Expression of multiple CPB genes encoding cysteine proteases is required for Leishmania mexicana virulence in vivo

Leishmania mexicana mutants deficient in the multicopy CPB gene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specific CPB2.8 or metacyclic stage-specific CPB2 gene into a CPB-deficient mutant L. mexicana failed to restore either a Th2 response or sustained virulence. However, reexpression of multiple CPB genes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm that L. mexicana cysteine proteases are important virulence factors and provide an explanation for the presence in L. mexicana of a multicopy tandem array of CPB genes.     

Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis

The cysteine proteinase B of Leishmania parasites is an important virulence factor. In this study we have expressed, isolated and characterized for the first time a recombinant CPB from Leishmania braziliensis, the causative agent of mucocutaneous leishmaniosis. The mature region of the recombinant CPB shares a high percentage identity with its Leishmania mexicana CPB2.8 (rCPB2.8DeltaCTE) counterpart (76.36%) and has identical amino acid residues at the S(1), catalytic triad and [Formula: see text] subsites. Nevertheless, when the kinetics of substrate hydrolysis was measured using a combinatorial library of internally quenched fluorescent peptides based upon the lead sequence Abz-KLRSSKQ-EDDnp, significant differences were obtained. These results suggest that the differences in substrate utilization observed between the L. mexicana and L. braziliensis CPs must be related to amino acid modifications outside the core of the active site cleft. Moreover, a potent inhibitor with Pro at P1 and high affinity for L. braziliensis recombinant CPB showed less affinity to L. mexicana CPB 2.8, which preferred Phe, Leu, and Asn at the same position.     

Characterisation of three groups of cysteine proteinases in the amastigotes of Leishmania mexicana mexicana

The multiple cysteine proteinases characteristic of the amastigote forms of Leishmania mexicana mexicana have been shown to be of three types. The groups of enzymes are distinguished by their substrate specificities and physical properties and have been purified free from other proteinases and most other proteins. One group (A) comprises at least four enzymes that bind to Con A. The cysteine proteinases comprising the other two groups (B and C) were separated by ion exchange chromatography. These last two groups of enzymes show different specificities towards a range of peptidyl aminomethylcoumarins. Notably, the two group C proteinases are more active towards compounds with a basic amino acid in the P1 position, whereas the three enzymes of group B are as active towards substrates with tyrosine in this position. All the cysteine proteinases show preference for compounds with bulky amino acids at positions P2 and P3 and all are equally susceptible to a range of inhibitors characteristically active against cysteine proteinases. Other proteinases present in amastigotes of L. m. mexicana were shown to differ significantly from the cysteine proteinases with respect to their substrate preferences and susceptibility to inhibitors.     

Analysis of the S(2) subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, using fluorescent substrates containing non-natural basic amino acids

We have explored the specificity of the S(2) subsite of recombinant cysteine proteinases from Leishmania mexicana (CPB2.8 Delta CTE) and from Trypanosoma cruzi (cruzain) employing a series of fluorogenic substrates based on the peptide Bz-F-R-MCA, in which Bz is the benzoyl group and the Phe residue has been substituted for by Arg, His and non-natural basic amino acids that combine a basic group with an aromatic or hydrophobic group at the side chain: 4-aminomethyl-phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). Bz-F-R-MCA was hydrolyzed well by CPB2.8 Delta CTE and cruzain, but all the substitutions of Phe resulted in less susceptible substrates for the two enzymes. CPB2.8 Delta CTE has a restricted specificity to hydrophobic side chains as with cathepsin L. However, the peptides with the residues Amf and Ama presented higher affinity to CPB2.8 Delta CTE, and the latter was an inhibitor of the enzyme. Although, cruzain accepts basic as well as hydrophobic residues at the S(2) subsite, it is more restrictive than cathepsin B and no inhibitor was found amongst the examined peptides.     

Sterols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes

Leishmania mexicana mexicana promastigotes grown with cholesterol, supplied in natural products as the free sterol and as cholesteryl esters, were exposed to [2-14C]mevalonate and to the antimycotic drug ketoconazole. Growth was inhibited and cholesterol and 14 alpha-methyl sterols accumulated in free and esterified forms (cholesterol much greater than 4 alpha,14 alpha-dimethylcholesta-8,24-dien-3 beta-ol much greater than 14 alpha-methylcholesta-8,24-dien-3 beta-ol congruent to 14 alpha-methylergosta-8,24(28)-dien-3 beta-ol much greater than 4 alpha,14 alpha-dimethylergosta-8,24(28)-dien-3 beta-ol; identified by capillary gas chromatography/mass spectrometry, and by 1H and 13C nuclear magnetic resonance spectrometry). The 14 alpha-methyl sterols were preferentially labelled with 14C. The cholesterol was unlabelled and substituted for a substantial fraction of the major product of sterol biosynthesis, ergosta-5,7, 24(28)-trien-3 beta-ol (5-dehydroepisterol), but did not replace it and did not offer remarkable protection against either growth inhibition or alteration of sterol biosynthesis. Promastigotes grown with [6-2H]cholesterol or [4-14C]cholesterol did not contain labelled forms of Leishmania sterols, or other sterols. The chromatographic and spectrometric sterol analyses and the isotopic tracer findings suggested that ketoconazole impaired the cytochrome P-450 dependent 14 alpha-demethylation of lanosterol, that cholesterol was neither biosynthesized nor metabolized, and that the physiological functions of 5-dehydroepisterol had sterol structural requirements not entirely met by cholesterol. In all these studies, L. mexicana mexicana demonstrated a sterol biochemistry remarkably similar to that of fungi. This recommends an increase in interest in antimycotic drugs as chemotherapeutic agents for leishmanial infections.     

Heme binding to Leishmania mexicana amazonensis

Leishmania mexicana amazonensis is a pathogenic parasite whose growth, due to a biosynthetic deficiency, is dependent on a supply of exogenous heme. Utilizing [55Fe]hemin, we have demonstrated that heme binding to non-dividing cultured promastigotes of L. m. amazonensis at 4 degrees C reaches equilibrium within 6 h, is 95% dissociable by 28 h and is elevated approximately 5-fold by decreasing the pH of the binding buffer to 5.4. Metalloporphyrins substituted either at the central metal atom or in the porphyrin ring all displaced [55Fe]hemin binding to varying extents. Scatchard analysis revealed the affinity of the interaction to be 0.03 nM-1 and the number of binding sites to be 400 per promastigote. These findings are remarkably similar to those demonstrated in murine erythroleukemia cells and are characteristic of a receptor-ligand interaction. During logarithmic growth, promastigote heme binding was increased approximately 10-fold compared to stationary phase organisms. The increase was caused by a 4-fold greater number of binding sites per promastigote with no significant change in affinity. These findings demonstrate not only that L. m. amazonensis promastigotes bind heme specifically, but that the binding may be regulated by the growth phase of the parasite.     

Phosphomonoesterases of Leishmania mexicana mexicana and other flagellates

Amastigotes and log-phase promastigotes of Leishmania mexicana mexicana contained distinct acid phosphatase, 3'-nucleotidase and 5'-nucleotidase activities, distinguishable by their response to pH and inhibitors. Both tartrate-sensitive and tartrate-resistant acid phosphatase were present in the two forms, amastigotes possessed less tartrate-resistant acid phosphatase than promastigotes. A tartrate-sensitive acid phosphatase was secreted into the medium in large amounts during the growth in vitro of L. m. mexicana promastigotes. The 5'-nucleotidase activity of both parasite forms was inhibited by ammonium molybdate, sodium tartrate and, to less extent, by sodium fluoride whereas 3'-nucleotidase was inhibited by EDTA. All three activities were shown to be present on the external surface of both amastigotes and promastigotes. The three phosphomonoesterase activities were also detected in extracts of L. m. amazonensis, L. donovani, L. tarentolae, Crithidia fasciculata, Herpetomonas muscarum muscarum, H.m. ingenoplastis and Trichomitus batrachorum whereas 5'-nucleotidase was not detected in Trypanosoma brucei brucei extract and 3'-nucleotidase was absent from extracts of Trichomonas vaginalis and Tritrichomonas foetus.     

Vaccination of BALB/c mice with Leishmania major amastigote-specific cysteine proteinase

Cellular immune mechanisms resulting in interferon-gamma (IFN-gamma) production are essential for protection against cutaneous leishmaniasis. Antigens of the intracellular amastigote form of the parasite, found in mammalian hosts, are likely to be good candidates for the induction of T cell response and protection from development of leishmaniasis. We purified a stage-specific antigen from amastigote soluble antigen (A-SLA) of Leishmania major by immunoaffinity chromatography. The purified protein was characterized as a cysteine proteinase with enzymatic activity which is inhibited by E-64, and it was named the amastigote cysteine proteinase (ACP). BALB/c mice were immunized by two intraperitoneal injections, at a month interval, of 5 microg of ACP or A-SLA in Freund's complete adjuvant (FCA). Animals were challenged 4 weeks later with 106 L. major promastigotes and examined 4 months after the last injection. The immunized animals developed significantly smaller or no lesions compared with controls. Spleen cells from immunized mice showed a significant proliferative response and produced a high level of IFN-gamma in response to ACP, suggesting the induction of Th1 cells after immunization. These results make 24-kD ACP a possible component for an eventual cocktail vaccine against L. major infection.     

Recombinant cysteine proteinase from Leishmania (Leishmania) chagasi implicated in human and dog T-cell responses

High in vitro lymphoproliferative responses were induced in humans and dogs by a recombinant Leishmania (Leishmania) chagasi cysteine proteinase, with secretion of IFN-gamma in asymptomatic subjects or of IFN-gamma, interleukin 4 (IL-4), and IL-10 in oligosymptomatic subjects. In contrast, responses of symptomatic patients and dogs were lower, with production of IL-4 and IL-10.     

Proteomic analysis of Leishmania mexicana differentiation

We have resolved the proteome of axenically differentiated Leishmania mexicana parasites by two-dimensional gel electrophoresis (2DE), employing optimised, robust and reproducible procedures, and visualised (by silver staining) approximately 2000 protein species in each of three developmental stages: procyclic promastigotes, metacyclic promastigotes and amastigotes. This analysis has used homogeneous populations of these parasite stages, characterised according to their morphology, protease and nuclease activity profiles and expression of stage-specific antigens. Following comparison of the whole proteome profiles between stages, 47 spots were found to be stage-specific, while a further 100 spots changed in intensity during differentiation. The majority of "unique" spots were expressed during the infective stages of parasite differentiation, metacyclic promastigotes and amastigotes. CapLC-QTOF mass spectrometry has allowed the identification of 47 protein species to date, including a number which are only detected in the amastigote stage. Proteins identified are members of eight functionally related groupings, some of which are implicated in infectivity and host-parasite interactions.     

Insights into the tracking of the cysteine proteinase B COOH-terminal polypeptide of Leishmania (Leishmania) amazonensis by surface plasmon resonance

Parasitology Research - Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily...     

Homologues of LMPK, a mitogen-activated protein kinase from Leishmania mexicana, in different Leishmania species

LMPK, a mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana, is essential for the proliferation of the amastigote, the mammalian stage of the protozoan parasite. This has been demonstrated using deletion mutant promastigotes, the insect stage of the parasite: first, in vitro after differentiation to amastigotes, which subsequently lost their potential to proliferate; second, by infection of peritoneal macrophages, which were able to cope with the infection and cleared the parasites; third, by infection of BALB/c mice, which showed no lesion development. The lmpk deletion mutant promastigotes are a potential live vaccine because they infect macrophages, transform to amastigotes and deliver amastigote antigens to raise an immune response without causing the disease. In addition, inhibition of LMPK in a wild-type infection is likely to resolve the disease and as such, is an ideal target for drug development against leishmaniasis. Here we investigated the presence and copy number of lmpk homologues in Leishmania amazonensis, L. major, L. tropica, L. aethiopica, L. donovani, L. infantum, and L. braziliensis and discuss the results with regard to drug development and vaccination using kinase deletion mutants.     

Identification of 'Old World' Leishmania by DNA recombinant probes

Leishmania are usually identified by iso-enzyme analysis. This method works well, but there is a need for an additional, more simple, method of identification. Here we present data that show that in a Southern blot analysis, recombinant DNA probes in combination with certain restriction enzymes can differentiate between taxa of Leishmania. Probes based on clones selected from a L. infantum cDNA library gave characteristic patterns on Southern blots for reference strains of the different types of Leishmania found in Europe, Africa and Asia. Within the different taxa little or no variation was observed. Although the L. infantum derived probes showed a somewhat stronger hybridization for strains of the L. donovani complex, the signal obtained with most probes was satisfactory for L. major, L. aethiopica and L. tropica. Within the L. donovani complex none of the selected probes differentiated between isolates belonging to L. infantum, L. chagasi or L. donovani. Probes containing kinetoplast DNA showed considerable variation in hybridization within a taxon.     

Interaction of different Leishmania mexicana morphotypes with plasminogen

The interaction of plasminogen with Leishmania mexicana promastigotes was found, using immunoperoxidase assays, to occur with a specific morphotype. In in vitro cultured promastigotes, the morphotype that possessed the plasminogen binding capacity had round to ovoid cell bodies. In contrast, neither slender nor metacyclic promastigotes showed this property. In vivo plasminogen immunofluorescence assays showed deposits of plasminogen exclusively on the cell surface of promastigotes. This was observed as intense patches spread around the cell, with higher intensities towards the flagellar pocket. Plasminogen binding capacity detected by plasmin activity increased with the age of the promastigote culture, at pH 7.2 and pH 5.5, as well as after heat shock. The total amastigote population, freshly isolated from a hamster lesion, also bound plasminogen in a lysine-dependent manner as detected by immunoperoxidase studies.     

Perturbation of sterol biosynthesis by itraconazole and ketoconazole in Leishmania mexicana mexicana infected macrophages

The azole antifungals ketoconazole and itraconazole possess in vitro antileishmanial activity against Leishmania mexicana mexicana amastigotes in macrophages (cell line J774G8). As in yeast and fungi, the activity is likely to be due to inhibition of the cytochrome P-450-dependent 14 alpha-demethylation of lanosterol and/or 24,25-dihydrolanosterol. Indeed, 50% inhibition of ergosterol synthesis was observed at 0.21 microM ketoconazole and 0.15 microM itraconazole. At 5 microM ketoconazole, traces of ergosterol could be found, whereas no ergosterol could be detected in cells treated with 5 microM itraconazole. The inhibition of ergosterol biosynthesis was concomitant with an accumulation of the 14 alpha-methylsterols lanosterol and 24,25-dihydrolanosterol. Fifty percent inhibition of cholesterol synthesis in uninfected macrophages was achieved at 0.95 microM and 1.5 microM itraconazole and ketoconazole, respectively. In infected macrophages all [14C]acetate was incorporated in ergosterol, suggesting an inhibition in cholesterol synthesis in the host cells. An inhibition of ergosterol synthesis coincided with increasing cholesterol synthesis. The latter synthesis was inhibited at concentrations greater than 1 microM. However, even at 5 microM cholesterol synthesis was higher than under control conditions.     

Differences in Lsh gene control over systemic Leishmania major and Leishmania donovani or Leishmania mexicana mexicana infections are caused by differential targeting to infiltrating and resident liver macrophage populations.

Earlier studies had shown that the viscerotropic NIH 173 strain of cutaneous Leishmania major fails to come under Lsh gene control. Visceral Leishmania donovani LV9 and another viscerotropic cutaneous strain, Leishmania mexicana mexicana LV4, are controlled by Lsh. The results of double-infection experiments presented here show that expression of Lsh resistance against L. mexicana mexicana was enhanced in the presence of L. donovani, whereas L. major still failed to come under Lsh gene control, even in the presence of L. donovani. Prior irradiation (850 rads) of mice showed that in the absence of infiltrating monocytes, Lsh did exert some influence over L. major. The presence of a higher infiltrate of fresh monocytes after L. major infection was confirmed in liver macrophage populations isolated from mice after infection in vivo and in liver cryosections immunostained with monoclonal antibody M1/70 directed against the type 3 complement receptor CR3. The results support the hypothesis that Lsh is expressed maximally in the resident tissue macrophages and poorly in the immature macrophages preferentially infected by L. major amastigotes.     

Isolation and analysis of a new developmentally regulated gene from amastigotes of Leishmania mexicana mexicana

Leishmania differentiates from the promastigote to the amastigote stage during its digenetic life cycle. Characterization of the developmentally regulated genes during that process would help to elucidate the mechanisms of gene regulation. In this study, specific fragments of mRNAs from the amastigote stage of L. mexicana mexicana were discriminated from those of the promastigote and metacyclic stages by differential display. This technique combined with spliced-leader polymerase chain reaction allowed isolation of the complete gene VG7A5. The sequence of this gene did not align with any published L. mexicana sequence. More than one copy of this gene was identified in the genome by Southern-blot analysis and was transcribed exclusively in the amastigote stage. At 20 bp upstream from the splice AG site it has a trans-splicing polypyrimidine tract. The gene encodes the subcellular localization motifs 5′-GGACT and AAGCT-3′ in the 3′ untranslated region of the mRNA. The open reading frame of the gene VG7A5 predicts a polypeptide of 587 amino acid residues that has a KGRR amidation motif near its carboxyl terminus, suggesting that in the mammalian host this protein may be involved in the process of acute inflammation. Received: 24 June 1999 / Accepted: 10 September 1999     

Theoretical and in vitro studies of a C-terminal peptide from PGKC of Leishmania mexicana mexicana

Trypanosomatids cause deadly diseases in humans. Of the various biochemical pathways in trypanosomatids, glycolysis, has received special attention because of being sequestered in peroxisome like organelles critical for the survival of the parasites. This study focuses on phosphoglycerate kinase (PGK) from Leishmania spp. which, exists in two isoforms, the cytoplasmic PGKB and glycosomal PGKC differing in their biochemical properties. Computational analysis predicted the likelihood of a transmembrane helix only in the glycosomal isoform PGKC, of approximate length 20 residues in the 62-residue extension, ending at, arginine residues R471 and R472. From experimental studies using circular dichroism and NMR with deuterated sodium dodecyl sulfate, we find that the transmembrane helix spans residues 448±2 to 476 in Leishmania mexicana PGKC. The significance of this observation is discussed in the context of glycosomal transport and substrate tunneling.     

Identification of acid cysteine proteinase inhibitor (cystatin A) in the human thymus

Background: Acid cysteine proteinase inhibitor (ACPI, also called cystatin A) is a protein that is present in the epithelial cells of the skin and in the dendritic reticulum cells of lymphoid tissues. In this study the presence and cellular localization of ACPI in the thymus was investigated. Methods: The cellular and topographical location of ACPI was immunohistochemically demonstrated in the normal thymus of man. Results: ACPI was found in the cells of the-Hassall's corpuscles and in many medullary cells. Most of these cells were epithelial cells, as shown by the results of immunohistochemical cytokeratin and epithelial membrane antigen stainings. Also, some individual cytokeratin negative but S-100 positive medullary reticular dendritic cells were stained with ACPI. Conclusions: The finding that ACPI is constantly present in the thymus at restricted and specific cellular locations leads to the suggestion that protease inhibitors may play a role in specific thymic functions. © 1994 Wiley-Liss, Inc.