Identification of Synthetic and Natural Host Defense Peptides with Leishmanicidal Activity.

Leishmaniaparasites are a major public health problem worldwide. Effective treatment of leishmaniasis is hampered by the high incidence of adverse effects to traditional drug therapy and the emergence of resistance to current therapeutics. A vaccine is currently not available. Host defense peptides have been investigated as novel therapeutic agents against a wide range of pathogens. Here we demonstrate that the antimicrobial peptide LL-37 and the three synthetic peptides E6, L-1018, and RI-1018 exhibit leishmanicidal activity against promastigotes and intramacrophage amastigotes ofLeishmania donovaniandLeishmania major We also report that theLeishmaniaprotease/virulence factor GP63 confers protection toLeishmaniafrom the cytolytic properties of alll-form peptides (E6, L-1018, and LL-37) but not thed-form peptide RI-1018. The results suggest that RI-1018, E6, and LL-37 are promising peptides to develop further into components for antileishmanial therapy.

Molecular cloning of the major surface antigen of leishmania.

The gene encoding gp63, the major surface glycoprotein of Leishmania promastigotes, was isolated from Leishmania major using a synthetic oligonucleotide probe based on the NH2-terminal protein sequence of purified gp63. DNA sequence analysis and the translated amino acid sequence indicate that gp63 is synthesized as precursor molecule having both an NH2-terminal preregion (signal peptide) and an adjacent proregion. This structure is consistent with the protease activity of gp63 since many other proteases are synthesized as precursor forms requiring processing for enzymatic activity. Hybridization studies demonstrated that there are multiple copies of the gp63 gene in the genome of L. major and other Leishmania species. The conservation of the coding sequence of gp63 amongst diverse species of Leishmania provides further support for the importance of gp63 during the life cycle of Leishmania.

Identification of Leishmania genes encoding proteins containing tandemly repeating peptides.

A genomic Leishmania major DNA expression library was screened using antibodies raised against L. major membranes. Two different clones were identified that encoded proteins containing regions of tandemly repeated peptides. Clone 20 encodes a repetitive peptide of 14 amino acids, while clone 39 encodes a repetitive peptide of 10 amino acids. DNA from clone 20 hybridized with two RNA species of 9,500 and 5,200 nucleotides in length, while DNA from clone 39 hybridized to a single RNA species of 7,500 nucleotides. Antibodies against clone 20 fusion protein recognized a series of L. major proteins of apparent mol wt 250,000. Regions of repetitive peptides is a characteristic shared by many malarial protein antigens and this feature has been implicated in immune evasion. Intracellular parasites such as Leishmania and Plasmodia, therefore, may have evolved similar mechanisms consisting of the expression of proteins containing tandemly repeating peptides that are involved in immune evasion.

Leishmania surface protein gp63 binds directly to human natural killer cells and inhibits proliferation.

Natural killer (NK) cells contribute to immunity as the first line of defence in numerous infections by early cytokine secretion and cytotoxicity. In Leishmania infection, NK cells contribute with interferon-gamma and may assist in directing the immune response towards T helper type 1, which is essential for successful control of the parasites. Thus, NK cells may play an important role in both resistance and control of the infection. However, during Leishmania infection NK cells show signs of suppression. To explore the reason for this suppression, we exposed naive and interleukin (IL)-2 activated NK cells directly to promastigotes of Leishmania major in vitro. As a rapid consequence of contact between naive NK cells and promastigotes, expression of NK cell receptors show significant changes. We identify one of the major surface molecules of promastigotes, glycoprotein (gp) 63, as an important agent for these suppressive effects by using promastigotes of a gp63ko strain of L. major. Furthermore, proliferation of IL-2-activated purified NK cells is suppressed after exposure to the wild-type but not to gp63ko promastigotes. However, gp63ko L. major induced no NK cell proliferation when NK cells were co-cultured with peripheral blood mononuclear cells populations such as CD14(+) monocytes or T cells.

Modulation of in vitro monocyte cytokine responses to Leishmania donovani. Interferon-gamma prevents parasite-induced inhibition of interleukin 1 production and primes monocytes to respond to Leishmania by producing both tumor necrosis factor-alpha and interleukin 1.

Cytokines produced by mononuclear cells are important regulatory and effector molecules and evidence has been presented to support a role at least for tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in host defense against Leishmania. In the present study, we examined the production of TNF-alpha and interleukin 1 (IL-1) by resting and IFN-gamma-primed peripheral blood monocytes infected in vitro with Leishmania donovani. Monocytes produced neither IL-1 nor TNF-alpha during challenge with Leishmania. Cells preinfected with Leishmania synthesized normal amounts of TNF-alpha, but had diminished production of IL-1 in response to stimulation with either S. aureus or lipopolysaccharide (LPS). The induction by S. aureus or LPS of IL-1 beta mRNA accumulation in infected cells was normal despite diminished intracellular or supernatant IL-1 protein and bioactivity. Thus, inhibition of IL-1 production by Leishmania most probably reflected diminished translation of IL-1 beta mRNA. Pretreatment of cells with IFN-gamma abrogated infection-induced inhibition of IL-1 production and primed cells for the production of both IL-1 and TNF-alpha upon subsequent exposure to Leishmania. These results indicate that L. donovani has evolved the capacity to infect mononuclear phagocytes, without stimulating the production of two potentially host-protective monokines. The ability of IFN-gamma to prime monocytes to produce TNF-alpha and IL-1 in response to infection with Leishmania and to prevent inhibition of IL-1 production may have implications for immunotherapy with this lymphokine.

Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana.

We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8156 predicted coding regions revealed 288 genes (3.5% of all genes) whose steady-state mRNA levels meet criteria for differential regulation between promastigotes and lesion-derived amastigotes. Interestingly, sample comparison of promastigotes to axenic amastigotes resulted in only 17 genes (0.2%) that meet the same statistical criteria for differential regulation. The reduced number of regulated genes is a consequence of an increase in the magnitude of the transcript levels in cells under axenic conditions. The expression data for a subset of genes was validated by quantitative PCR. Our studies show that interspecies hybridization on microarrays can be used to analyze closely related protozoan parasites, that axenic culture conditions may alter amastigote transcript abundance, and that there is only a relatively modest change in abundance of a few mRNAs between morphologically distinct promastigote and amastigote cultured cells. Leishmania may represent an alternative paradigm for eukaryotic differentiation with minimal contributions from changes in mRNA abundance.

Analysis of the active site and activation mechanism of the Leishmania surface metalloproteinase GP63.

The major surface glycoprotein of Leishmania promastigotes, referred to as GP63, is a zinc metalloproteinase of 63,000 M(r) containing a glycosylphosphatidylinositol (GPI) membrane anchor. Recent studies demonstrated that recombinant GP63 (rGP63) expressed by the baculovirus insect cell system was secreted as a glycosylated latent proteinase that required activation for full proteinase activity (Button et al. (1993) Gene 134, 75-81). To extend these studies, the active site of L. major GP63 was characterized by site-directed mutagenesis and the activation mechanism of latent rGP63 was studied using both secreted and cell surface expression systems. To determine whether the proposed active site of L. major GP63 conforms to other well characterized zinc metalloproteinases, the proposed GP63 catalytic Glu-265, corresponding to catalytic Glu-147 of thermolysin, was changed to Asp-265. Using a transient expression system in COS-7 cells, expression of the Asp-265 mutant GP63 gene resulted in rGP63 with no detectable proteinase activity, whereas expression of the wild-type GP63 gene resulted in rGP63 with a level of proteinase activity similar to native GP63. Thus, the mechanism of GP63 proteinase activity is predicted to be homologous to that of other well characterized zinc metalloproteinases. NH2-Terminal sequence analysis revealed that activation with HgCl2 resulted in removal of the pro region, ultimately generating the mature NH2-terminus. This processing included the removal of a conserved Cys residue (Cys-48) and occurred by a cis mechanism, since the addition of previously activated rGP63 did not lead to an enhancement of latent rGP63 proteinase activation. The mechanism of activation of GP63 is consistent with the cysteine switch mechanism proposed for matrix metalloproteinases and thus has been conserved from protozoa to mammals.

Purification of an in vitro inhibitor of normal myelopoiesis using a monoclonal antibody directed to a common antigen of myelogenous leukemia (CAMAL).

A monoclonal antibody, CAMAL-1, has been previously shown to react specifically and at high frequencies with cells of patients with acute myelogenous leukemia (AML) and chronic granulocytic leukemia (CGL). High expression of this antigen in remission patients' bone marrow cells was shown to correlate with both relapse and lower survival times. Preliminary studies showed that material extracted from leukemic cells and eluted from CAMAL-1 immunoadsorbent columns profoundly inhibited the formation of normal colony-forming units (CFU) but had no effect on formation of such colonies from cells of patients with CGL. We have used CAMAL-1 affinity chromatography in combination with FPLC gel filtration to purify the inhibitory material from leukemic cell extracts. We have successfully isolated a 30-kd component (P30) that functions as an inhibitor of normal myelopoiesis in vitro; when P30 was added to normal progenitor cell assays it significantly inhibited the growth of normal CFU but had no inhibitory effect on the growth of CGL progenitor cells at equivalent concentrations. The inhibitory effect is preferentially directed to granulocytic progenitors. Antibodies raised to P30 reacted in Western blot analyses with affinity purified material from patients' cells as well as with a 30-kd component in the cell lysates and supernatants of the leukemic cell lines HL60 and K562; this finding suggested that the P30 inhibitory component might be produced by leukemic cells.

Recombinant Leishmania surface glycoprotein GP63 is secreted in the baculovirus expression system as a latent metalloproteinase.

A gene encoding the Leishmania surface metalloproteinase, GP63, was modified using the polymerase chain reaction to obtain effective secretion of recombinant GP63 (reGP63) in the baculovirus insect cell expression system. The coding region for the N-terminal signal peptide (SP) of GP63 was modified to resemble the SP for the GP67 envelope protein from the budded virus form of Autographa californica nuclear polyhedrosis virus. To prevent processing at the C-terminus with a glycosyl phosphatidylinositol anchor and the subsequent membrane anchoring of reGP63 in insect cells, the coding region for a putative SP at the C-terminus of GP63 was deleted. The reGP63 protein was glycosylated and secreted as a latent metalloproteinase in the baculovirus expression system. The reGP63 protein was purified from serum-free medium using concanavalin A lectin affinity chromatography, with a yield of 1 mg/l. The purified Leishmania reGP63 was secreted as a latent proteinase. Treatment of reGP63 with HgCl2 resulted in activation of full proteinase activity and a concomitant decrease in M(r). The mechanism of the activation of Leishmania reGP63 is consistent with that of other members of the family of matrix-degrading metalloproteinases.

The gene encoding streptothricin acetyltransferase (sat) as a selectable marker for Leishmania expression vectors.

The pLEX series of vectors was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. These pUC-based constructs contain one of three independent selectable markers and a multiple cloning site inserted between the upstream and downstream untranslated regions of the previously cloned Leishmania major HEXBP gene. Selection was based on resistance to the aminoglycosides, hygromycin B and neomycin, and to nourseothricin, a novel independent selectable marker for transfection of Leishmania. The vectors were introduced into Leishmania promastigotes by electroporation and were maintained as extrachromosomal circular concatemers containing between four and eight repeat units of the pLEX monomer. To demonstrate the efficient expression of cloned exogenous genes using the pLEX system, promastigotes were transfected with a pLEX construct that contained a second drug-resistant selectable marker gene cloned into the expression site, and clones were obtained that grew on media containing two antibiotics. These vectors, together with the novel selectable marker, will further facilitate the molecular analysis of gene expression in Leishmania.

Leishmania major HEXBP deletion mutants generated by double targeted gene replacement.

The Leishmania major single-stranded DNA binding protein HEXBP contains nine 'CCHC' zinc finger motifs and binds to oligodeoxynucleotides derived from the antisense strand of the GP63 gene 5' flanking region in gel mobility shift assays and UV-crosslinking assays. In the present study a HEXBP-deficient clone of L. major was generated by double targeted gene replacement. The two HEXBP alleles were sequentially replaced with genes encoding resistance to the aminoglycoside antibiotics hygromycin B and G418 and drug-resistant clones were selected by plating on semi-solid drug-containing media. Successful deletion of both copies of the HEXBP gene implies that HEXBP is a not essential for growth of Leishmania promastigotes. Characterization HEXBP-deficient promastigotes revealed that HEXBP deficiency had no effect on the abundance of GP63 mRNA and protein in in vitro cultivated promastigotes and that HEXBP-deficient promastigotes were capable of lesion formation in BALB/c mice.

Heterogeneity of the genes encoding the major surface glycoprotein of Leishmania donovani.

The major surface glycoprotein of Leishmania (GP63) is present on all known species of Leishmania and likely plays an integral role during the infection of macrophages in the mammalian host. To identify regions of GP63 which may be of functional significance, the nucleotide sequence of a gene encoding GP63 of Leishmania donovani was determined and compared to the sequences reported for GP63 genes of Leishmania major and Leishmania chagasi. The GP63 nucleotide and predicted protein sequence was highly conserved among the 3 species despite their diverse geographical distribution. L. donovani GP63 is encoded by a multigene family and the gene locus contains at least 7 tandemly repeated genes and at least 3 genes which are dispersed from the tandem array. In addition, polymerase chain reaction and Southern blot analyses demonstrated that there was size heterogeneity within the pro-peptide coding regions of the multiple GP63 genes of L. donovani and that such genes were expressed concurrently in the promastigote life stage.

Modification of GP63 genes from diverse species of Leishmania for expression of recombinant protein at high levels in Escherichia coli.

Toward the future development of a defined subunit vaccine against leishmaniasis is, high levels of recombinant GP63 for diverse species of Leishmania were produced in Escherichia coli. Several features of Leishmania GP63 genes were simultaneously modified with the polymerase chain reaction (PCR) using either cloned genes or total genomic DNA from Leishmania as template DNA for the PCR amplification reactions. The PCR products included only the coding region for the predicted mature form of GP63 that occurs on the surface of Leishmania, flanked by the appropriate translation signals and cloning sites for the production of recombinant GP63 as nonfusion protein in E. coli. When the codon usage in the GP63 gene was modified to reduce the guanine and cytosine content for the codons adjacent to the ATG initiation codon, rGP63 represented about 50% of total protein in E. coli. Mouse monoclonal antibodies raised against purified Leishmania major rGP63 had equivalent immunoblotting characteristics for native GP63 and recombinant GP63 with respect to linear determinants on GP63 expressed in diverse species of Leishmania. Human T cell lines and clones were derived from a patient infected with Leishmania braziliensis panamensis using rGP63 purified from an L. major GP63 expression clone as antigen.

The major surface glycoprotein (GP63) is present in both life stages of Leishmania.

Leishmania exist as extracellular promastigotes which multiply in the gut of the sandfly insect vector and as intracellular amastigotes which divide in the phagolysosome of mononuclear phagocytic cells of the mammalian host. Promastigotes express a major surface glycoprotein of 63 kDa, referred to as GP63. The expression of GP63 in both Leishmania life stages was studied using rabbit antibodies against native GP63 as well as rabbit antibodies against recombinant GP63 that was synthesized in an Escherichia coli expression system. Immunofluorescence staining detected GP63 in intracellular amastigotes contained within a macrophage cell line and within freshly isolated lesion amastigotes. Western blot analysis using anti-recombinant GP63 antibodies also demonstrated that amastigotes synthesize GP63 which may undergo differential post-translational processing as compared to promastigote GP63.

Differentially expressed Leishmania major gp63 genes encode cell surface leishmanolysin with distinct signals for glycosylphosphatidylinositol attachment.

The Leishmania cell surface metalloproteinase, leishmanolysin or GP63, is expressed in all stages of Leishmania major. Initial studies reported that in L. major the gp63 genes were arranged as five homologous, tandemly repeated genes (gp63 genes 1-5) and a sixth, less conserved gp63 gene located 8 kb downstream of gp63 gene 5. This study compared the sequences of L. major gp63 gene 1 and gp63 gene 6 and identified a seventh L. major gp63 gene located downstream from gp63 gene 6. The L. major gp63 genes exhibited stage-specific differences in their expression: gp63 genes 1-5 were expressed in promastigotes only, gp63 gene 6 was expressed in promastigotes and amastigotes, while gp63 gene 7 was expressed predominantly in stationary phase promastigotes and in amastigotes. Analysis of the predicted protein sequence of gp63 gene 6 (GP63-6) and gp63 gene 1 (GP63-1) showed that these two proteins were homologous in terms of overall predicted domain structure. L. major GP63-1 has been reported to contain a glycosylphosphatidylinositol (GPI) membrane anchor while sequence analysis predicted that GP63-6 contained a different hydrophobic C-terminus that may act as a transmembrane region. Transfection studies using L. major gp63 gene 1 and gp63 gene 6 expressed in L. donovani promastigotes showed that GP63-6 was expressed at the cell surface and that the distinct GP63-6 C-terminus was capable of mediating GPI anchor attachment.

Genes encoding the major surface glycoprotein in Leishmania are tandemly linked at a single chromosomal locus and are constitutively transcribed.

The major surface glycoprotein of Leishmania, gp63, is encoded by a small multi-gene family of tandemly linked genes which map to a single chromosome. For Leishmania major, there are five 3.1 kilobasepair (kb) direct repeat units which include a 1.8-kb open reading frame and a 1.3-kb intergenic or spacer region. In addition, there is a single gene copy linked as a direct repeat but separated from the tandem array of gp63 genes by about 8 kb. The restriction enzyme map of the repeat unit is highly conserved among the gene copies. The regions which flank the tandemly repeated genes diverge outside of the 3.1-kb repeat unit. Transcription of the gp63 gene locus is constitutive as the 3-kb transcript was present in promastigotes as well as in amastigotes. A minor 6-kb gp63 gene transcript was also detected in Northern blot analysis which could signify the transcription of the genes as a polycistronic or multigene precursor RNA.

Extensive polymorphism at the Gp63 locus in field isolates of Leishmania peruviana.

Genetic diversity within and between tandemly arrayed copies of the Gp63 gene occurs in laboratory isolates of Leishmania spp., but the extent to which this represents natural genetic diversity has not been assessed. Here, the Gp63 locus is examined in 58 fresh isolates of L. peruviana, and clones derived from them, collected throughout the Peruvian Andes. Extensive polymorphism is observed, both in size of Gp63 containing chromosomes, and for restriction-fragment-length polymorphisms (RFLPs) at the Gp63 locus. All clones within an isolate are identical, including those with two distinct Gp63-hybridising chromosomal-sized pulsed-field gel electrophoresis (PFGE) bands, consistent with diploidy but with size differences in homologous chromosomes. For RFLP analysis, three enzymes were selected to cut within the coding region (PstI), in the intergenic region (SalI) and outside (EcoRI) the Gp63 gene cluster. PstI gave identical banding patterns across all isolates/clones. For EcoRI and SalI, all clones within an isolate were identical, but isolates were polymorphic for fragments at 13 (2-30 kb) and 8 (2.6-8.8 kb) different molecular mass locations generating 19 and 16 distinct RFLP patterns or genotypes for each enzyme, respectively. EcoRI restriction patterns, analysed by PFGE, were consistent with the presence of two clusters of Gp63 genes on each homologous chromosome, one contained within EcoRI fragments large enough to carry from 3 to 10 copies of the Gp63 gene, the second on fragments which could carry 1 or 2 copies of the gene. SalI patterns indicated variable restriction sites within clusters, but not within every intergenic region. A hierarchical analysis of variance of allele frequencies, expressed in terms of Wright's F-statistic, indicated significant barriers to gene flow at all levels, valleys within regions (north/south), villages within valleys, and individuals within villages. This extreme polymorphism at the Gp63 locus of L. peruviana demonstrates the great potential for generation of genetic diversity in parasite populations.

Genetic heterogeneity in Peruvian Leishmania isolates.

Leishmania were isolated from Peruvian patients with uta or espundia; genomic DNA was examined for restriction fragment length differences by Southern blot analysis using DNA probes for beta-tubulin and for the major surface antigen gp63. Using 5 different restriction endonucleases, Peruvian isolates show homogeneity when examined at the beta-tubulin locus. In contrast, the organisms demonstrated heterogeneity both within and between disease groups when examined for restriction site differences within the gp63 locus. The differences observed did not correlate with the 2 disease groups. Comparison of these Peruvian isolates to New World reference strains of the Leishmania braziliensis complex reveals no consistent pattern of identity with either L. b. guyanensis, L. b. panamensis, or L. b. braziliensis.

Vaccination of vervet monkeys against cutaneous leishmaniosis using recombinant Leishmania 'major surface glycoprotein' (gp63).

Vervet monkeys (Cercopithicus aethiops) were shown to give a positive delayed-type hypersensitivity (DTH) reaction to gp63, a major surface glycoprotein of Leishmania parasites, and also produce antibodies to the molecule following a triple vaccination with a total dose of 150 micrograms of recombinant gp63 mixed with Bacille Calmette Guerin (BCG). However, peripheral blood leucocytes (PBL) from these animals neither proliferated nor produced any interferon-gamma (IFN-gamma) following in vitro stimulation with the antigen. Analysis of lymphocyte subsets following vaccination did not reveal any striking phenotypic alteration of cellular sub-populations in PBL. When vaccinated animals were rechallenged, via the needle, with virulent Leishmania major promastigotes containing salivary gland extracts from vector sandflies, only partial protection was achieved. We concluded from these studies that rgp63 produced in Escherichia coli is a safe vaccine molecule which gives only partial protection following vaccination in the vervet monkey host. The molecule requires further improvement for vaccine and/or immunodiagnosis application.