Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31 000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67 000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67 000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.     

Evidence for a vertical displacement of intramembranous particles on the plasma membrane of Tetrahymena cells by a local anesthetic

We examined the effect of a local anesthetic, dibucaine, on the plasma membrane of Tetrahymena pyriformis strain NT-1 using freeze-fracture electron microscopy. Intramembranous particles (IMPs) were distributed homogeneously on the plasma membrane of untreated cells. But, when Tetrahymena cells had been treated with 1.3 mM dibucaine for 5 min at growth temperature, freeze-fracture micrographs of the plasma membrane showed marked alterations. Although IMPs showed an almost homogeneous distribution, their density was elevated markedly on the protoplasmic fracture (PF) face but greatly reduce on the exoplasmic fracture (EF) face. Areas around deciliated portions had a reverse IMP density distribution for the PF and EF faces. These results suggest that dibucaine induced vertical displacement of the IMPs in the plasma membrane.     

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 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.     

Leishmania mexicana: distribution of intramembranous particles and filipin sterol complexes in amastigotes and promastigotes

The density and distribution of intramembranous particles was analyzed in freeze fracture replicas of the plasma membrane of amastigotes, and infective as well as noninfective promastigotes of Leishmania mexicana amazonensis. The density of intramembranous particles on both protoplasmic and extracellular faces was higher in infective than in noninfective promastigotes and it was lower in amastigotes than in promastigotes. Amastigotes purified immediately after tissue homogenization were surrounded by a membrane which corresponded to the membrane which lined the endocytic vacuoles where the parasites were located within the tissue macrophages. Aggregation of the particles was seen in the flagellar membrane at the point of emergence of the flagellum from the flagellar pocket. Differences in the organization of the particles were seen in the membrane which lined the flagellar pocket of amastigotes and promastigotes. The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the plasma membrane of L. m. amazonensis. The effect of filipin in the parasite's structure was analyzed by scanning electron microscopy and by transmission electron microscopy of thin sections and freeze fracture replicas. Filipin sterol complexes were distributed throughout the membrane which lined the cell body, the flagellar pocket, and the flagellum. No filipin sterol complexes were seen in the cell body-flagellar adhesion zone. The density of filipin sterol complexes was lower in the membrane lining the flagellum than in that lining the cell body of promastigotes.     

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.     

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.     

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.     

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 mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages

Macrophages of the cell line J774 were used in a comparative study of virulence involving amastigote stages of Leishmania mexicana pifanoi isolated from macrophages (AMA-M) of the aforementioned cell line, amastigote forms grown in the UM-54-cell-free medium (AMA-C), and promastigote stages. The macrophage cultures were inoculated with AMA-M and AMA-C at the culture cell to parasite ratios of 1:3, 1:5, and 1:10. The macrophages were exposed to either kind of amastigotes for 24, 48, and 72 h. At the end of each of these periods, and for each dilution, the percentages of macrophages harboring the parasites within their cytoplasm and the mean numbers of intracellular parasite/macrophage were estimated on the basis of examination of 200 phagocytes. When either AMA-M or AMA-C were employed, after 24 h, the percentages of infected macrophages were, respectively, 84.5%, 89.0%, and 94.5% for the three aforementioned dilutions, the majority of the phagocytes containing 1-5 parasites. After 48- and 72-h exposures, the macrophages harbored 6-11 and 11-20 amastigotes/cell, respectively. Evidently intracellular multiplication of the amastigotes has taken place. In contrast to the results obtained with amastigote forms, after inoculations of the macrophages cultures with promastigotes at the dilutions previously used for amastigotes, only 48-78 phagocytes were found to contain intracellular stages within their cytoplasm. Many macrophages were parasite-free, especially when exposed to fewer promastigotes. Experiments in which 5 X10(6) promastigotes, AMA-M, or AMA-C were inoculated into the footpads of hamsters yielded the following results with regard to terminal footpad volumes: 1.57, 3.31, and 3.32 cm3, respectively. Evidently both kinds of amastigotes had equal virulence for hamsters; however, the promastigote stages were much les virulent for these experimental hosts.     

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: Energy metabolism of amastigotes and promastigotes

The utilisation of substrates by Leishmania mexicana amastigotes and promastigotes differed significantly. The rates of uptake and catabolism of nonesterified fatty acids were up to 10-fold higher with amastigotes. Almost all the available exogenous fatty acids were consumed during amastigote transformation and by stationary phase of promastigote growth. The results suggest that fatty acids are important energy substrates for amastigotes, whereas promastigote utilisation may reflect the requirement for these substrates in anabolism. Glucose was utilised by amastigotes and promastigotes but the rate of catabolism was up to 10-fold higher in promastigotes. Uptake of glucose occurred throughout amastigote transformation and growth in vitro of promastigotes. High-subpassage promastigotes exhibited markedly lower glucose but higher amino acid utilisation than low-subpassage promastigotes. Asparagine, glutamine, glutamate, leucine, lysine, methionine, and threonine were consumed in large quantities by amastigotes and promastigotes, whereas alanine and glycine were excreted. Proline was catabolised to CO₂ by amastigotes and promastigotes but only at a low rate, and it was excreted in large amounts throughout promastigote growth. The major end products of energy metabolism were found to be CO₂ and succinate with both forms of the parasite and there was a secretion of up to 12 and 16% of the total protein synthesised by transforming amastigotes and growing promastigotes, respectively. Catabolism in amastigotes and promastigotes was found to be sensitive to cyanide and amytal, whereas 2-mercaptoacetate and 4-pentenoate primarily affected β-oxidation in the amastigote.     

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.     

Subcellular localisation of purine-metabolising enzymes in Leishmania mexicana mexicana

Leishmania mexicana mexicana cultured promastigotes were fractionated by isopycnic centrifugation on linear sucrose gradients. Guanine, hypoxanthine and xanthine phosphoribosyltransferase activities were found to be associated with glycosomes, whereas adenine phosphoribosyltransferase was cytosolic. 3'- and 5'-nucleotidases and IMP dehydrogenase were shown to be particulate, the former two possibly being associated with the plasma membrane, IMP dehydrogenase with the endoplasmic reticulum. Nucleosidases and deaminases were found to be cytosolic. The results demonstrate that intracellular separation of enzymes could play a part in the regulation of the parasite's purine metabolism.     

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.     

Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.     

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.     

Salt effects on membranes of the hypodermis and mesophyll cells of Avicennia germinans (Avicenniaceae): a freeze-fracture study

Freeze-fracture electron microscopy was used to investigate intramembranous particle (IMP) densities and particle distributions in the plasma membrane and tonoplast of the cells of secreting and nonsecreting leaves of Avicennia germinans (L.) Steam. Intramembranous particle densities of the protoplasmic (P) and exoplasmic (E) face of the plasma membrane and tonoplast were significantly higher in hypodermal cells of secreting leaves than of nonsecreting leaves. In contrast, no significant differences in the frequency of intramembranous particles were found in any membrane faces of secreting or nonsecreting mesophyll cells. However, particle densities were higher in the plasma membrane and tonoplast of the mesophyll cells, compared to the hypodermal cells, with the exception of the P-face of hypodermal plasma membranes of secreting tissue, which had the highest particle density measured. Particle distributions were dispersed and no discernible patterns such as paracrystalline arrays or other multi-IMP structures were observed. Results support the hypothesis that secretion is coupled to changes in membrane ultrastructure, and the possibility that salt secretion is an active process driven by integral membrane proteins such as the H⁺/ATPase. Additionally, the hypodermal cells of the leaf may function as storage reservoirs for salt as well as water, suggesting a regulatory role in salt secretion.