Hydrolysis of L-leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases.

The main cysteine proteinases of the amastigote form of Leishmania mexicana mexicana were partially purified by gel filtration and ion exchange chromatography. The latter procedure resulted in the separation of some individual cysteine proteinases, as demonstrated by gelatin-sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Fractions containing the partially purified proteinases rapidly hydrolysed L-leucine methyl ester to leucine. The activity towards this compound co-eluted with and resembled the parasite's cysteine proteinase activity. The results suggest that amastigotes of L.m.mexicana are susceptible to L-leucine methyl ester because this compound is rapidly hydrolysed by cysteine proteinases that occur in abundance in the megasomes of this stage.     

Expression of Cysteine Proteinases by Metacyclic Promastigotes of Leishmania mexicana

ABSTRACT. The expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana was investigated using gelatin polyacrylamide gel electrophoresis. Two prominent bands were detected which distinguished metacyclics from multiplicative promastigotes, lacking detectable cysteine proteinase activity, and amastigotes, with a distinct banding pattern composed of multiple enzymes. A correlation between relative activity of the metacyclic-specific bands and the prevalence of metacyclics was found both during the growth cycle in vitro as metacyclogenesis occurred, and by comparison of stationary phase populations from consecutive subpassages in vitro. Irreversible inhibition of the metacyclic activities using N-benzyloxycarbonyl-phenylalanyl-alanyl diazomethane did not inhibit metacyclic to amastigote transformation in vitro. These activities provide a useful biochemical marker for the metacyclic promastigotes of L. mexicana .     

Leishmania mexicana: amastigote hydrolases in unusual lysosomes

Leishmania mexicana mexicana (M379) amastigotes were found to contain much higher activities than cultured promastigotes of five putative lysosomal enzymes: cysteine proteinase; arylsulfatase (EC 3.1.6.1); beta-glucuronidase (EC 3.2.1.31); DNase (EC 3.1.22.1), and RNase (EC 3.1.27.1). The release profiles of the first three of these enzymes from digitonin-permeabilized amastigotes suggests that they are located within organelles. Cytochemical staining for cysteine proteinase, using gold labeled antibodies and arylsulfatase, showed that both were present in large organelles previously named "megasomes." Comparative studies with L. mexicana amazonensis (LV78), L. donovani donovani (LV9), and L. major (LV39) revealed that L. mexicana amazonensis was similar to L. mexicana mexicana in possessing both high amastigote cysteine proteinase activity and large numbers of megasome organelles in amastigotes, whereas the other two species lacked both these features. The results suggest that the presence of numerous lysosome-like organelles in the amastigote is a characteristic of the L. mexicana group of parasites.     

Stage-specific proteinases of Leishmania mexicana mexicana promastigotes

Four distinct bands of cysteine proteinase activity were detected when stationary-phase populations of Leishmania mexicana mexicana were subjected to gelatin-SDS-PAGE. The highest mobility band contained at least three isoforms separable by mono Q anion exchange chromatography. These high mobility activities were distinct from all the major amastigote enzymes. Stationary-phase promastigote populations also contained two acid-activable precursor forms of the promastigote-specific band. It is suggested that these promastigote-specific activities occur in the infective metacyclic stage of the parasite and may have a role in parasite survival upon inoculation into a mammal.     

Leishmania mexicana: subcellular distribution of enzymes in amastigotes and promastigotes

Glycosomes and mitochondrial vesicles from cultured promastigotes of Leishmania mexicana mexicana have been separated using isopycnic centrifugation on linear sucrose gradients. Hexokinase (EC 2.7.1.2), glucose phosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were recovered largely in association with glycosomes (density; 1.215 g/ml). Phosphoglycerate kinase (EC 2.7.2.3) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) had some small glycosomal activity, but were mostly recovered in the soluble fractions. Malate dehydrogenase (EC 1.1.1.37) showed a broad peak corresponding to that of the mitochondrial marker oligomycin-sensitive ATPase (EC 3.6.1.4) (density; 1.190 g/ml). Glutamate dehydrogenase (EC 1.4.1.3) and alanine aminotransferase (EC 2.6.1.2) both showed small mitochondrial peaks, but most of the activities were recovered elsewhere on the gradient and in the soluble fractions. The subcellular location of enzymes in L.m. mexicana amastigotes was investigated by following the release of soluble enzymes from digitonin-treated amastigotes. This revealed distinct cytosolic, mitochondrial, and glycosomal compartments. The findings give an insight into the organization and control of L.m. mexicana promastigote and amastigote energy metabolism.     

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.     

A developmentally regulated cysteine proteinase gene of Leishmania mexicana

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (lmcpa) [corrected]. has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective metacyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPheValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.     

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana.

lmcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucei and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. lmcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity.     

Leishmania mexicana pifanoi: analysis of the antigenic relationships between promastigotes and amastigotes by gel diffusion, immunoelectrophoresis, and immunoprecipitation

The antigenic relationships between Leishmania mexicana pifanoi promastigotes, axenically grown amastigotes, and amastigotes isolated from the footpads of infected hamsters or from a J774 macrophage cell line were studied by three serologic methods. Amastigote and promastigote antigens were disrupted by freeze-thawing of intact cells in a lysis buffer. Antisera were prepared in rabbits by repeated subcutaneous inoculations of the parasite antigens in complete Freund's adjuvant and were tested against the homologous and heterologous antigens in a series of gel diffusion experiments. Negative results were obtained in all control experiments. In each instance, the homologous antigen-antiserum reactions yielded the largest numbers of precipitin lines. A pattern of cross-reactivity was also observed in the heterologous systems. Results indicated that the amastigote and promastigote forms had unique and common antigens. The two parasite antigen-antiserum systems were also examined by immunoelectrophoresis. Qualitative and quantitative differences between the promastigote and amastigote antigens were readily demonstrable by this technique. Results indicated that each parasite form had specific and many common antigens. In the homologous system, major proteins, with molecular weights (MW) of 23, 52, and 68 kd, were demonstrated in the promastigotes by immunoprecipitation of lactoperoxidase-catalyzed radioiodinated cells. In a similar (homologous) system, axenically grown amastigotes were found to contain three proteins with MW of 38, 70, and 74 kd and, therefore, different from those demonstrated for the promastigotes. All the results suggested that the three amastigote stages of different origins are antigenically similar to one another, but differ from the promastigote forms.     

Purification and characterization of a metallo-endoproteinase from mouse kidney.

A metallo-endoproteinase was purified from mouse kidney. The enzyme was solubilized from the 100 000 g sediment of kidney homogenates with toluene and trypsin, and further purified by fractionation with (NH4)2SO4. DEAE-cellulose chromatography and gel filtration. The molecular weight of the metalloproteinase was estimated by gel filtration on Sepharose 6B to be 270 000--320 000. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of 2-mercaptoethanol, a single major protein with a mol.wt. of 81 000 was observed. Thus the active enzyme is an oligomer, probably a tetramer. It is a glycoprotein and has an apparent isoelectric point of 4.3. Kidney homogenates and purified preparations of the metalloproteinase degraded azocasein optimally at pH 9.5 and at I 0.15--0.2. The activity was not affected by inhibitors of serine proteinases (di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride), cysteine proteinases (4-hydroxymercuribenzoate, iodoacetate), aspartic proteinases (pepstatin) or several other proteinase inhibitors from actinomycetes (leupeptin, antipain and phosphoramidon). Inhibition of the enzyme was observed with metal chelators (EDTA, EGTA, 1,10-phenanthroline), and thiol compounds (cysteine, glutathione, dithioerythritol, 2-mercaptoethanol). The metalloproteinase degraded azocasein, azocoll, casein, haemoglobulin and aldolase, but showed little or no activity against the synthetic substrates benzoylarginine 2-naphthylamide, benzoylglycylarginine, benzyloxycarbonylglutamyltyrosine or acetylphenylalanyl 2-naphthyl ester. This metalloproteinase from mouse kidney appears to be distinct from previously described kidney proteinases.     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.     

Leishmania mexicana pifanoi: Analysis of the Antigenic Relationships Between Promastigotes and Amastigotes by Gel Diffusion,Immunoelectrophoresis, and Immunoprecipitation1

ABSTRACT. The antigenic relationships between Leishmania mexicana pifanoi promastigotes, axenically grown amastigotes, and amastigotes isolated from the footpads of infected hamsters or from a J774 macrophage cell line were studied by three serologic methods. Amastigote and promastigote antigens were disrupted by freeze-thawing of intact cells in a lysis buffer. Antisera were prepared in rabbits by repeated subcutaneous inoculations of the parasite antigens in complete Freund's adjuvant and were tested against the homologous and heterologous antigens in a series of gel diffusion experiments. Negative results were obtained in all control experiments. In each instance, the homologous antigen-antiserum reactions yielded the largest numbers of precipitin lines. A pattern of cross-reactivity was also observed in the heterologous systems. Results indicated that the amastigote and promastigote forms had unique and common antigens. The two parasite antigen-antiserum systems were also examined by immunoelectrophoresis. Qualitative and quantitative differences between the promastigote and amastigote antigens were readily demonstrable by this technique. Results indicated that each parasite form had specific and many common antigens. In the homologous system, major proteins, with molecular weights (MW) of 23, 52, and 68 kd, were demonstrated in the promastigotes by immunoprecipitation of lactoperoxidase-catalyzed radioiodinated cells. In a similar (homologous) system, axenically grown amastigotes were found to contain three proteins with MW of 38, 70, and 74 kd and, therefore, different from those demonstrated for the promastigotes. All the results suggested that the three amastigote stages of different origins are antigenically similar to one another, but differ from the promastigote forms.     

Leishmania amazonensis: involvement of cysteine proteinases in the killing of isolated amastigotes by L-leucine methyl ester

L-leucine-methyl ester (Leu-OMe) kills Leishmania mexicana amazonensis amastigotes by a mechanism which requires proteolytic cleavage of the ester. N-Benzyloxycarbonyl-phenylalanyl-alanyl diazomethane (Z-Phe-AlaCHN2), a specific and irreversible inhibitor of cysteine proteinases, was used to characterize the enzymes involved in parasite destruction. It was shown that (1) amastigotes preincubated with micromolar concentrations of Z-Phe-AlaCHN2 survived challenge with Leu-OMe concentrations lethal to control parasites; (2) the proteolytic activity of 25- to 33-kDa cysteine proteinases in parasite lysates subjected to electrophoresis in gelatin-containing acrylamide gels was selectively inhibited in parasites pretreated with Z-Phe-AlaCHN2 and chased in inhibitor-free medium; and (3) cysteine proteinase activity was also inhibited in gels incubated with amino acid and dipeptide esters, possibly because the compounds were acting either as substrates (e.g., Leu-Leu-OMe) or as inhibitors (e.g., Ile-OMe) of the enzyme. The results support the involvement of low molecular weight cysteine proteinases in the destruction of amastigotes by Leu-OMe. Characterization of the structure and substrate specificity of the enzymes may permit the rational development of more selectively leishmanicidal amino acid derivatives.     

Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana

In the past, ultrastructural investigations of Leishmania mexicana amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/CPB-deficient mutants transform to amastigotes very poorly and lack virulence in macrophages and mice.     

Purification and substrate specificity of two cysteine proteinases of Giardia lamblia.

The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.     

Leishmania mexicana: purine-metabolizing enzymes of amastigotes and promastigotes

Cultured promastigote and isolated amastigote forms of Leishmania mexicana mexicana have been surveyed for the presence of enzymes involved in purine metabolism. Quantitative but not qualitative differences between the enzymes of two forms were discovered. There were found to be significant differences between the enzyme content of L. m. mexicana and that reported for L. donovani. Extracts of both parasite forms of L. m. mexicana were found to have higher levels of adenine deaminase (EC 3.5.4.2) and guanine deaminase (EC 3.5.4.3) than adenosine deaminase (EC 3.5.4.4). There appeared to be two distinct nucleosidases (EC 3.2.2.1), one active on nucleosides, the other on deoxynucleosides. Phosphorylase (EC 2.4.2.1) could be detected only in the catabolic direction. Nucleotidases were present, but were more active on 3' (EC 3.1.3.6)- than 5' (EC 3.1.3.5)-nucleotides. Phosphoribosyltransferase (EC 2.4.2.7,.8 and .22) and nucleoside kinase (EC 2.7.1.20) activities were detected in both forms. Nucleotide-interconverting enzymes were found to be present, with IMP dehydrogenase (EC 1.2.1.14) being the most active. Cell fractionation experiments revealed that, in the promastigote, enzyme separation within the parasite may play an important part in regulating cellular purine metabolism.     

Leishmania tropica: cysteine proteases are essential for growth and pathogenicity

Leishmania parasites are responsible for a diverse collection of diseases of humans and other animals. Cysteine proteases are putative virulence factors of leishmania parasites. There are differences in the susceptibility of specific stages in different Leishmania species to cysteine protease inhibitors. Here, we establish a key role of cysteine proteases in growth, viability, and pathogenicity of Leishmania tropica by using a specific cysteine protease inhibitor (N-Pip-F-hF-VS Phenyl). Reduction or arrest of promastigote growth occurred at inhibitor concentration of 5 and 100 microM, respectively. This shows an essential role for cysteine proteases in viability and growth of L. tropica promastigotes. It confirms that the promastigote stage of L. tropica more closely resembles that of Leishmania major than that of Leishmania mexicana, which is refractory to this inhibitor. Pathogenicity of L. tropica amastigotes in mice, as assessed by footpad swelling, was also reduced by treatment with the cysteine protease inhibitor. This suggests that cysteine proteases are essential for pathogenicity of L. tropica amastigote in mammalian host, similar to both L. major and L. mexicana.     

A novel metalloproteinase associated with brain myelin membranes. Isolation and characterization

Brain myelin membrane preparations contain a metalloproteinase activity which degrades myelin basic protein (MBP). The activity was associated with lentil lectin-binding glycoproteins solubilized from myelin and could be detected in the presence of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS). The metalloproteinase represented about 5% of this glycoprotein fraction and was isolated from it by chromatography on DEAE-Sephacel, CM-Sepharose, and Superose 6. The proteinase had an apparent relative molecular weight (Mr) of approximately 58,000 both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr value was unaffected by the presence of reducing agents but was diminished to about 52,000 by treating the proteinase with endoglycosidase F. The purified proteinase cleaved many bonds in MBP but did not generate trichloroacetic acid-soluble products. Two major polypeptides, putatively MBP1-73 and MBP74-170, were prominent in digests of MBP by either the purified enzyme or myelin membranes. The proteinase was active between pH 7 and 9 and was inhibited by phenanthroline and dithiothreitol but not phosphoramidon or inhibitors of serine or cysteine proteinases. Histones, but not azocasein, also served as substrates for the proteinase. From its enzymic and molecular characteristics the myelin-derived metalloproteinase appears distinct from previously described enzymes.     

In vitro and in vivo antileishmanial activity of 2-amino-4,6-dimethylpyridine derivatives against Leishmania mexicana

Leishmania mexicana promastigote and intracellular amastigote growths were inhibited by the water-soluble furan-2-carboxamide issued from the pharmacophore 2-amino-4,6-dimethylpyridine with IC50 values of 69 +/- 2 and 89 +/- 9 microM, respectively. This compound was also tested against established L. mexicana infection in susceptible BALB/c mice; an intraperitoneal administration of 10 mg/Kg/day during five consecutive days induced a high reduction in the amastigote burden of the poplitea lymph node (81 +/- 6.4%), the spleen (80 +/- 1.6%) and the liver (73 +/- 9%). Approach of the mechanism of antileishmanial activity of this compound, assessed by the flow cytometry, showed a reduction in the protein and DNA synthesis. Finally, an actual increase of the in vitro antileishmanial activity was obtained by replacement of the amidic function by an imidazolidin-2-one moiety. In this new series, two of the N-substituted derivatives showed IC50 values of 13 +/- 0.5 and 7 +/- 3 microM in intracellular amastigotes constituting new promising compounds for further studies.