Internal and surface subpopulations of the major surface protease (MSP) of Leishmania chagasi

Major surface protease (MSP) facilitates Leishmania promastigote evasion of complement-mediated lysis in the mammalian host and enhances host macrophage phagocytosis of the promastigotes. We previously showed that the steady-state abundance of MSP protein increases 14-fold during in vitro cultivation of L. chagasi promastigotes from logarithmic to stationary phase, despite the fact that the total amount of MSP mRNA does not increase. Furthermore, 10 major MSP isoforms are differentially expressed in different promastigote growth phases, and attenuation of parasites by long-term in vitro cultivation influences MSP isoform expression. Herein, we report that although about two-thirds of newly synthesized MSP becomes surface localized, the rest of the MSP does not reach the promastigote surface. This internal MSP is stable without detectable decrease in abundance up to 6 days after biosynthesis. Furthermore, surface-localized MSP is released at different rates from logarithmic and stationary phase virulent Leishmania promastigotes. These data are consistent with the hypothesis that the major mechanism regulating MSP abundance is the rate of loss of surface-localized MSP from the promastigote surface, and that internally localized MSP is very stable.     

Serine protease activities in Leishmania (Leishmania) chagasi promastigotes

The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-l-arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.     

Regulation of genes encoding the major surface protease of Leishmania chagasi via mRNA stability

The intercoding regions between many Leishmania sp. genes regulate their mRNA expression. The MSPL mRNA, encoding a subclass of the major surface protease (MSP) of Leishmania chagasi, increases in abundance, when protein synthesis is arrested, while alpha-tubulin (alpha-TUB) mRNA and most other mRNAs do not. We found that the intercoding region between MSPL-coding regions, when cloned downstream of the beta-galactosidase reporter gene (beta-GAL), caused beta-GAL mRNA to increase 8- to 10-fold after inhibiting protein synthesis with cycloheximide. Stable L. chagasi transfectants containing hybrid MSPL/alpha-TUB intercoding regions cloned downstream of beta-GAL were made. The alpha-TUB intercoding region induced high-level baseline beta-GAL mRNA that increased only 1.3-fold after incubation with cycloheximide. In contrast, the MSPL intercoding region, as well as constructs containing nucleotides 303-505 from the MSPL 3'UTR, caused steady-state beta-GAL mRNA levels in the absence of cycloheximide that were approximately 10% of alpha-TUB constructs. These levels increased between 4.4- and 13.2-fold after cycloheximide was added. Constructs containing half of this region (303-394 or 395-505) produced intermediate levels of beta-GAL mRNA and intermediate levels of cycloheximide induction. The kinetics of cycloheximide induction of beta-GAL mRNA was similar with region 303-505 constructs as with constructs bearing the entire endogenous MSPL intercoding region. Furthermore, region 303-505 increased reporter mRNA abundance after cycloheximide by increasing mRNA half-life. Hence, we have identified a 202-nucleotide region within the MSPL 3'UTR that is in part responsible for cycloheximide induction. We hypothesize that this region may interact with labile regulatory protein factor(s).     

Targeted gene deletion in Leishmania major identifies leishmanolysin (GP63) as a virulence factor

Leishmanolysin, the Leishmania surface metalloproteinase of 63 kDa (GP63) has been described as a parasite virulence factor and is involved in the direct interaction of promastigotes and host macrophage receptors and interaction with the complement cascade. To study the role of leishmanolysin in the pathogenesis and virulence of Leishmania major, targeted gene replacement was used to delete the entire 20 kb region containing all seven leishmanolysin genes (gp63 genes 1-7). The resulting L. major leishmanolysin deficient mutants showed normal development inside the sand fly vector, however, promastigotes recovered from sand flies or from culture showed an increase in sensitivity to complement-mediated lysis and a delay in lesion formation in BALB/c animals. The phenotypic differences could be significantly improved by expression of a cloned leishmanolysin gene. These results demonstrate that leishmanolysin is a vital virulence factor in Leishmania pathogenesis.     

Acid protease activity of a major surface membrane glycoprotein (gp63) from Leishmania mexicana promastigotes

A unique protease with activity optimal at pH 4.0 and trailing toward the alkaline pH spectrum was detected with intact glutaraldehyde-fixed promastigotes of Leishmania mexicana amazonensis, indicating surface localisation of the enzyme. That this surface protease may be a virulence factor is suggested by its apparent roles in multiple steps during leishmanial infections of macrophages. Indeed, its specific activity was 2–2.5 fold higher on virulent cells than on avirulent cells. Several lines of evidence indicate that this acid protease activity is expressed by the major surface glycoprotein (gp63) of L. m. amazonensis. Monoclonal antibody affinity purified gp63 degraded serum albumin, hemoglobin, complement C3, immunoglobulin G and purified rat liver lysosomal proteins in their native forms. The specific activity is about 20-fold higher at pH 4.0 than at pH 7.5 and is about four-fold higher at the body temperature of the mammalian host (37°C) than at that of the insect host (27°C). The protease activity is sodium dodecyl sulphate-sensitive. Among various protease inhibitors tested, only heavy metal ions (1 mM), 1,10-orthophenanthroline (1 mM) and bestatin (100 ng ml⁻¹) significantly inhibited gp63 acid protease activity by up to 80%. N-linked oligosaccharides of gp63 appear to be important for the stability of this molecule, possibly by preventing its autodegradation. Purified gp63 effected limited proteolysis of human complement C3 molecules at the physiological serum pH of 7.5 in a manner, which supports the idea of its participation in complement-receptor mediated endocytosis of promastigotes by macrophages.     

Immunological analysis of the zinc-binding peptides of surface metalloproteinase (gp63) of Leishmania major.

The surface metalloproteinase, gp63, is highly conserved and immunogenic. A peptide spanning the zinc-binding region of the molecule is immunogenic and can induce protective immunity in mice against Leishmania major infection. We report here that the minimum length of the immunogenic peptide in this region is a heptapeptide, VVTHEMA, corresponding to residues 161-167. Optimal immunogenicity is conferred by a decapeptide, LVTVVTHEMA, corresponding to residues 158 to 167, where H and E are consensus zinc-binding residues. These two residues determine the specificity of the peptide. The next two residues, M and A are necessary for the immunogenicity of the peptide. These results suggest that the zinc-binding residues are recognized by the T-cell receptor complex, while the two adjacent residues are involved in the peptide presentation by the major histocompatibility complex (MHC) molecule.     

Identification and characterization of host-protective T-cell epitopes of a major surface glycoprotein (gp63) from Leishmania major

By using a series of overlapping synthetic peptides that cover more than 75% of the amino acid sequence of the major surface glycoprotein (gp63) from Leishmania major, 11 T-cell epitopes in CBA and BALB/c mice have been identified. Six of the peptides were recognized by T cells of CBA mice recovered from L. major infection, while one was recognized by the T cells from BALB/c mice recovered from the infection following sublethal doses of gamma-irradiation. Lymph node cells from mice immunized with the peptides also responded to a number of the same peptides (seven in CBA and one in BALB/c). Peptide p10-28 induced proliferative T-cell responses in both CBA and BALB/c mice. Five of the peptides (p10-28, p22-40, p289-309, p459-471 and p467-482) induced vigorous T-cell response in CBA mice but were not recognized by T cells from recovered mice. Four other peptides (p321-336, p364-476, p372-385 and p378-396) were recognized by T cells from recovered CBA mice but could not induce a T-cell response in normal CBA mice. Three peptides (p146-171, p289-309 and p395-414) were both able to induce a T-cell response and were recognized by T cells from recovered mice. However, only two peptides (p146-171 and p467-482) were able to activate T cells, which also recognized epitopes expressed by antigen-presenting cells infected with promastigotes. T cells induced by p146-171 and p467-171 or a mixture of these two peptides were mainly CD4+ and produced interleukin (IL-2) and interferon-gamma (IFN-gamma) but not IL-4 upon antigen stimulation in vitro. These two peptides also induced a classical delayed type hypersensitivity (DTH) response in CBA mice. Furthermore, CBA mice immunized with a mixture of the two peptides in Coryne parvum or entrapped in liposomes induced significant resistance against L. major infection. The implications of these results in terms of a synthetic vaccine against leishmaniasis and the mechanism of the induction of Th1 and Th2 cells are discussed.     

The LPG1 gene family of Leishmania major

In Leishmania major, the core of the abundant surface lipophosphoglycan (LPG) is structurally related to that of the smaller glycosylinositolphospholipids (GIPLs) in containing galactosylfuranose (Gal(f)) residues in a Gal(f) (beta1, 3)Man motif. However, deletion of the putative Gal(f)-transferase (Gal(f)T) LPG1 affected Gal(f) incorporation in LPG but not GIPLs. We hypothesized that the presumptive GIPL Gal(f)-transferases could be homologous to LPG1, and identified three related genes in the L. major genome. These were termed LPG1L, LPG1R, and LPG1G, the latter of which was found in three identical copies located at the telomeres of chromosomes 5, 19, and 32 based on Leishmania genome project data. Neither LPG1 nor its homologues LPG1L and LPG1R were involved in the biosynthesis of GIPLs, as an lpg1(-)/lpg1l(-)/lpg1r(-) triple knockout (the first such in Leishmania) grew normally and made wild-type levels of Gal(f) -containing GIPLs. In contrast, overexpression of these three led to elevated galactose incorporation in glycoproteins. Gal(f)-containing glycoproteins had not been described in Leishmania but occur at high levels in other closely related trypanosomatids including Trypanosoma cruzi, Crithidia, Leptomonas, and Endotrypanum, and LPG1L and LPG1R homologs were detected in these species. These data suggest that the glyco-synthetic capabilities of Leishmania and perhaps other trypanosomatids may be larger than previously thought, with some activities being 'cryptic' in different lineages and potentially serving as reservoirs for glycoconjugate variation during evolution. Future tests will address whether the LPG1G family encodes the hypothesized GIPL-specific Gal(f)T.     

The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage

The expression, processing and localization of the promastigote surface glycoprotein, gp63, in the amastigote form of Leishmania mexicana was examined. Metabolically labeled protein was immunoprecipitated from promastigotes and amastigotes. The isolated proteins were subjected to deglycosylation and partial peptide mapping. The cleavage products generated migrated similarly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the proteins were closely related. The majority of gp63 in amastigotes was inaccessible to surface-labeling procedures, and lacked the phosphatidylinositol membrane anchor. Immunolocalization of this subpopulation of gp63 revealed it to be present within the parasite's flagellar pocket. Despite the relative paucity of 'membrane-form' gp63, isolation and analysis of surface proteins from lesion amastigotes indicated that gp63 was the most abundant protein on the amastigote surface.     

Involvement of the GP63 protease in infection of Trichomonas vaginalis

There are 48 members of the GP63 protease family in Trichomonas vaginalis according to our annotations; 37 of them are predicted to be transmembrane proteins. Because the GP63 protease family is the largest surface protease family and the second largest surface protein family, they are most likely to be involved in the interactions between T. vaginalis and the host cell surfaces, or otherwise participate in infection. We performed a preliminary study on the functions of GP63 in T. vaginalis (TvGP63). We demonstrated the cell surface localization of one highly expressed member of TvGP63 using indirect immunofluorescence assays in both isolate T016 and isolate 30236. The specific inhibitor of TvGP63 protease, 1,10-phenanthroline, was found to significantly inhibit the destruction of HeLa cells, whereas another chelator, EDTA, could not. Further tests showed that 1,10-phenanthroline did not inhibit the adherence of T. vaginalis cells to HeLa cells. The results presented in here suggest that GP63 protease plays a vital role in T. vaginalis infection process, but may not be related to the adherence of parasitic cells to their hosts.     

In vitro cytocidal effects on Trypanosoma brucei and inhibition of Leishmania major GP63 by peptidomimetic metalloprotease inhibitors

Peptidomimetic inhibitors of mammalian zinc metalloproteases have been tested as potential agents for intervention in disease caused by kinetoplastid protozoa. Certain metalloprotease inhibitors were able to inhibit the release of variant surface glycoprotein from cultured transgenic procyclic Trypanosoma brucei, confirming our previous identification of a cell surface zinc metalloprotease activity in this stage of the trypanosome lifecycle [Bangs, JD et al. Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion, EMBO J. 1997;16:4285]. Selected peptidomimetics were also found to be toxic for cultured bloodstream trypanosomes with IC50 values in the low micromolar range. The paradigm for zinc metalloproteases in kinetoplastids are the GP63 surface enzymes of Leishmania. Peptidomimetics at low micromolar concentrations were able to inhibit in vitro cleavage of a synthetic peptide substrate by purified GP63 from L. major. Our results suggest that zinc metalloproteases perform essential functions in different stages of the trypanosome lifecycle and we hypothesize that these activities may be affected by the recently discovered trypanosomal homologues of GP63 [El-Sayed, NMA and Donelson, JE. African trypanosomes have differentially expressed genes encoding homologues of Leishmania GP63 surface protease, J. Biol. Chem. 1997;272:26742]. Development of higher affinity metalloprotease inhibitors may provide a novel avenue for treatment of parasitic diseases.     

Comparison of the effects of Leishmania major or Leishmania donovani infection on macrophage gene expression

The intracellular parasite Leishmania causes a wide spectrum of human disease, ranging from self-resolving cutaneous lesions to fatal visceral disease, depending on the species of Leishmania involved. The mechanisms by which different Leishmania species cause different pathologies are largely unknown. We have addressed this question by comparing the gene expression profiles of bone marrow-derived macrophages infected with either Leishmania donovani or L. major promastigotes. We found that the two species had very similar effects on macrophage gene expression. Both species caused a small (<2.5-fold) but statistically significant repression of several hundred genes. In addition, both species strongly induced and repressed about 60 genes. Comparing the effects of the two species showed that only 26 genes were regulated differently by L. major as opposed to L. donovani, including those for metallothioneins 1 and 2, HSP70 and -72, CCL4, Gadd45β, Dsp1, matrix metalloprotease 13, T-cell death-associated gene 51 (Tdag51), RhoB, spermine/spermidine N1-acyl transferase 1 (SSAT), and Cox2. L. donovani-infected macrophages were also found to express higher levels of Cox2 protein and prostaglandin E synthase mRNA than L. major-infected macrophages. While both species have previously been shown to trigger prostaglandin E synthesis by bystander cells, this study suggests that infected macrophages themselves express prostaglandin E-synthesizing genes only in response to L. donovani.     

Relationship between canine visceral leishmaniosis and the Leishmania (Leishmania) chagasi burden in dermal inflammatory foci

The skin is the first point of contact with organisms of the genus Leishmania from sand fly vectors, and apparently normal skin of sick dogs harbours amastigote forms of Leishmania chagasi. In relation to canine visceral leishmaniosis (CVL), the ear skin was examined in 10 uninfected dogs (UDs) and in 31 dogs dogs naturally infected with L. chagasi. The infected animals consisted of 10 symptomless dogs (SLDs), 12 mildly affected dogs (MADs) and nine affected dogs (ADs). A higher parasite burden was demonstrated in ADs than in SLDs by anti-Leishmania immunohistochemistry (P<0.01), and by Leishman Donivan Unit (LDU) indices (P=0.0024) obtained from Giemsa-stained impression smears. Sections stained with haematoxylin and eosin demonstrated a higher intensity of inflammatory changes in ADs than in SLDs (P<0.05), and in the latter group flow cytometry demonstrated a correlation (P=0.05/r=0.7454) between the percentage of CD14(+) monocytes in peripheral blood and chronic dermal inflammation. Extracellular matrix assessment for reticular fibres by staining of sections with Masson trichrome and Gomori ammoniacal silver demonstrated a decrease in collagen type I and an increase in collagen type III as the clinical signs increased. The data on correlation between cellular phenotypes and histological changes seemed to reflect cellular activation and migration from peripheral blood to the skin, mediated by antigenic stimulation. The results suggested that chronic dermal inflammation and cutaneous parasitism were directly related to the severity of clinical disease.     

The role of LPD-nanoparticles containing recombinant major surface glycoprotein of Leishmania (rgp63) in protection against leishmaniasis in murine model

Context: Leishmaniasis is a major public health problem. Despite numerous attempts, yet there is no effective vaccine against human leishmaniasis, mainly due to a lack of an effective vaccine delivery system as well as adjuvant.

Objective(s): The aim of this study was to evaluate the ability of recombinant glycoprotein 63 (rgp63) as a model of Leishmania antigen, entrapped in liposome–polycation–DNA (LPD) complexes nanoparticles in inducing cell mediated immune (CMI) response and protecting against L. major in BALB/c mice.

Materials and methods: To this end, the abundant leishmania promastigote cell surface glycoprotein, gp63, was entrapped in nano-sized LPD (CpG) particles, (LPD (CpG)-rgp63), and BALB/c mice were immunized three times with either (LPD (CpG)-rgp63) or rgp63-CpG DNA or LPD (CpG) or free rgp63 and dextrose 5%. Various parameters including footpad thickness, splenic load of L. major parasites, rgp63-binding IgGs and also cytokine levels of rgp63-reactive T lymphocytes were then compared among different vaccinated animals.

Results: The lowest number of parasites in spleen, the higher levels of IgG2a after challenge infection, the minimal footpad swelling and high level of IFN-γ secretion, all indicated that adjuvants and antigen-delivery systems are essential in modifying immune responses; as mice received LPD (CpG)-rgp63 induced immune response stronger than the other groups.

Conclusions: This study demonstrates that LPD nanoparticle is a promising and adaptable delivery system which could be modified towards specific vaccine targets to induce a more potent immune response in combination with rgp63.