Structure-activity relationships of acetylcholine derivatives with Lucilia cuprina nicotinic acetylcholine receptor α1 and α2 subunits in chicken ß2 subunit hybrid receptors in comparison with chicken nicotinic acetylcholine receptor α4/ß2.

Insect nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several insecticide classes. In the present study, we report the gene identification and cloning of nAChR α1 and α2 subunits (Lcα1 and Lcα2) from the sheep blowfly Lucilia cuprina. Xenopus oocytes voltage clamp experiments as hybrids with the chicken ß2 nAChR (Ggß2) subunit resulted in ACh-gated ion channels with distinct dose-response curves for Lcα1/Ggß2 (effective concentration 50% [EC50 ] = 80 nM; nH = 1.05), and Lcα2/Ggß2 (EC50 = 5.37 µM, nH = 1.46). The neonicotinoid imidacloprid was a potent agonist for the α-bungarotoxin-sensitive Lcα1/Ggß2 (EC50 ∼ 20 nM), while the α-bungarotoxin-resistant Lcα2/Ggß2 showed a 30-fold lower sensitivity to this insecticide (EC50 = 0.62 µM). Thirteen close derivatives of ACh were analysed in EC50 , Hill coefficient and maximum current (relative to ACh) determinations for Lcα1/Ggß2 and Lcα2/Ggß2 and the chicken Ggα4/Ggß2 nAChRs, and comparisons relative to ACh allowed the definition of novel structure-activity and structure-selectivity relationships. In the case of N-ethyl-acetylcholine, the EC50 of the chicken Ggα4/Ggß2 rose by a factor of 1000, while for both Lcα1/Ggß2 and Lcα2/Ggß2, potency remained unchanged. Further derivatives with insect nAChR selectivity potential were acetyl-α-methylcholine and trimethyl-(3-methoxy-3-oxopropyl)ammonium, followed by acetylhomocholine and trimethyl-(4-oxopentyl) ammonium. Our results may provide guidance for the identification or design of insect-specific nAChR agonists using structure-based or in silico methods.

Trehalose-6-phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster: gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening.

Trehalose phosphate synthase (EC 2.4.1.15; TPS) is the crucial enzyme for the biosynthesis of trehalose, the main haemolymph sugar of insects, and therefore a potential insecticidal molecular target. In this study, we report the functional heterologous expression of Drosophila melanogaster TPS, the gene identification, full length cDNA cloning and functional expression of cat flea (Ctenocephalides felis) TPS, and the Michaelis-Menten constants for their specific substrates glucose-6-phosphate and uridinediphosphate-glucose. A novel high throughput screening-compatible TPS assay and its use for the identification of the first potent insect TPS inhibitors from a large synthetic compound collection (>115 000 compounds) is described. One compound class that emerged in this screening, the 4-substituted 2,6-diamino-3,5-dicyano-4H-thiopyrans, was further investigated by analysing preliminary structure-activity relationships. Here, compounds were identified that show low µM to high nM half maximal inhibitory concentrations on insect TPS and that may serve as lead compounds for the development of insecticides with a novel mode of action.

Molecular cloning, partial genomic structure and functional characterization of succinic semialdehyde dehydrogenase genes from the parasitic insects Lucilia cuprina and Ctenocephalides felis.

The enzyme succinic semialdehyde dehydrogenase (SSADH; EC1.2.1.24) is a component of the gamma-aminobutyric acid degradation pathway in mammals and is essential for development and function of the nervous system. Here we report the identification, cDNA cloning and functional expression of SSADH from the parasitic insects Lucilia cuprina and Ctenocephalides felis. The recombinant proteins possess potent NAD+-dependent SSADH activity, while their catalytic efficiency for other aldehyde substrates is lower. A genomic copy of the L. cuprina SSADH gene contains two introns, while a genomic gene version of C. felis is devoid of introns. In contrast to the single copy SSADH genes in Drosophila melanogaster and mammals, in L. cuprina and C. felis, multiple SSADH gene copies are present in the genome.

Highly water-soluble prodrugs of anthelmintic benzimidazole carbamates: synthesis, pharmacodynamics, and pharmacokinetics.

Highly water-soluble prodrugs 1a- g of anthelmintic benzimidazole carbamates 2a- g were synthesized. These prodrugs combine high aqueous solubility and stability with high lability in the presence of alkaline phosphatases. The veterinary utility of 1a was shown by a pharmacodynamic and pharmacokinetic study performed in swine. Comparable anthelmintic efficacy was observed with prodrug 1a or the parent fenbendazole 2a. The pharmacokinetic results showed that 2a is better absorbed when derived from 1a than when applied as such.

Live Leishmania promastigotes can directly activate primary human natural killer cells to produce interferon-gamma.

Natural killer (NK) cells have been implicated in the natural protection and healing of leishmaniasis by their ability to secrete the macrophage activating cytokine interferon (IFN)gamma. Previous studies have demonstrated that early production of interleukin (IL)-12 triggers IFN gamma secretion by NK cells. Here we report that live Leishmania promastigotes (the form that is injected by the vector) can directly induce human peripheral blood NK cells from healthy donors to IFN gamma secretion in the absence of IL-12 and professional antigen presenting cells. Killing of promastigotes abolishes this property. This novel mechanism of activation of the innate immune response may be relevant for establishment of infection and thus also the design of vaccines against leishmaniasis.

Glycosylation defects and virulence phenotypes of Leishmania mexicana phosphomannomutase and dolicholphosphate-mannose synthase gene deletion mutants.

Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (DeltaPMM and DeltaDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicana DeltaDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana DeltaPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.

Disruption of mannose activation in Leishmania mexicana: GDP-mannose pyrophosphorylase is required for virulence, but not for viability.

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDPMP) is essential for the formation of GDP-mannose, the central activated mannose donor in glycosylation reactions. Deletion of its gene is lethal in fungi, most likely as a consequence of disrupted glycoconjugate biosynthesis. Furthermore, absence of GDPMP enzyme activity and the expected loss of all mannose-containing glycoconjugates have so far not been observed in any eukaryotic organism. In this study we have cloned and characterized the gene encoding GDPMP from the eukaryotic protozoan parasite Leishmania mexicana. We report the generation of GDPMP gene deletion mutants of this human pathogen that are devoid of detectable GDPMP activity and completely lack mannose-containing glycoproteins and glycolipids, such as lipophosphoglycan, proteophosphoglycans, glycosylphosphatidylinositol protein membrane anchors, glycoinositolphospholipids and N-glycans. The loss of GDPMP renders the parasites unable to infect macrophages or mice, while gene addback restores virulence. Our study demonstrates that GDP-mannose biosynthesis is not essential for Leishmania viability in culture, but constitutes a virulence pathway in these human pathogens.

Phosphoglycan repeat-deficient Leishmania mexicana parasites remain infectious to macrophages and mice.

The human pathogen Leishmania synthesizes phosphoglycans (PGs) formed by variably modified phosphodisaccharide [6-Galbeta1-4Manalpha1-PO(4)] repeats and mannooligosaccharide phosphate [(Manalpha1-2)(0-5)Manalpha1-PO(4)] caps that occur lipid-bound on lipophosphoglycan, protein-bound on proteophosphoglycans, and as an unlinked form. PG repeat synthesis has been described as essential for survival and development of Leishmania throughout their life cycle, including for virulence to the mammalian host. In this study, this proposal was investigated in Leishmania mexicana using a spontaneous mutant that was fortuitously isolated from an infected mouse, and by generating a lmexlpg2 gene deletion mutant (Deltalmexlpg2), that lacks a Golgi GDP-Man transporter. The spontaneous mutant lacks PG repeats but synthesizes normal levels of mannooligosaccharide phosphate caps, whereas the Deltalmexlpg2 mutant is deficient in PG repeat synthesis and down-regulates cap expression. In contrast to expectations, both L. mexicana mutants not only retain their ability to bind to macrophages, but are also indistinguishable from wild type parasites with respect to colonization of and multiplication within host cells. Moreover, in mouse infection studies, the spontaneous L. mexicana repeat-deficient mutant and the Deltalmexlpg2 mutant showed no significant difference to a wild type strain with respect to the severity of disease caused by these parasites. Therefore, at least in Leishmania mexicana, PG repeat synthesis is not an absolute requirement for virulence.

The role of phosphomannose isomerase in Leishmania mexicana glycoconjugate synthesis and virulence.

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates. Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa. To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype. Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose. The mutants are unable to synthesize phosphoglycan repeats [-6-Gal beta 1-4Man alpha 1-PO(4)-] and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose. The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development. L. mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function.

Lipophosphoglycan of the protozoan parasite Leishmania: stage- and species-specific importance for colonization of the sandfly vector, transmission and virulence to mammals.

Leishmaniasis is a major health problem to the human population of the tropics, subtropics and Mediterranean regions. This disease is caused by the parasitic protozoa Leishmania, which have adapted to survive in several hostile environments such as the vector insect midgut, blood and the mammalian macrophage phagolysosome. Several Leishmania glycoconjugates have been implicated as key molecules for these remarkable capabilities. This review summarizes the current knowledge on potential and proven functions of the most prominent of the Leishmania glycoconjugates, the lipophosphoglycan.

Proteophosphoglycans of Leishmania.

Proteophosphoglycans are an expanding family of highly glycosylated Leishmania proteins with many unusual and some unique structural features. The novel protein-glycan linkage in proteophosphoglycans - phosphoglycosylation of Ser by lipophosphoglycan-like structures - emerges as a major form of protein glycosylation in Leishmania. Here, Thomas Ilg reviews the chemical structure, the ultrastructure, the genes and the potential functions of different members of this novel family of parasite glycoproteins.

Lipophosphoglycan is not required for infection of macrophages or mice by Leishmania mexicana.

Cell surface lipophosphoglycan (LPG) is commonly regarded as a multifunctional Leishmania virulence factor required for survival and development of these parasites in mammals. In this study, the LPG biosynthesis gene lpg1 was deleted in Leishmania mexicana by targeted gene replacement. The resulting mutants are deficient in LPG synthesis but still display on their surface and secrete phosphoglycan-modified molecules, most likely in the form of proteophosphoglycans, whose expression appears to be up-regulated. LPG-deficient L.mexicana promastigotes show no significant differences to LPG-expressing parasites with respect to attachment to, uptake into and multiplication inside macrophages. Moreover, in Balb/c and C57/BL6 mice, LPG-deficient L.mexicana clones are at least as virulent as the parental wild-type strain and lead to lethal disseminated disease. The results demonstrate that at least L. mexicana does not require LPG for experimental infections of macrophages or mice. Leishmania mexicana LPG is therefore not a virulence factor in the mammalian host.

Subunit vaccination of mice against new world cutaneous leishmaniasis: comparison of three proteins expressed in amastigotes and six adjuvants.

A mixture of well-defined recombinant antigens together with an adjuvant that preferentially stimulates specific gamma interferon (IFN-gamma)-secreting helper type 1 CD4(+) T cells (Th1 cells) presents a rational option for a vaccine against leishmaniasis. The potential of this approach was investigated in murine infections with Leishmania mexicana, which are characterized by the absence of a parasite-specific Th1 response and uncontrolled parasite proliferation. A mixture of three antigens (glycoprotein 63, cysteine proteinases, and a membrane-bound acid phosphatase), which are all expressed in amastigotes, the mammalian stage of the parasite, were used for the immunization of C57BL/6 mice in combination with six adjuvants (interleukin 12 [IL-12], Detox, 4'-monophosphoryl lipid A, QS-21, Mycobacterium bovis BCG, and Corynebacterium parvum). All six vaccine formulations containing the mixture of recombinant antigens were protective against challenge infections with promastigotes, the insect stage of the parasite, in that mice controlled and healed infections but developed transient and, in certain cases, accentuated disease. The most effective adjuvants were IL-12 followed by Detox. Further studies using these two adjuvants showed that a similar protective effect was observed with a mixture of the corresponding native proteins, and mice which had controlled the infection showed a preponderance of IFN-gamma-secreting CD4(+) T cells in the lymph nodes draining the lesion. Using the recombinant proteins individually, it is shown that the relatively abundant cysteine proteinases and glycoprotein 63, but not the acid phosphatase, are able to elicit a protective response. The results are discussed in comparison to previous studies with subunit vaccines and with respect to cell biological aspects of antigen presentation in Leishmania-infected macrophages.

Leishmania major proteophosphoglycan is expressed by amastigotes and has an immunomodulatory effect on macrophage function.

Proteophosphoglycan (PPG) is a newly described mucin-like glycoprotein found on the surface of Leishmania major promastigotes and secreted in the culture supernatant. We show here that antigenically similar PPGs are present in several Leishmania species. PPG could also be detected on the surface of amastigotes and in small, parasite-free vesicles in infected macrophages. Because of the similarity of its carbohydrate chains to lipophosphoglycan, a parasite receptor for host macrophages, PPG was tested for binding to macrophages. PPG bound to macrophages and was internalized in a time-dependent manner. PPG inhibited the production of tumor necrosis factor-alpha and synergized with interferon-gamma to stimulate the production of nitric oxide by macrophages. PPG may contribute to the binding of Leishmania to host cells and may play a role in modulating the biology of the infected macrophage at the early stage of infection.

Proteophosphoglycans of Leishmania mexicana. Molecular cloning and characterization of the Leishmania mexicana ppg2 gene encoding the proteophosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes.

Intracellular amastigotes of the pathogenic protozoon Leishmania mexicana secrete an extensively phosphoglycosylated proteophosphoglycan (aPPG) into the phagolysosome of mammalian host macrophages, that appears to fulfil important functions for the parasites. Promastigotes (the sandfly vector forms) of the same species secrete a proteophosphoglycan with identical protein backbone but exhibiting stage-specific phosphoglycosylation patterns [Klein, Göpfert, Goehring, Stierhof and Ilg (1999) Biochem. J. 344, 775-786]. In this study we report the cloning of the novel repeat-containing proteophosphoglycan gene ppg2 by antibody screening of a Leishmania mexicana amastigote cDNA expression library. ppg2 is equally expressed in promastigotes and amastigotes at the mRNA level. Targeted gene replacement of both alleles of the single copy gene ppg2 results in the loss of pPPG2 expression in promastigotes. Antisera against Escherichia coli-expressed ppg2 recognize the deglycosylated forms of aPPG as well as pPPG2. These results confirm that ppg2 encodes the protein backbones of aPPG and pPPG2. An unusual finding is that ppg2 exhibits two stable allelic forms, ppg2a and ppg2b. Their main difference lies in the number of central 72 bp DNA repeats (7 versus 8). ppg2a and ppg2b encode polypeptide chains of 574 and 598 amino acids, respectively, that show no homology to known proteins. The novel 24 amino acid Ser-rich peptide repeats encoded by the 72 bp DNA repeats are targets for Ser phosphoglycosylation in Leishmania mexicana.

Proteophosphoglycans of Leishmania mexicana. Identification, purification, structural and ultrastructural characterization of the secreted promastigote proteophosphoglycan pPPG2, a stage-specific glycoisoform of amastigote aPPG.

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans that appear to be important for successful colonization of the sandfly and for virulence in the mammalian host. A hallmark of these molecules is extensive phosphoglycosylation by phosphoglycan chains via the unusual linkage Manalpha1-PO(4)-Ser. In this study we have identified and purified to apparent homogeneity a novel proteophosphoglycan (pPPG2) which is secreted by Leishmania mexicana promastigotes (sandfly stage). Amino acid analysis and immunoblots using polypeptide-specific antisera suggest that pPPG2 shares a common protein backbone with a proteophosphoglycan (aPPG) secreted by Leishmania mexicana amastigotes (mammalian stage). Both pPPG2 and aPPG show a similar degree of Ser phosphoglycosylation (50. 5 mol% vs. 44.6 mol%), but the structure of their phosphoglycan chains is developmentally regulated: in contrast to aPPG which displays unique, complex and highly branched glycan chains [Ilg, Craik, Currie, Multhaup, and Bacic (1998) J. Biol. Chem. 273, 13509-13523], pPPG2 contains short unbranched structures consisting of >60 mol% neutral glycans, most likely (Manalpha1-2)(0-5)Man and Galbeta1-4Man, as well as about 40 mol% monophosphorylated glycans of the proposed structures PO(4)-6Galbeta1-4Man and PO(4)-6(Glcbeta1-3)Galbeta1-4Man. The major differences between pPPG2 and aPPG with respect to their apparent molecular mass, their ultrastructure and their proteinase sensitivity are most likely a consequence of this stage-specific glycosylation of their common protein backbone.

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors.

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.

Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.

Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions.

Proteophosphoglycan, a major secreted product of intracellular Leishmania mexicana amastigotes, is a poor B-cell antigen and does not elicit a specific conventional CD4+ T-cell response.

Secreted and surface-exposed antigens of intracellular pathogens are thought to provide target structures for detection by the host immune system. The major secreted product of intracellular Leishmania mexicana amastigotes, a proteophosphoglycan (aPPG), is known to contribute to the establishment of the parasitophorous vacuole and is able to activate complement. aPPG belongs to a novel class of serine- and threonine-rich Leishmania proteins that are extensively modified by phosphodiester-linked phosphooligosaccharides and terminal mannooligosaccharides. Here we show that mice chronically infected with L. mexicana generally do not produce antibodies or Th cells specific for aPPG. Similarly, antibody titers are very low in mice vaccinated with aPPG, and specific CD4+ T cells are undetectable. Comparative analyses of other Leishmania glycoconjugates indicate that L. mexicana-specific carbohydrate structures are poorly immunogenic in mice and that the proteophosphoglycan aPPG behaved immunologically like a carbohydrate. The latter observation is explained by the lack of induction of aPPG-specific CD4+ T cells. In contrast, recombinant aPPG peptides stimulate CD4+ T-cell responses and high titers of specific antibodies are found in the sera of mice vaccinated with these peptides. Native aPPG is highly resistant to proteinases and apparently cannot be degraded by macrophages. It is concluded that conventional CD4+ T cells against the polypeptide backbone of aPPG are not induced because the molecule resists antigen processing due to its extensive and complex carbohydrate modification. The complex glycan chains of aPPG, which exhibit important biological functions for the parasite, may therefore also have evolved to evade detection by the immune system of the host organism.