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.

Leishmania mexicana metacaspase is a negative regulator of amastigote proliferation in mammalian cells.

Metacaspases (MCAs) are caspase family cysteine peptidases that have been implicated in cell death processes in plants, fungi and protozoa. MCAs have also been suggested to be involved in cell cycle control, differentiation and clearance of aggregates; they are virulence factors. Dissecting the function of MCAs has been complicated by the presence in many organisms of multiple MCA genes or limitations on genetic manipulation. We describe here the creation of a MCA gene-deletion mutant (Δmca) in the protozoan parasite Leishmania mexicana, which has allowed us to dissect the role of the parasite's single MCA gene in cell growth and cell death. Δmca parasites are viable as promastigotes, and differentiate normally to the amastigote form both in in vitro macrophages infection and in mice. Δmca promastigotes respond to cell death inducers such as the drug miltefosine and H(2)O(2) similarly to wild-type (WT) promastigotes, suggesting that MCAs do not have a caspase-like role in execution of L. mexicana cell death. Δmca amastigotes replicated significantly faster than WT amastigotes in macrophages and in mice, but not as axenic culture in vitro. We propose that the Leishmania MCA acts as a negative regulator of amastigote proliferation, thereby acting to balance cell growth and cell death.

The efficacy of aerosol treatment with non-ionic surfactant vesicles containing amphotericin B in rodent models of leishmaniasis and pulmonary aspergillosis infection.

Amphotericin B (AMB) is used to treat both fungal and leishmanial infections, which are of major significance to human health. Clinical use of free AMB is limited by its nephrotoxicity, whereas liposomal AMB is costly and requires parenteral administration, thus development of novel formulations with enhanced efficacy, minimal toxicity and that can be applied via non-invasive routes is required. In this study we analysed the potential of non-ionic surfactant vesicles (NIV) given by nebulisation to deliver AMB to the lungs, liver and skin. Treatment with AMB-NIV resulted in significantly higher drug levels in the lungs and skin (p<0.05) compared to similar treatment with AMB solution but significantly lower plasma levels (p<0.05). Treatment with AMB-NIV resulted in a significant reduction in fungal lung burdens in a rat model of invasive pulmonary aspergillosis (p<0.05) compared to treatment with the carrier alone. Treatment with AMB-NIV but not AMB solution significantly suppressed Leishmania donovani liver parasite burdens (p<0.05) but could not inhibit the growth of cutaneous Leishmania major lesions. The results of this study indicate that aerosolised NIV enhanced pulmonary and hepatic delivery whilst minimising systemic exposure and toxicity.

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.

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.

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.

Differences in enzyme activities between two species of Hematodinium, parasitic dinoflagellates of crustaceans.

Parasitic dinoflagellates of the genus Hematodinium infect several commercially important decapod crustaceans. Different species of Hematodinium have different levels of virulence in their respective hosts. Enzyme activities were studied from two species of Hematodinium, one isolated from the Norway lobster (Nephrops norvegicus) and the other from the American blue crab (Callinectes sapidus). We report the identification of differences in secretion of acid phosphatase (AP) and leucine arylamidase from two parasite species. Leucine arylamidase was only contained and secreted by the species infecting the blue crab. Both parasite species contained AP, but only the species infecting the Norway lobster secreted this enzyme. In this species, AP activity was predominantly in the soluble fraction (69.5%). AP activity was localized to cytoplasmic granules and on the membranes surrounding the cell nucleus. In addition to providing information on the cellular metabolism of the parasite, the pattern of activities of these enzymes may also be useful in distinguishing among different species of Hematodinium.

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.

Vaccination with a preparation based on recombinant cysteine peptidases and canine IL-12 does not protect dogs from infection with Leishmania infantum.

Cysteine peptidases (CPs) have been implicated in various processes central to the pathogenicity of Leishmania parasites, and are thought to be key factors in the host-parasite interaction. In order to fully evaluate the potential of the CPs as vaccine candidates, studies in natural host species are required. In the study we report here, recombinant L. infantum CPs CPA and CPB were used to vaccinate dogs. In order to induce an appropriate response against the antigens, recombinant canine IL-12 was added as an adjuvant either by itself or in combination with Quil A. After vaccination, dogs were given an intravenous challenge with promastigotes of L. infantum JPC strain. In both vaccinated groups (CPs with IL-12 or CPs with IL-12 and Quil A) CP-specific antibodies were detected after vaccination, indicating that there was a reaction to the vaccine. However, all dogs were found parasite-positive and all developed some degree of clinical leishmaniosis. The observed lack of efficacy of the candidate vaccines could be due, completely or in part, to a number of factors associated with the vaccine antigen, the adjuvant or host-parasite interactions. When compared to results from other studies, it seems less likely that the molecular conformation of the rCPs or rIL-12 caused this lack of efficacy. More plausible explanations are the dose and timing of the IL-12 application and the potentially different effects IL-12 induces as an adjuvant in either the murine or the canine leishmaniosis model.

A parasitic scuticociliate infection in the Norway lobster (Nephrops norvegicus).

A histophagous ciliate infection was discovered in a number of Norway lobsters (Nephrops norvegicus) from the Clyde Sea Area, Scotland. Silver-carbonate staining of cultured ciliates revealed an oral apparatus and additional structural features that are morphologically similar to scuticociliates in the genus Mesanophrys, which are known to parasitize crustaceans. However, ribosomal DNA sequences (ITS1/5.8S/ITS2) of the ciliate were identical to Orchitophyra stellarum, a parasitic scuticociliate of sea stars with a different morphology from Mesanophrys spp. and to the ciliate from N. norvegicus. Associated pathology included degeneration and necrosis of the myocardial heart muscle, and large numbers of ciliates in the gill filaments.

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.

Biochemical and genetic evidence for a family of heterotrimeric G-proteins in Trichomonas vaginalis.

We have cloned a single copy gene from the human parasite Trichomonas vaginalis that encodes a putative protein of 402 amino acids with approximately 35% sequence identity to known alpha subunits of heterotrimeric G-proteins. It contains the characteristic GTP binding domains G-1 to G-5 with the key residues conserved. The new sequence has an unusual N-terminal extension of approximately 70 residues that cannot be aligned to reference G-proteins and which is characterised by proline-rich repeats. To investigate the expression and cellular localisation of the protein we produced specific antisera against a recombinant fusion protein. The antisera recognised a protein of an apparent molecular mass of 51 kDa in protein extracts from T. vaginalis and immunofluorescent microscopy established that the protein is localised to discrete endomembranes. Using a protocol designed to purify mammalian heterotrimeric G-proteins incorporating a GTPgammaS binding assay, we isolated two proteins from Trichomonas that are recognised by an heterologous GA/1 antisera raised to a peptide of the conserved G-1 domain of G-protein alpha subunits. These two proteins have an apparent molecular mass of 61 and 48 kDa, respectively, larger and smaller than the translation product of the cloned gene. Consistent with these results, the GA/1 antisera did not cross-react with the fusion protein produced from the gene we have cloned. These data suggest T. vaginalis possesses more than one heterotrimeric G-protein alpha subunit. Based on the sequence features of the cloned gene and the biochemical properties of the purified proteins, we suggest that these alpha subunits are likely to be part of classic heterotrimeric G-protein complexes.

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.

Recent advances in the search for new anti-coccidial drugs.

Coccidia provide a rich hunting ground for drug-designers, as there are significant biochemical differences between the parasites and their hosts. Recent years have brought the discovery of the plastid and its possible metabolic machinery, characterisation of acidocalcisomes, reports on the apparent absence from some coccidia of a typical mitochondrion, and the discovery of the mannitol cycle and shikimate pathway in the parasites. Moreover, modern technologies such as genomics and proteomics are bringing new insights into the biochemistry of coccidia and highlighting possible drug targets in abundance. A major issue for would-be drug discoverers is to decide upon the targets to prioritise. This review provides an update on recent findings on how coccidia differ biochemically from vertebrates. It includes discoveries within coccidian parasites themselves but also uses findings in Plasmodium to provide an overview of biochemical features that may be characteristics of many apicomplexan parasites and so potential targets for broad-spectrum drugs.

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.

Detection of the parasitic dinoflagellate Hematodinium in the Norway lobster Nephrops norvegicus by ELISA.

Norway lobsters Nephrops norvegicus from the coastal waters of Scotland are seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of the parasite in the haemolymph of N. norvegicus. The ELISA is simple to perform with a detection limit of 5 x 10(4) parasites ml(-1) haemolymph. The ELISA is currently being used to study the prevalence and seasonality of Hematodinium infection in N. norvegicus and other crustacean hosts.

Development and application of an immunoassay diagnostic technique for studying Hematodinium infections in Nephrops norvegicus populations.

Patent Hematodinium infections of the Norway lobster Nephrops norvegicus can be detected with a morphological method (pleopod diagnosis), but this fails to identify low-level haemolymph (sub-patent) and any tissue-based (latent) infections. The current study describes the development and application of an immunoassay for the detection of antigens of the parasite Hematodinium in the Norway lobster N. norvegicus. Infected tissue and haemolymph samples were detected as multiple-band reactions to a polyclonal antibody (anti-Hematodinium). The sensitivity limit of the method was 204 parasites mm(-3), approximately 10 times more sensitive than the pleopod diagnosis method. Use of the immunoassay on tissue samples taken from catches in the Clyde Sea area, Scotland, UK, showed that the pleopod method considerably under-diagnosed infection prevalence in the early part of the season, though this under-diagnosis decreased as infected lobsters in the field progressed from latent and sub-patent to patent infections. However, the immunoassay failed to detect any infected lobsters during the summer months, suggesting that infection may not be carried over from one season to the next. The data presented suggest that this immunoassay allows for the accurate estimation of Hematodinium infection prevalence in the field and should be employed, where possible, for the routine monitoring of infection prevalence in N. norvegicus.

The stage-regulated expression of Leishmania mexicana CPB cysteine proteases is mediated by an intercistronic sequence element.

The tandemly arranged CPB genes of Leishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific; CPB1 and CPB2 are expressed predominantly in metacyclics, whereas CPB3-CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into a CPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2 with CPB2.8 (typical of CPB3-CPB18) revealed two major regions of divergence as follows: one of 258 base pairs (bp) corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totaling 57 bp, localized to the intercistronic region downstream of CPB1 and CPB2. Cell lines expressing CPB2.8 or CPB2 with the 3'-untranslated region and intercistronic sequence of CPB2.8 showed up-regulation in amastigotes. Conversely, metacyclic-specific expression occurred with CPB2 or CPB2.8 with the 3'-untranslated region and intercistronic sequence of CPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into the CPB locus. It is proposed that the InS mediates metacyclic-specific stage-regulated expression of CPB by affecting the maturation of polycistronic pre-mRNA. This is the first well defined cis-regulatory element implicated in post-transcriptional stage-specific gene expression in Leishmania.