble, serine protease inhibitor [4-(2-aminoethyl)benzenesulfonylfluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent cations present in sea water, on the gelatin-cleavage activity. Calcium and magnesium competed for binding to the 41-kDa collagenase/gelatinase, and prebound calcium was displaced by magnesium. Cleavage activity was inhibited by magnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may regulate the proteolytic activity of the 41-kDa species.     

Reelin is a serine protease of the extracellular matrix.

Reelin is an extracellular matrix protein that plays a pivotal role in development of the central nervous system. Reelin is also expressed in the adult brain, notably in the cerebral cortex, where it might play a role in synaptic plasticity. The mechanism of action of reelin at the molecular level has been the subject of several hypotheses. Here we show that reelin is a serine protease and that proteolytic activity is relevant to its function, since (i) Reelin expression in HEK 293T cells impairs their ability to adhere to fibronectin-coated surfaces, and adhesion to fibronectin is restored by micromolar concentrations of diisopropyl phosphorofluoridate, a serine hydrolase inhibitor; (ii) purified Reelin binds FP-Peg-biotin, a trap probe which irreversibly binds to serine residues located in active catalytic sites of serine hydrolases; (iii) purified Reelin rapidly degrades fibronectin and laminin, while collagen IV is degraded at a much slower rate; fibronectin degradation is inhibited by inhibitors of serine proteases, and by monoclonal antibody CR-50, an antibody known to block the function of Reelin both in vitro and in vivo. The proteolytic activity of Reelin on adhesion molecules of the extracellular matrix and/or receptors on neurons may explain how Reelin regulates neuronal migration and synaptic plasticity.     

The Hageman factor-dependent system in the vascular permeability reaction

The mechanism by which the Hageman factor-dependent system induces vascular permeability has been analyzed. The Mr-28,000 active fragment of guinea pig Hageman factor (beta-HFa), injected intradermally, induces an increase in local vascular permeability. Inhibition of vascular permeability resulted from pretreatment of the beta-HFa with immunopurified anti-Hageman factor F(ab')2 antibody at concentrations of 10(-6)-10(-7) M as well as by incubation with corn and pumpkin seed inhibitors of beta-HFa. To determine whether prekallikrein and kallikrein participated in the permeability induced by beta-HFa, circulating prekallikrein was depleted by intra-arterial injections of anti-prekallikrein F(ab')2 antibody. This resulted in about 80% diminution of the vascular permeability response to beta-HFa, without affecting the permeability reaction to bradykinin. Soybean trypsin inhibitor (10(-6) M), injected at the same cutaneous site as the beta-HFa, inhibited the vascular permeability response to beta-HFa by more than 90%. This concentration of soybean inhibitor blocked more than 90% of the activity of guinea pig plasma kallikrein, but did not inhibit the amidolytic capacity of beta-HFa. The permeability activity of beta-HFa (but not its amidolytic activity) was augmented 10-fold by simultaneous injection of a synthetic kinin potentiator, SQ 20,881 (Glu-Tyr-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OH), and was almost completely inhibited by the simultaneous injection of a kinin-destroying enzyme, carboxypeptidase B. These results support the hypothesis that the greatest proportion of vascular permeability induced by beta-HFa is produced by the activation of prekallikrein followed by the release of kinin in the cutaneous tissue. These data offer the first in vivo evidence that the Hageman factor-dependent system by itself can induce inflammatory changes.     

Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.     

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found.     

Degradation of Rat Brain Cholinergic Muscarinic Receptors In Vitro: Enhancement by Agonists and Inhibition by Antagonists

Cholinergic muscarinic receptors undergo proteolytic degradation in vitro under physiological conditions as shown by a loss in [3H]quinuclidinylbenzilate binding activity. The serine protease inhibitor phenylmethylsulfonyl fluoride was very effective in diminishing the receptor loss. Soybean trypsin inhibitor was less effective. Both EDTA and EGTA were also effective in abolishing receptor degradation, suggesting the involvement of metallopeptidases in the process. Calcium-dependent neutral proteases requiring sulfhydryl reducing agents did not seem to be involved in receptor degradation. Dithiothreitol failed to enhance receptor degradation and iodoacetamide, leupeptin, and antipain, inhibitors of this enzyme class, failed to alter receptor loss as measured by radioligand binding. Most of the proteolytic activity occurred in the cytosol and was readily resolved from the receptor in the membrane fraction. We found that [3H]quinuclidinylbenzilate, an antagonist, inhibited the rate of receptor loss. On the other hand, agonists (acetylcholine, methacholine, and muscarine) appeared to enhance the rate of receptor loss. We postulate that these opposite effects are due to differences in receptor conformation in response to ligand binding. Susceptibility to proteolysis may therefore serve as a probe for receptor conformation.     

Effects of calcium and magnesium on a 41‐kDa serine‐dependent protease possessing collagen‐cleavage activity

We report here the continued characterization of a 41‐kDa protease expressed in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin‐cleavage activity and an echinoderm‐specific collagen‐cleavage activity. In the experiments reported here, we have explored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10‐phenanthroline (a metalloprotease inhibitor), and E‐64 (a thiol protease inhibitor) were without effect on the gelatin‐cleavage activity of the 41‐kDa species. Using a gelatin substrate gel zymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamide appeared to partially inhibit gelatin‐cleavage activity. This result was confirmed in a quantitative gelatin‐cleavage assay using the water soluble, serine protease inhibitor [4‐(2‐aminoethyl)benzenesulfonylfluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent cations present in sea water, on the gelatin‐cleavage activity. Calcium and magnesium competed for binding to the 41‐kDa collagenase/gelatinase, and prebound calcium was displaced by magnesium. Cleavage activity was inhibited by magnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may regulate the proteolytic activity of the 41‐kDa species. J. Cell. Biochem. 80:139–145, 2000. © 2000 Wiley‐Liss, Inc.     

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.     

Peptidyl boronates inhibit Salmonella enterica serovar Typhimurium Lon protease by a competitive ATP-dependent mechanism

Lon is a homo-oligomeric ATP-dependent serine protease that functions in the degradation of damaged and certain regulatory proteins. This enzyme has emerged as a novel target in the development of antibiotics because of its importance in conferring bacterial virulence. In this study, we explored the mechanism by which the proteasome inhibitor MG262, a peptidyl boronate, inhibits the peptide hydrolysis activity of Salmonella enterica serovar Typhimurium Lon. In addition, we synthesized a fluorescent peptidyl boronate inhibitor based upon the amino acid sequence of a product of peptide hydrolysis by the enzyme. Using steady-state kinetic techniques, we have shown that two peptidyl boronate variants are competitive inhibitors of the peptide hydrolysis activity of Lon and follow the same two-step, time-dependent inhibition mechanism. The first step is rapid and involves binding of the inhibitor and formation of a covalent adduct with the active site serine. This is followed by a second slow step in which Lon undergoes a conformational change or isomerization to increase the interaction of the inhibitor with the proteolytic active site to yield an overall inhibition constant of 5-20 nM. Although inhibition of serine and threonine proteases by peptidyl boronates has been detected previously, Lon is the first protease that has required the binding of ATP in order to observe inhibition.     

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii

A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry.     

The role of proteases in the differentiation of Acanthamoeba castellanii

Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii . Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.     

Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.     

Protease activity in brook trout (Salvelinus fontinalis) follicle walls demonstrated by substrate-polyacrylamide gel electrophoresis

Proteolytic activity was demonstrated in the follicle wall surrounding oocytes of brook trout (Salvelinus fontinalis) by an assay system that incorporated protein substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (substrate-SDS-PAGE). At least six proteolytic enzymes (78, 70, 67, 59, 22 and 20 kDa) were present when follicle wall extracts were electrophoresed and incubated in gels containing gelatin. Of these six enzymes, only two enzymes (20 and 22 kDa) were present when follicle wall extracts were resolved and incubated in gels containing casein. The activities of the 78 and 70 kDa enzymes were completely inhibited with metallo- and collagenolytic protease inhibitors and partially inhibited with serine protease inhibitors. The activities of the 67 and 59 kDa enzymes were completely blocked with metallo- and collagenolytic protease inhibitors. The activities of the 22 and 20 kDa enzymes were only slightly decreased with a serine protease inhibitor.     

Proteases and Their Involvement in the Infection and Immobilization of Nematodes by the Nematophagous Fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora produced extracellular proteases when grown in a liquid culture, as revealed by measuring the hydrolysis of the chromogenic substrate Azocoll. The extracellular protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and other serine protease inhibitors and partly inhibited by the aspartate protease inhibitor pepstatin and by a cysteine protease inhibitor [l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, or E-64]. Substrate gel electrophoresis showed that the fungus produced several different proteases, including multiple serine proteases. The function of proteases in the infection of nematodes was examined by treating the fungus with various protease inhibitors. None of the inhibitors tested affected the adhesion of nematodes to the traps, but incubating trap-bearing mycelium with a serine protease inhibitor, PMSF, antipain, or chymostatin, or the metalloprotease inhibitor phenanthroline significantly decreased the immobilization of nematodes captured by the fungus. Inhibitors of cysteine or aspartic proteases did not affect the immobilization of captured nematodes. The effects of PMSF on the immobilization of nematodes were probably due to serine proteases produced by the fungus, since the effects were observed when unbound inhibitor was washed away from the fungus before the nematodes were added to the system. No effects were observed when the nematodes only were pretreated with PMSF.