Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania

Species of the subgenus Leishmania (Leishmania) cause the debilitating disease leishmaniasis on four continents. Species grouped within the Leishmania donovani complex cause visceral leishmaniasis, a life-threatening disease, often associated with poverty, and affecting some 0.5 million people each year. The Leishmania glycoprotein GP63, or major surface protease, is a metalloprotease involved in parasite survival, infectivity and virulence. Here, we show that evolution of the gp63 multigene family is influenced by mosaic or fragmental gene conversion. This is a major evolutionary force for both homogenisation and for generating diversity, even in the absence of sexual reproduction. We propose here that the high GC content at the third codon position in the gp63 family of Old World Leishmania may be higher in multicopy regions, under the biased gene conversion model, because increased copy numbers may lead to increased rates of recombination. We confirm that one class of gp63 genes with an extended 3'end signal, gp63(EXT), reveals genetic groups within the complex and gives insights into evolution and host associations. Gp63(EXT) genes can also provide the basis for rapid and reliable genotyping of strains in the L. donovani complex. Our results confirmed that a more stringent definition of Leishmania infantum is required and that the species Leishmania archibaldi should be suppressed.     

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.     

The effect of tunicamycin on the protease activity of GP63 from Leishmania major

The protease activity of gp63 from L. major was studied in relation to tunicamycin induced N-deglycosylation. It was found that after tunicamycin treatment, a N-deglycosylated product of gp63 with protease activity is present at the cell surface of Leishmania promastigote.     

Extracellular release of the glycosylphosphatidylinositol (GPI)-linked Leishmania surface metalloprotease, gp63, is independent of GPI phospholipolysis: implications for parasite virulence.

The major zinc metalloprotease of Leishmania (gp63), an important determinant of parasite virulence, is attached to the parasite surface via a glycosylphosphatidylinositol anchor. Here we report the spontaneous release of proteolytically active gp63 from a number of Leishmania isolates, causing cutaneous and visceral disease. To investigate the mechanism(s) of gp63 release, we transfected a gp63-deficient variant of Leishmania amazonensis with constructs expressing gp63 and various mutants thereof. Surprisingly, approximately half of wild type gp63 was found in the culture supernatant 12 h post-synthesis. Biochemical analysis of the extracellular gp63 revealed two forms of the protein, one that is released from the cell surface, and another, that apparently is directly secreted. Release of cell surface gp63 was significantly reduced when the proteolytic activity of the protein was inactivated by site-specific mutagenesis or inhibited by zinc chelation, suggesting that release involves autoproteolysis. The extracellular gp63 does not contain a glycosylphosphatidylinositol moiety or ethanolamine, indicating that phospholipolysis is not involved in the release process. Release of gp63 is also independent of glycosylation. The finding of proteolytically active, extracellular gp63 produced by multiple Leishmania isolates suggests a potential role of the extracellular enzyme in substrate degradation relevant to their survival in both the mammalian host and the insect vector.     

Secondary structure of the promastigote surface protease of Leishmania

By Raman spectroscopic analysis we have determined the secondary structure of the promastigote surface protease, named PSP or gp63, of Leishmania major. It consist of nearly 50% antiparallel beta-strand, and less than 20% alpha-helix. These results are contrasted with the predominantly alpha-helical VSGs of the African trypanosomes and the alpha-helical metalloprotease thermolysin. The PSP of Leishmania thus represents a novel kind of membrane-anchored protease.     

Cloning and expression of truncated form of tissue plasminogen activator in Leishmania tarentolae

An expression cassette containing kringle 2 and serine protease domains (K2S), tissue plasminogen activator (tPA), together with a signal sequence derived from Leishmania tarentolae and two fragments of the small subunit ribosomal RNA locus, was introduced into L. tarentolae. The transfected cells produced recombinant K2S (rK2S) protein extracellularly with serine protease activity. Expression and enzyme activity of rK2S in the supernatant was 930 i.u./ml. The specific activity of purified rK2S was 7.4 U/mg of protein. Replacement of the human signal sequence tPA with the signal sequence derived from Leishmania increased the secretion of recombinant protein up to 30 times.     

Insights from the analysis of a predicted model of gp63 in Leishmania donovani

Leishmaniasis is a protozoal disease of human that occurs in most parts of the world. By considering the progress of bioinformatics in molecular modeling, major surface glycoprotein of Leishmania donovani (gp63) structure was modeled using homology modeling with high accuracy based on the X-ray crystal structure of the Leishmania major gp63 as a template, and then analyzed 3D structure of gp63 which can reveal exact facts about its structure and interaction. The objective of this study was to find folding and three dimensional structure of the gp63 as potent antigen for human. In this project, we applied the theory of evolution method, including comparative modeling and threading. This study presented a simple protocol for rapid and precise finding 3D structure of gp63 and investigation of its structural properties. The translated amino acid sequence showed that Leishmania donovani gp63 contains 590 amino acids precursor protein consisting of an NH₂-terminal signal peptide of 39 amino acids for membrane targeting, a pro region of 48 amino acids, the mature protein of 478 amino acids containing glycosylation and putative catalytic sites, and a COOH-terminal signal peptide of 25 amino acids for GPI attachment. Based on our model, the protein consists of three domains: the N-terminal, central and C-terminal domains. Additionally, these results could guide future structure-function analyses of gp63 protein.     

Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae

Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.     

Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening

Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.     

Cultivation of Leishmania donovani and Leishmania braziliensis in Defined Media: Nutritional Requirements

SYNOPSIS Nutritional requirements of promastigotes of Leishmania donovani and Leishmania braziliensis were studied in modifications of a simple defined culture medium. "Continuous growth," considered as propagation through 10 successive passages, was supported by inorganic salts, 14 l -amino acids (arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine), glucose, adenosine, and a mixture of 11 vitamins and related growth factors. Purified defatted bovine serum albumin proved beneficial. The nutritional needs of the above species of Leishmania differ from those of 2 other hemoflagellate species, Leishmania tarentolae and Crithidia fasciculata , for which glucose, proline and glutamine were found to be nonessential. It is suggested that lower hemoflagellates may be capable of synthesizing these substrates de novo. Leishmania donovani and L. braziliensis required higher levels of folic acid than L. tarentolae , probably due to the fact that folates are involved as cofactors in the biosyntheses of pyrimidines and serine. Although the mixtures reported here cannot be regarded as "minimal essential" media, they are considerably less complex than the ones employed so far for cultivating hemoflagellates, and are therefore well suited for studies related to nutrition and biosynthetic capabilities of Trypanosomatids.     

Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis

Two proteins from Leishmania donovani, dp72 and gp70-2, have been previously utilized to specifically serodiagnose patients with visceral leishmaniasis. The proteins were shown by ELISA and Western blotting with monoclonal and polyclonal antibodies to be present in both stages of the parasite. Antibodies to gp70-2 recognize in promastigotes multiple discrete bands of similar m.w. which are common to several isolates of L. donovani. The total amount of Ag and number of bands observed per isolate is not constant. Lectin blots with Con A show gp70-2 to be a glycoprotein. Dp72 shows pronounced microheterogeneity between isolates of L. donovani. The Brazilian isolates examined appear to possess a lower m.w. form (64,000 or 68,000) of this molecule. No reactions were observed with dp72 and lectins in Western blots; and neither tunicamycin, N-glycanase, endoglycosidase H nor F affected the migration of [35S]-methionine-labeled protein on SDS-PAGE. A mAb against dp72 also cross-reacted in Western blots with a 60-kDa protein in Leishmania major, Leishmania aethiopica, and Leishmania tropica. No reaction was observed between the purified promastigote surface protease (gp63) and either monoclonal or polyclonal antibodies produced to dp72 or gp70-2. The ability of the pure proteins to provide protection against a challenge by L. donovani amastigotes was examined. BALB/c mice were immunized with gp70-2 and/or dp72 by using Corynebacterium parvum as an adjuvant. Mice immunized with gp70-2 were not protected; however, mice receiving dp72 showed a 81.1% reduction in the liver parasitemia compared with the adjuvant controls.     

Three distinct RNAs for the surface protease gp63 are differentially expressed during development of Leishmania donovani chagasi promastigotes to an infectious form.

Leishmania sp. protozoa contain an abundant surface protease (gp63) that is important for the virulence of the parasite. We found that the average amount of gp63 expressed by Leishmania donovani chagasi promastigotes increases 6-11-fold as they develop from a less infectious form in logarithmic phase to a highly infectious form during stationary phase of cultivation in vitro. The predominant gp63 RNA switches from a 2.7 to a 3.0 kilobase (kb) RNA during the transition from log to stationary phase. Sequence analysis of gp63 cDNAs reveals that three different classes of gp63 RNAs, containing unique 3'-untranslated regions (3' UTRs), are expressed during growth to stationary phase. The predominant 2.7-(log) and 3.0-kb (stationary) class gp63 RNAs possess nearly identical coding regions, but they diverge in their 3' UTRs. A third class, consisting of 3.1- and 2.6-kb (constitutive) gp63 RNAs, is expressed at low levels throughout cultivation. This latter class encodes a gp63 with an additional 41 amino acids at its C terminus, replacing a potential signal for attachment of a glycolipid membrane anchor with a sequence that could be a transmembrane region. These findings are consistent with the regulated expression of different gp63 genes, resulting in different amounts of gp63 protein, during the promastigote's in vitro development to an infectious form.     

Leishmania major: production of recombinant gp63, its antigenicity and immunogenicity in mice

The Mr 63,000 membrane polypeptide (gp63) is one of the Leishmania receptors for host macrophages and has been shown to protect mice from infection. The gene encoding gp63, the major Mr 63,000 surface glycoprotein of L. major promastigotes, has been expressed as a fusion protein with the enzyme glutathione S- transferase encoded by the parasitic helminth Schistosoma japonicum. This fusion protein was recognized by polyclonal antibodies to the native Leishmania gp63 polypeptide. The insoluble gp63 fusion protein was purified by SDS-PAGE and electroelution and was used to raise antibodies in rabbits. These rabbit anti-gp63 antibodies recognized the fusion protein and the denatured parasite gp63 on immunoblots and by immunofluorescence on fixed promastigotes, but did not recognize the native molecule on live organisms. However, antibodies raised against native promastigote glycoproteins, affinity purified on solid-phase gp63 fusion protein, recognized both native and denatured gp63, suggesting the presence of native determinants in the recombinant protein. The gp63 fusion protein did not protect mice of either healer or nonhealer phenotype from challenge infection with live promatigotes. The implications of these results for the engineering of recombinant DNA-produced molecular vaccines are discussed.     

Leishmania major: expression and gene structure of the glycoprotein 63 molecule in virulent and avirulent clones and strains

Two Leishmania membrane glycoconjugates, gp63 and lipophosphoglycan, have been implicated in parasite attachment and uptake into the host macrophage. Moreover, recent data suggest that parasite virulence is associated with high expression of gp63. In this study we have surveyed gp63 gene copy number, in addition to the level of expression of gp63 mRNA and protein in several Leishmania major isolates, as well as virulent and avirulent strains and clones. The highest level of gp63 expression was found in the avirulent cloned line LRC-L119.3G7, which expresses about a 15-fold higher level of gp63 RNA and protein than the virulent cloned line LRC-L137/7/V121, suggesting that large amounts of gp63 are not sufficient for infectivity and do not correlate with virulence. L119.3G7 has eight copies of the gp63 gene compared to five copies in the virulent cloned line V121 and its parental virulent isolate LRC-L137. A series of avirulent clones derived from LRC-L137 also had five copies of the gene, suggesting that gp63 copy number is maintained among closely related parasites. Different virulent isolates of L. major from different geographic regions exhibited six copies of the gp63 gene. The variation in total gene copy number is due to different numbers of the tandemly repeated gp63 isogene in different strains. Our data show that there is wide variability between strains of L. major in the copy number of gp63 genes as well as in the amount of RNA and protein expressed.     

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.