The serine peptidase inhibitor N-ρ-tosyl-l-phenylalanine chloromethyl ketone (TPCK) affects the cell biology of Candida haemulonii species complex.

Candida haemulonii species complex (C. haemulonii, C. haemulonii var. vulnera and Candida duobushaemulonii) is composed by emerging and multidrug-resistant (MDR) yeasts. Candidiasis, the disease caused by these species, is difficult to treat and culminates in clinical failures and patient death. It is well-known that Candida peptidases play important roles in the fungus-host interactions, and hence these enzymes are promising targets for developing new antifungal drugs. Recently, serine-type peptidases were described in clinical isolates of C. haemulonii complex with the ability to cleave relevant key host proteins. Herein, the effects of serine peptidase inhibitors (SPIs) on the cell biology of this fungal complex were evaluated. Initially, eight distinct SPIs (phenylmethylsulfonyl fluoride - PMSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride - AEBSF, N-α-tosyl-l-lysine chloromethyl ketone hydrochloride - TLCK, N-p-tosyl-l-phenylalanine chloromethyl ketone - TPCK, simeprevir, boceprevir, danoprevir and telaprevir) were tested on the fungal growth. TPCK showed the best efficacy in controlling cell proliferation, being selected for the following experiments. This SPI induced changes in the architecture of yeast cells, as observed by scanning electron microscopy, besides injuries at the plasma membrane and reduction in the ergosterol content. TPCK also diminished the ability of yeasts to adhere to abiotic (polystyrene and glass) and biotic (murine macrophages) surfaces in a typically concentration-dependent manner. In addition, the 24 h-treatment of the mature biofilm promoted a decrease in biomass, viability and extracellular matrix. Altogether, our results highlight that SPIs may be promising new therapeutic agents in the treatment of candidiasis caused by emergent, opportunistic and MDR species forming the C. haemulonii complex.

Cysteine peptidases in Herpetomonas samuelpessoai are modulated by temperature and dimethylsulfoxide-triggered differentiation.

Cysteine peptidases of protozoa have been implicated in a variety of biological events, and the expression of these enzymes is modulated in response to distinct stimuli, including environmental changes and differentiation. In the present work, we have examined the expression of cysteine peptidases from Herpetomonas samuelpessoai grown at distinct temperatures and during dimethylsulfoxide (DMSO)-elicited differentiation. We demonstrated that a 45 kDa cysteine peptidase had its activity reduced during the parasite growth at 37 degrees C in comparison to 26 degrees C, and when cultured up to 72 h in the presence of DMSO. The modulation in the 45 kDa cysteine peptidase expression is connected to the differentiation process, since both temperature and DMSO are able to trigger the promastigote to paramastigote transformation in H. samuelpessoai. The possible immunological similarity of H. samuelpessoai proteins with well-known cysteine peptidases produced by trypanosomatid pathogens, including cruzipain (Trypanosoma cruzi) and cysteine peptidase b (cpb) from Leishmania mexicana, was also investigated, as well as with calpain molecules. The protein cellular lysate of H. samuelpessoai reacted with antibodies raised against cpb of L. mexicana and calpain of Drosophila melanogaster; however, no reaction was observed against cruzipain. The 35 kDa cpb-like protein had its expression diminished in DMSO-treated parasites, while the 80 kDa calpain-like molecule was enhanced and an additional 30 kDa calpain-related polypeptide was exclusively observed in these cells. Fluorescence microscopy and flow cytometry analyses corroborated these data. The results described above add H. samuelpessoai to the list of parasites whose differentiation seems to be correlated with cysteine peptidase expression.

Arrested growth of Trypanosoma cruzi by the calpain inhibitor MDL28170 and detection of calpain homologues in epimastigote forms.

In this paper, we aimed to explore the effects of the calpain inhibitor III (MDL28170) and to detect calpain-like molecules (CALPs) in epimastigote forms of Trypanosoma cruzi isolate Dm28c. MDL28170 at 70 microM promoted a powerful reduction in the growth rate after 48 h. The IC50 value was calculated to be 31.7 microM. This inhibitor promoted an increase in the cellular volume, but not cell lysis, resulting in a trypanostatic effect. T. cruzi CALPs presented a strong cross-reactivity with anti-Drosophila melanogaster calpain and anti-cytoskeleton-associated protein from Trypanosoma brucei antibodies, and labelling was found mainly intracellularly. Furthermore, an 80 kDa reactive protein was detected by Western blotting assays. No significant cross-reactivity was found with anti-human brain calpain antibody. The expression of CALPs was decreased in cells kept for long periods in axenic cultures in comparison to a strain recently isolated from mice, as well as in MDL28170-treated cells, the latter being paralleled by an increased expression of cruzipain. Different levels of CALPs expression were also detected in distinct phylogenetic lineages, like Y strain (lineage TcII), Dm28c (lineage TcI) [corrected] and INPA6147 strain (Z3 zymodeme). These results may contribute for the investigation of the functions of CALPs in trypanosomatids.

Novel antimony(iii) hydroxamic acid complexes as potential anti-leishmanial agents.

Reaction of benzohydroxamic acid (Bha), 2-pyridinehydroxamic acid (2-pyha), 2-amino-phenylhydroxamic acid (2-NH2-pha) and salicylhydroxamic acid (Sha) with SbCl3 in ethanol gave the corresponding novel hydroxamato Sb(iii) complexes, [Sb(Bha-1H)2Cl] 1, [SbCl2(2-pyha-1H)] 2, [Sb(2-NH2-pha-1H)(2-NH3-pha-1H)]Cl23 and [SbCl(Sha-1H)2] 4. In all cases the hydroxamic acids coordinate to the Sb centres in the typical bidentate hydroxamato (O,O') coordination mode, via the carbonyl oxygen and deprotonated hydroxyl group. Reaction of the histone deacetylase inhibitor (HDACi) suberoylanilidehydroxamic acid (SAHA) with Sb(OEt)3 gave the Sb(iii) hydroxamato/hydroximato complex, [Sb(SAHA-1H)(SAHA-2H)] 6. All test complexes significantly inhibited the promastigote proliferation of Leishmania amazonensis and L. chagasi and induced substantial changes in the general morphology of the parasites, including reduction in size and loss of flagellum, when compared to the untreated promastigotes. A dose-response approach using the test complexes showed a decreased in plasma membrane permeability and the mitochondrial dehydrogenase activities of the Leishmania species. [Sb(Bha-1H)2Cl] exhibited the best activity and was superior to the Sb HDACi complex 6. Though 1 exhibited noteworthy anti-leishmanial activity, the selectivity indexes determined suggest that [Sb(2-NH2-pha-1H)(2-NH3-pha-1H)]Cl23 is the test complex that merits further investigation as a potential anti-leishmanial agent.

Cruzipain: An Update on its Potential as Chemotherapy Target against the Human Pathogen Trypanosoma cruzi.

Chagas' disease is one of the most impactful and prevalent neglected tropical diseases in the Americas, specially affecting the poor and underdeveloped areas in Latin America. Aggravating this scenario, the medicines used in the current chemotherapy are old, toxic and present a low efficacy to treat the chronic stage of this disease. In addition, resistant strains of Trypanosoma cruzi, the etiological agent, are frequently reported. So, there is an imperative requirement for novel chemotherapeutic options to treat this debilitating disease. In this context, peptidases have emerged as potential targets and, consequently, proteolytic inhibitors have confirmed to be valuable drugs against several human pathologies. In this line of thinking, T. cruzi produces a major multifunctional cysteine peptidase, named cruzipain, which directly and/or indirectly orchestrates several physiological and pathological processes, which culminate in a successful parasitic infection. Taken together, these findings point out that cruzipain is one of the most important targets for driving a chemotherapy approach against the human pathogen T. cruzi. The present review summarizes some of the recent advances and failures in this area, with particular emphasis on recently published studies.

Structural studies on the polar glycoinositol phospholipids of Trypanosoma (Schizotrypanum) dionisii from bats.

The polar glycoinositol phospholipids (GIPLs) of a Trypanosoma species that belongs to the Schizotrypanum subgenus were purified by reversed-phase and normal-phase liquid chromatography and analysed by negative-ion mode electrospray-mass spectrometry (ES-MS). The phosphatidylinositol moieties were released by nitrous acid deamination and identified as ceramide- and alkylacylglycerol-containing species. The structures of the GIPLs were determined using chemical treatments, sequential exoglycosidase digestions and positive-ion mode ES-MS-MS. All of the GIPLs were based on the same Man alpha1-2Man alpha1-2Man alpha1-6Man alpha1-4(NH2-CH2CH2-HPO3-)GlcN-PI core with single terminal Galf residue substitutions either on the terminal nonreducing Man or on the second alphaMan residue from the inositol and with either ethanolamine phosphate or 2-aminoethylphosphonate on the third alphaMan residue from the inositol. The T. (S.) dionisii GIPLs are compared with those of T. (S.) cruzi, a closely related species of the Schizotrypanum subgenus.

Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species.

Crithidia oncopelti, Crithidia deanei and Crithidia desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Gelatin-SDS-PAGE analysis was used to characterize cell-associated and extracellular proteinases in these organisms. Our survey indicates that the proteolytic profiles of C. deanei and C. desouzai are identical; that C. oncopelti displays a distinct zymogram; and that species naturally lacking endosymbionts have a more complex extracellular proteolytic activity, which illustrates the heterogeneity of this genus. This is the first report on the presence of cysteine proteinases in the culture supernatant of monoxenic trypanosomatids, and by the use of wild and aposymbiotic strains from C. deanei we also demonstrated that the prokaryote endosymbiont somehow alters quantitatively the expression of extracellular proteinases in this trypanosomatid.

Characterization of proteinases in trypanosomatids.

Proteinases are important factors in the pathogenicity of many parasitic diseases. In this study, the proteolytic activities of 10 trypanosomatids from five different genera (Crithidia, Phytomonas, Endotrypanum, Trypanosoma and Leishmania) were determined by SDS-PAGE containing copolymerized gelatin as substrate. In almost all species we could detect two proteolytic classes, cysteine- and metalloproteinases, based on the inhibition of their activities by E-64 and 1,10-phenanthroline, respectively. In all cases, the metalloproteinase activities did not change over a broad pH range (from 5.5 to 10). E. schaudinni, T. mega, T. dionisii, C. luciliae, C. fasciculata, C. oncopelti and C. guilhermei expressed one or two metalloproteinases of 45-66 kDa, whereas in P. serpens and P. hyssopifolia a double band of this endopeptidase was detected at 94 kDa. In contrast, no metalloproteinase activity was observed in L. tarentolae. The optimal pH for the cysteine-proteinase activities was acidic (about 5.5). In E. schaudinni, T. mega and in Crithidia sp., these proteinases had an apparent molecular weight of 66-94 kDa, while L. tarentolae expressed a broad band from 29 to 45 kDa. In Phytomonas sp., this class of endopeptidase showed a unique feature, in that major cysteine-proteinases were found at 29-66 kDa, but multiple, low-activity bands were detected from 116 to 200 kDa. The most striking characteristic, however, was the very intense cysteine-proteinase activity expressed by T. dionisii (29-66 kDa). We conclude that these differences in the proteolytic profiles could be useful markers to characterize and compare trypanosomatids.

Aspartic peptidases of human pathogenic trypanosomatids: perspectives and trends for chemotherapy.

Aspartic peptidases are proteolytic enzymes present in many organisms like vertebrates, plants, fungi, protozoa and in some retroviruses such as human immunodeficiency virus (HIV). These enzymes are involved in important metabolic processes in microorganisms/virus and play major roles in infectious diseases. Although few studies have been performed in order to identify and characterize aspartic peptidase in trypanosomatids, which include the etiologic agents of leishmaniasis, Chagas' disease and sleeping sickness, some beneficial properties of aspartic peptidase inhibitors have been described on fundamental biological events of these pathogenic agents. In this context, aspartic peptidase inhibitors (PIs) used in the current chemotherapy against HIV (e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) were able to inhibit the aspartic peptidase activity produced by different species of Leishmania. Moreover, the treatment of Leishmania promastigotes with HIV PIs induced several perturbations on the parasite homeostasis, including loss of the motility and arrest of proliferation/growth. The HIV PIs also induced an increase in the level of reactive oxygen species and the appearance of irreversible morphological alterations, triggering parasite death pathways such as programed cell death (apoptosis) and uncontrolled autophagy. The blockage of physiological parasite events as well as the induction of death pathways culminated in its incapacity to adhere, survive and escape of phagocytic cells. Collectively, these results support the data showing that parasites treated with HIV PIs have a significant reduction in the ability to cause in vivo infection. Similarly, the treatment of Trypanosoma cruzi cells with pepstatin A showed a significant inhibition on both aspartic peptidase activity and growth as well as promoted several and irreversible morphological changes. These studies indicate that aspartic peptidases can be promising targets in trypanosomatid cells and aspartic proteolytic inhibitors can be benefic chemotherapeutic agents against these human pathogenic microorganisms.

Calpains: potential targets for alternative chemotherapeutic intervention against human pathogenic trypanosomatids.

The treatment for both leishmaniasis and trypanosomiasis, which are severe human infections caused by trypanosomatids belonging to Leishmania and Trypanosoma genera, respectively, is extremely limited because of concerns of toxicity and efficacy with the available anti-protozoan drugs, as well as the emergence of drug resistance. Consequently, the urgency for the discovery of new trypanosomatid targets and novel bioactive compounds is particularly necessary. In this context, the investigation of changes in parasite gene expression between drug resistant/sensitive strains and in the up-regulation of virulence-related genes in infective forms has brought to the fore the involvement of calpain-like proteins in several crucial pathophysiological processes performed by trypanosomatids. These studies were encouraged by the publication of the complete genome sequences of three human pathogenic trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which allowed in silico analyses that in turn directed the identification of numerous genes with interesting chemotherapeutic characteristics, including a large family of calpain-related proteins, in which to date 23 genes were assigned as calpains in T. brucei, 40 in T. cruzi and 33 in L. braziliensis. In the present review, we intend to add to these biochemical/biological reports the investigations performed upon the inhibitory capability of calpain inhibitors against human pathogenic trypanosomatids.

Antimicrobial action of chelating agents: repercussions on the microorganism development, virulence and pathogenesis.

Infections caused by resistant microorganisms often fail to respond to conventional therapy, resulting in prolonged illness, increased treatment costs and greater risk of death. Consequently, the development of novel antimicrobial drugs is becoming more demanding every day since the existing drugs either have too many side-effects or they tend to lose effectiveness due to the selection of resistant strains. In view of these facts, a number of new strategies to obstruct vital biological processes of a microbial cell have emerged; one of these is focused on the use of metal-chelating agents, which are able to selectively disturb the essential metal metabolism of the microorganism by interfering with metal acquisition and bioavailability for crucial reactions. The chelation activity is able to inhibit the biological role of metal-dependent proteins (e.g., metalloproteases and transcription factors), disturbing the microbial cell homeostasis and culminating in the blockage of microbial nutrition, growth and development, cellular differentiation, adhesion to biotic (e.g., extracellular matrix components, cell and/or tissue) and abiotic (e.g., plastic, silicone and acrylic) structures as well as controlling the in vivo infection progression. Interestingly, chelating agents also potentiate the activity of classical antimicrobial compounds. The differences between the microorganism and host in terms of the behavior displayed in the presence of chelating agents could provide exploitable targets for the development of an effective chemotherapy for these diseases. Consequently, metal chelators represent a novel group of antimicrobial agents with potential therapeutic applications. This review will focus on the anti-fungal and anti-protozoan action of the most common chelating agents, deciphering and discussing their mode of action.

Differential influence of gp63-like molecules in three distinct Leptomonas species on the adhesion to insect cells.

Parasites belonging to the Leptomonas genus have been used as model organisms for studying biochemical, cellular, and genetic processes unique to members of the Trypanosomatidae family. In the present study, the cell-associated and extracellular peptidases of three Leptomonas species, Leptomonas collosoma, Leptomonas samueli, and Leptomonas wallacei, were assayed and characterized by gelatin-sodium dodecyl sulfate polyacrylamide gel electrophoresis. All parasites released metallopeptidases, whereas no cell-associated proteolytic activity could be detected in the cellular extracts from L. collosoma. Western blotting probed with a polyclonal antibody raised against gp63 from Leishmania amazonensis revealed two major reactive polypeptides of apparent molecular masses of 63 and 52 kDa, with different intensities in cellular extracts and released proteins from the studied trypanosomatids. Flow cytometry and fluorescence microscopy analyses showed that the gp63-like molecules have a surface location. This is the first report on the presence of gp63-like molecules in L. collosoma, L. samueli, and L. wallacei. The pretreatment of L. samueli and L. wallacei with anti-gp63 antibody significantly diminished their association index to Aedes albopictus cell line (C6/36), suggesting a potential involvement of the gp63-like molecules in the interaction process of these insect trypanosomatids with the vector.

Proteolytic expression in Blastocrithidia culicis: influence of the endosymbiont and similarities with virulence factors of pathogenic trypanosomatids.

Blastocrithidia culicis is an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of the B. culicis proteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids, Leishmania spp. and Trypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.

Monohexosylceramides of Trypanosoma dionisii.

A detailed knowledge of the primary structure of neutral glycosphingolipids isolated and purified from Trypanosoma dionisii has been elucidated using a combination of techniques--as column chromatography, HPTLC and GC-MS together with fast atom bombardment spectrometry. The ceramide monohexoside fraction (CMH) contained both glucosyl- and galactosylceramides in a ratio of 1:1, sphingosine and as fatty acyl groups mainly C-24 saturated and 2-hydroxy fatty acids. A close similarity between Trypanosoma cruzi and T. dionisii monohexosylceramides was reported.

Glycolipid and protein profiles in trypanosomatids.

A comparative study of glycolipid and protein composition in Trypanosoma cruzi and non-pathogenic trypanosomatids was carried out using Triton X-114 extraction. Protein profiles were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE), and glycolipids were detected using high-performance thin-layer chromatography (HPTLC). Hydrophilic protein profiles were similar in non-pathogenic protozoa. Endotrypanum schaudinni, Crithidia luciliae and T. mega showed five characteristic protein bands ranging between 30 and 66 kDa. In the hydrophobic phase, a band of 50 kDa was present only in T. mega. Strain-specific protein distribution was detected in T. cruzi clone Dm28c and T. cruzi G and Y strains; clone Dm28c had five typical hydrophilic proteins at between 24 and 45 kDa, the G strain had two bands at 45 kDa in the hydrophilic phase and the Y strain had a major protein band at 24 kDa in both phases. T. dionisii and T. cruzi clone Dm28c showed a characteristic distribution of three hydrophilic proteins of approx. 45 kDa. Qualitative analysis of glycolipid composition showed that the T. cruzi strains and Dm28c clone and T. dionisii had four orcinol-positive spots, whereas in the other non-pathogenic trypanosomatids only three glycolipids were detected.

Use of glycoconjugates for trypanosomatid taxonomy.

Glycoconjugates from five trypanosomatid genera--Crithidia, Herpetomonas, Endotrypanum, Leishmania, and Trypanosoma--were extracted with Triton X-114 and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by periodic acid-Schiff staining. Most of the glycoconjugates were detected in the hydrophobic phase, indicating the presence of anchored glycoconjugates. All the trypanosomatids expressed a glycoconjugate with a low molecular weight (below 20 kDa) in this phase. In each species, however, a characteristic and specific pattern of glycoconjugates was also observed in both phases. In the hydrophobic phase: 14-29 kDa glycoconjugates in C. guilhermei; 24-70 kDa in C. fasciculata, C. luciliae, E. schaudinni, and T. cruzi Y and G strains; 45-66 kDa in C. oncopelti and H. samuelpessoai; above 36 kDa in T. dionisii; 20-24 kDa, 36-45 kDa, and 70 kDa in L. tarentolae and T. mega. In the hydrophilic phase, typical glycoproteins were observed in some trypanosomatids: 60 kDa in T. mega and T. cruzi Y strain; 70 kDa in H. samuelpessoai; 66 kDa in C. oncopelti; 20-70 kDa in C. luciliae. These findings suggest that Triton X-114-extracted glycoconjugates could be useful markers for trypanosomatid taxonomy.

Ubiquity of cysteine- and metalloproteinase activities in a wide range of trypanosomatids.

We have analysed the proteinase profiles of 11 species from 7 different genera of trypanosomatids by in situ detection of enzyme activities on SDS-PAGE gels containing co-polymerized gelatin as substrate, and the use of specific proteinase inhibitors. Our survey indicates that while cysteine- and metalloproteinases are distributed ubiquitously among trypanosomatids, there are marked differences between the enzyme profiles from the monogenetic (Crithidia, Herpetomonas, Leptomonas) and digenetic (Trypanosoma, Endotrypanum, Phytomonas, Leishmania) species. The detected metalloproteinase activities, ranging in size from 50-100 kDa, partitioned into the detergent-phase after Triton X-114 extraction, while most of cysteine proteinases, of three distinct molecular mass ranges (30-50 kDa, 80-100 kDa and 116-205 kDa), partitioned into the aqueous phase. Thus, within this group of organisms, the metalloproteinase activities seem to be predominantly membrane-associated proteins. We also show that the plant parasites of the genus Phytomonas exhibit a distinctive cysteine proteinase profile that might be exploited further as a criterion for taxonomy of the genus.

Extracellular serine-proteinases isolated from Streptomyces alboniger: partial characterization and effect of aprotinin on cellular structure.

Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI) medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4% and 10.4% yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate, was 9-10 and the optimum temperature was 37 degrees C. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth.

Detection of extracellular proteases from microorganisms on agar plates.

We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

Crithidia guilhermei: purification and partial characterization of a 62-kDa extracellular metalloproteinase.

An extracellular metalloproteinase from Crithidia guilhermei, a monoxenic trypanosomatid of insects, was purified 11-fold by ammonium sulfate precipitation, gel filtration on a Shinpack Diol-150 column, and anion-exchange chromatography in a MONO Q column, both using the HPLC system. The proteinase appeared as a single band with an apparent molecular mass of 62 kDa in SDS-PAGE, under reducing conditions, and was optimally active at 37 degrees C and pH 6.0. The enzyme showed 62% residual activity at 50 degrees C for 30 min. The proteinase was completely inhibited by 1, 10-phenanthroline, indicating that the enzyme belongs to the metalloproteinase class. This is the first report of the purification of an extracellular metalloproteinase from the Crithidia species. The possible role of this enzyme in the digestive tract of the insect host is discussed.