Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains.

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 µM at the promastigote stage and 32 and 195 µM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 µM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.

Characterization of Leishmania infantum thiol-dependent reductase 1 and evaluation of its potential to induce immune protection.

The need to develop an effective vaccine against leishmaniasis to prevent the 2 million new cases each year led to the search for antigens able to elicit protection against infection with Leishmania. In this study, we have characterized a parasite-specific protein of Leishmania infantum named thiol-dependent reductase 1 (TDR1). The protein is present in both life cycle stages of L. infantum with a notable higher expression in the amastigote forms, suggesting a role in the interaction between the parasite and the mammalian host. Thiol-dependent reductase 1 is localized in the cytosol, although we were able to detect the protein in the culture medium of both promastigotes and axenic amastigotes, and consequently, TDR1 is considered an excreted/secreted molecule of the parasite. Therefore, we have evaluated the potential of TDR1 recombinant protein to protect against experimental challenge with L. infantum parasites using a murine model. Despite a reduction in spleen parasite load in the chronic phase of disease, TDR1 administration was not effective in the protection of Balb/c mice against visceral leishmaniasis and thus TDR1 do not have a crucial role in the modulation of mammalian host immune response, as observed with its protein counterpart Tc52 of Trypanosoma cruzi.

Molecular cloning and localization of a calpain-like protease from the abdominal muscle of Norway lobster Nephrops norvegicus.

Calpains are ubiquitous cysteine-proteases found in many, if not all, living organisms and their roles within these organisms are diverse, ranging from the mediation of cytoskeletal remodeling to the regulation of gene expression. In crustaceans calpains have so far been shown to be important mainly during moulting and growth. In the present study we report the expression of a calpain in the abdominal muscle of Norway lobster (Nephrops norvegicus) using degenerate primer, rapid amplification of cDNA ends (5'-3'-RACE), reverse transcriptase-PCR and RNA in situ hybridization approaches. The full-length mRNA sequence (2,774 bp) was found to include an open reading frame (bp 225-1,940) encoding a 572 amino acid polypeptide with a predicted mass of 65.9 kDa and a predicted pI of 5.17. The calpain was found to be an arthropod M-class calpain homologue to Homarus americanus Calpain M (Ha-CalpM) and has thus been termed Nephrops norvegicus calpain M (Nn-CalpM). When its expression pattern in abdominal muscle of adult intermoult Nephrops norvegicus was investigated an exclusive expression in a thin layer of connective tissue cells surrounding muscle fibres was found. This localization suggests a role in tenderizing connective tissue networks during growth and moulting.

An essential role for the Leishmania major metacaspase in cell cycle progression.

Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

Differential inhibition of high and low Mr thioredoxin reductases of parasites by organotelluriums supports the concept that low Mr thioredoxin reductases are good drug targets.

Thioredoxin reductase (TrxR), a NADPH-dependent disulfide oxidoreductase, is vital in numerous cellular processes including defence against reactive oxygen species, cell proliferation and signal transduction. TrxRs occur in 2 forms, a high Mr enzyme characterized by those of mammals, the malaria parasite Plasmodium falciparum and some worms, and a low Mr form is present in bacteria, fungi, plants and some protozoan parasites. Our hypothesis is that the differences between the forms can be exploited in the development of selective inhibitors. In this study, cyclodextrin- and sulfonic acid-derived organotelluriums known to inhibit mammalian TrxR were investigated for their relative efficacy against P. falciparum TrxR (PfTrxR), a high Mr enzyme, and Trichomonas vaginalis TrxR (TvTrxR), a low Mr form of TrxR. The results suggest that selective inhibition of low Mr TrxRs is a feasible goal.

Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins.

The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (DeltaGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other non-protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the DeltaGPI8 mutant. Significantly, the DeltaGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

Roles of cysteine proteinases of trypanosomes and Leishmania in host-parasite interactions.

Trypanosomes and Leishmania contain an abundance of stage-regulated cysteine proteinases encoded by several gene families. Analysis of parasites rendered defective in cysteine proteinase function, either through genetic manipulation or through the use of specific inhibitors, has revealed roles for the enzymes in parasite virulence, in modulation of the host's immune response and in parasite differentiation.

Molecular detection of Hematodinium spp. in Norway lobster Nephrops norvegicus and other crustaceans.

The Norway lobster Nephrops norvegicus (L.) from the coastal waters of Scotland is seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. Methods used to detect infection include a morphological index (pleopod diagnosis) and several immunoassays. The present study describes the development and application of a set of Hematodinium-specific polymerase chain reaction (PCR) primers and DNA probes based on Hematodinium ribosomal DNA (rDNA). In the PCR assay, a diagnostic band of 380 bp was consistently amplified from total genomic DNA isolated from Hematodinium-infected N. norvegicus. The sensitivity of the assay was 1 ng DNA, which is equivalent to 0.6 parasites. The primer pair also detected Hematodinium DNA in preparations of the amphipod Orchomene nanus, indicating that the amphipod may be infected with the same Hematodinium sp. infecting N. norvegicus. DNA probes detected Hematodinium parasites in heart, hepatopancreas and gill tissues from N. norvegicus, and hepatopancreas and gill tissues from Carcinus maenas, confirming Hematodinium infection in the latter.

Recent advances in identifying and validating drug targets in trypanosomes and leishmanias.

The unique aspects of the biochemistry of trypanosomatids make rational drug design an attractive approach, but targets must be selected carefully. Genetic manipulation provides a valuable means of mimicking loss of function attributable to therapeutic intervention, but caution must be exercised when interpreting such data with respect to target validation.

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana.

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.

The glycoinositolphospholipids from Leishmania panamensis contain unusual glycan and lipid moieties.

The cell surface of Leishmania parasites is coated by glycosylphosphatidylinositol (GPI)-anchored macromolecules (glycoproteins and a lipophosphoglycan) and a polymorphic family of free GPI glycolipids or glycoinositolphospholipids (GIPLs). Here we show that GIPLs with unusual glycan and lipid moieties are likely to be major cell surface components of L. panamensis (subgenus Viannia) promastigotes. These glycolipids were purified by high performance thin layer chromatography and their structures determined by gas-liquid chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, methylation analysis and chemical and enzymatic sequencing of the glycan headgroups. The major GIPLs contained two glycan core sequences, Manalpha1-3Manalpha1-4GlcN-phosphatidylinositol (type-2 series) or Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1- 4GlcN-phosphatidylinosit ol (hybrid series), which were elaborated with Galalpha1-2Galbeta1- or Galalpha1-2/3Galalpha1-2Galbeta1- extensions that were attached to the 3-position of the alpha1-3 linked mannose. The phosphatidylinositol moiety contained exclusively diacylglycerol with palmitoyl, stearoyl and heptadecanoyl chains. Non-galactosylated GIPL species with the same core structures were also found. The galactose extensions and the presence of diacylglycerol in the lipid moieties are novel features for the GIPLs of Leishmania spp. The implications of these structures for the biosynthesis of leishmanial GIPLs and their putative function in the mammalian host are discussed.

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus.

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.

Targeting polyamines of parasitic protozoa in chemotherapy.

All parasitic protozoa contain polyamines and in recent years they, and their associated enzymes, have attracted attention as drug targets because they might reveal novel antiparasite therapies. How justified is this approach to drug discovery? In this review, Sylke Müller, Graham Coombs and Rolf Walter summarize the current status of research into drugs that exploit polyamine metabolism of trypanosomatid and malaria parasites, and propose priorities for research into such drugs. This review was inspired by an Expert Meeting entitled 'Polyamine Metabolism of Parasitic Protozoa as a Drug Target'.

Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets.

All parasitic protozoa contain multiple proteases, some of which are attracting attention as drug targets. Aspartic proteases are already the targets of some clinically useful drugs (e.g. chemotherapy of HIV infection) and a variety of factors make these enzymes appealing to those seeking novel antiparasite therapies. This review provides a critical analysis of the current knowledge on Plasmodium aspartic proteases termed plasmepsins, proposes a definitive nomenclature for this group of enzymes, and compares these enzymes with aspartic proteases of humans and other parasitic protozoa. The present status of attempts to obtain specific inhibitors of the parasite enzymes that will be useful as drugs is outlined and suggestions for future research priorities are proposed.

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana.

lmcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucei and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. lmcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity.

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension.

A cDNA for a Trypanosoma brucei cysteine proteinase has been cloned and sequenced. The deduced protein can be divided into four domains, based on homologies with other cysteine proteinases: the pre-, pro- and central regions show considerable homology to the cathepsin L class of mammalian enzymes, whilst the long C-terminal extension distinguishes the trypanosome enzyme from all mammalian cysteine proteinases reported. This 108 amino acid extension, which includes 9 contiguous prolines near the junction with the central domain, appears likely to be processed in part to produce the mature enzyme, and may be involved in targeting the protein within the cell. The trypanosome genome contains more than 20 copies of the cysteine proteinase gene arranged in a long tandem array.

Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens.

Cysteine peptidase inhibitor genes (ICP) of the chagasin family have been identified in protozoan (Leishmania mexicana and Trypanosoma brucei) and bacterial (Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.

The effects of carbon dioxide and oxygen upon the growth and in vitro transformation of Leishmania mexicana mexicana.

Growth of Leishmania mexicana promastigotes is highly dependent upon O2 tension. There was a strong positive correlation between the level of O2, growth rate and maximum parasite density. Promastigotes under low oxygen tension decreased in size, protein content and motility, and deaths occurred. Changes in the carbon dioxide concentration (0.1-5.0%) had little effect on promastigote growth. Transformation in vivo of L. mexicana amastigotes to promastigotes also required oxygen, but a low level (0.4%) was sufficient for a high percentage of the amastigotes to transform. At high O2 concentrations, transformation was a little speedier but the number of parasites transforming was little affected. A greater effect was found with CO2. At 5%, transformation was much more rapid than at 0.1% and also an even greater percentage of amastigotes transformed within 48 h. The results give some indication that amastigotes are adpated for growth at low oxygen tensions encountered in vivo and that high carbon dioxide levels may act as a trigger for transformation of the amastigote to promastigote after it is taken up by the sandfly.

Proteinases of Leishmania mexicana amastigotes and promastigotes: analysis by gel electrophoresis.

The proteinases of Leishmania mexicana mexicana amastigotes and promastigotes have been analysed by electrophoresis on polyacrylamide gels containing denatured haemoglobin. Eleven bands of activity were detected indicating multiple proteinases. These were significant quantitative and qualitative differences between the proteinases of the two developmental forms. Four, B-E, were present in both forms but were of much higher activity in the amastigote. There were two major activities in promastigotes, A and D. The other proteinases, F-K, were of lower activity; I and K were not detected in promastigotes. All proteinases were active optimally at pH 4.0. Most of them, including the major proteinases A-E, were thiol proteinases since they were stimulated by 1 mM dithiothreitol and were sensitive to inhibitors such as HgCl2, leupeptin, antipain and iodoacetic acid.