Increased transmission potential of Leishmania major/Leishmania infantum hybrids

Development of Leishmania infantum/Leishmania major hybrids was studied in two sand fly species. In Phlebotomus papatasi, which supported development of L. major but not L. infantum, the hybrids produced heavy late-stage infections with high numbers of metacyclic promastigotes. In the permissive vector Lutzomyia longipalpis, all Leishmania strains included in this study developed well. Hybrids were found to express L. major lipophosphoglycan, apparently enabling them to survive in P. papatasi midgut. The genetic exchange of the hybrids thus appeared to have enhanced their transmission potential and fitness. A potentially serious consequence is the future spread of the hybrids using this peridomestic and antropophilic vector.     

A proposed role for Leishmania major carboxypeptidase in peptide catabolism

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids—the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes—the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.     

Genomic DNA macroarrays as a tool for analysis of gene expression in Leishmania

Gene-array technologies have been applied in a wide number of organisms to study gene expression profiling under several physiological and experimental conditions. Gene-array implementations combined with the information arising from emerging genome sequencing projects are expected to be in the near future a major tool to characterize genes involved in different processes. So far, gene expression profile studies in trypanosomatids have been performed in microarrays that use a glass support to immobilize fragments of genomic DNA followed by fluorescent detection. Here, we wanted to test the potential of genomic DNA macroarrays of Leishmania infantum using nylon membranes and radioactive detection. Nylon macroarrays present a number of advantages since the processing of the membranes is based on standard Southern blotting protocols familiar to molecular biologists, and the data acquisition equipment is available to most research institutions. Nylon macroarrays were employed to search for genes showing increased mRNA abundance during an axenic differentiation of L. infantum promastigotes to amastigotes. Several clones were rescued and, after validation by Northern blot assays, these L. infantum sequences were used to screen the Leishmania major gene database. The L. major contigs with high homology to the L. infantum sequences allowed a consistent identification of the regulated genes.     

Intradermal inoculations of low doses of Leishmania major and Leishmania amazonensis metacyclic promastigotes induce different immunoparasitic processes and status of protection in BALB/c mice

In order to simulate the natural long term parasitisms which may occur in mammals infected with Leishmania, cutaneous leishmaniases due to Leishmania major or Leishmania amazonensis were induced using a model based on the inoculation of 10-1000 metacyclic promastigotes into the ear dermis of BALB/c mice. The final outcome of these parasitisms was dependent upon the number of inoculated parasites. Only some of the mice inoculated with ten parasites displayed cutaneous lesions, whereas most mice infected with 100 metacyclics and all mice infected with 1000 metacyclics developed progressive lesions. We found, using the latter experimental conditions, that the onset of the pathology was associated with: (a) parasite multiplication in the inoculation site and the draining lymph node correlating with an increase of the lymph node cell number, especially in L. major-infected mice; and (b) the detection of lymph node cells, at least in part CD4(+) T lymphocytes, able to produce high levels of interferon-gamma, interleukin (IL)-4, IL-10 and IL-13. Thereafter, mice infected by L. major harboured few parasites in the ear and had a 100-fold reduction in lymph node parasite load between 23 and 40 weeks post-inoculation. In contrast, the parasite loads of L. amazonensis-infected mice remained stable in the ear and increased in nodes during the same period of time. Only L. major-infected mice that exhibited cutaneous lesions in the primary site were resistant to the re-inoculation of 1000 metacyclic promastigotes, whereas all L. amazonensis-primary infected mice remained susceptible to a second homologous challenge. These results are the first to document that a status of resistance to re-infection, referred to concomitant immunity, is coupled to the development of primary progressive lesions in L. major-infected BALB/c mice. Such a protective status is absent in L. amazonensis-infected BALB/c mice.     

Glycosome turnover in Leishmania major is mediated by autophagy

Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite''s autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ∼20 glycosomes per cell, whereas amastigotes contain ∼10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ∼15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes.     

LaRbp38: a Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.     

Detection of Leishmania DNA in phlebotomines captured in Campo Grande, Mato Grosso do Sul, Brazil

Over the past years, leishmaniases have become a public health issue in the Brazilian state of Mato Grosso do Sul, particularly in Campo Grande, the state capital. The purpose of this study was to detect the presence of Leishmania DNA in the population of phlebotomine sandflies using DNA amplification by PCR. Insect captures were carried out from 4 pm. to 7 am for 4 consecutive days each month from October 2005 to September 2006 in 16 neighborhoods located in 7 urban regions of Campo Grande. Traps were placed indoors and in the vicinity of households. As many as 971 males and 203 females were collected. One hundred and five naturally fed females were identified and grouped as 1- to 4-specimen pools. DNA extraction was carried out using whole insects. Lutzomyia longipalpis predominated, accounting for 99.15% of the phlebotomines captured. Also found was Nyssomyia whitmani, the vector of tegumentary leishmaniasis. Abundance was greatest in the vicinity of households (69.8% of the phlebotomines captured). As revealed by PCR, parasites were present in 1.9% of the Leishmania spp. specimens investigated and confirmed for visceral leishmaniasis.     

Dynamic association of topoisomerase II to the mitotic chromosomes in live cells of Aspergillus nidulans

DNA topoisomerase II (Topo II) is an essential enzyme that catalyzes topological changes of DNA and consists of a major member of mitotic chromosomes. To investigate the dynamic localization of Topo II in nuclei, we engineered the strain of Aspergillus nidulans expressing Topo II fused with green fluorescent protein (GFP). Time-lapse microscopy revealed that the distribution of Topo II-GFP in nuclei varied depending on the cell cycle. In interphase, Topo II-GFP distributed evenly in the nucleoplasm and at the onset of G2 phase became concentrated into nucleolus. During mitosis, Topo II-GFP accumulated on chromosomes, when the chromosomes condensed. In the early mitosis, the Topo II also showed a single or two brighter spots among the fluorescence of clumped chromosomes. The spots once divided into several spots and then concentrated again into a spot per nucleus in the dividing nuclei of anaphase. Along with the subsequent decondensation of chromosomes, Topo II diffused back into nucleoplasm.     

Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.     

Inhibitors of casein kinase 1 block the growth of Leishmania major promastigotes in vitro

Casein kinase 1 (CK1) is a family of multifunctional Ser/Thr protein kinases that are ubiquitous in eukaryotic cells. Recent studies have demonstrated the existence of, and role for, CK1 in protozoan parasites such as Leishmania, Plasmodium and Trypanosoma. The value of protein kinases as potential drug targets in protozoa is evidenced by the successful exploitation of cyclic guanosine monophosphate-dependent protein kinase (PKG) with selective tri-substituted pyrrole and imidazopyridine inhibitors. These compounds exhibit in vivo efficacy against Eimeria tenella in chickens and Toxoplasma gondii in mice. We now report that both of these protein kinase inhibitor classes inhibit the growth of Leishmania major promastigotes and Trypanosoma brucei bloodstream forms in vitro. Genome informatics predicts that neither of these trypanosomatids codes for a PKG orthologue. Biochemical studies have led to the unexpected discovery that an isoform of CK1 represents the primary target of the pyrrole and imidazopyridine kinase inhibitors in these organisms. CK1 from extracts of L. major promastigotes co-fractionated with [(3)H]imidazopyridine binding activity. Further purification of CK1 activity from L. major and characterization via liquid chromatography coupled tandem mass spectrometry identified CK1 isoform 2 as the specific parasite protein inhibited by imidazopyridines. L. major CK1 isoform 2 expressed as a recombinant protein in Escherichia coli displayed biochemical and inhibition characteristics similar to those of the purified native enzyme. The results described here warrant further evaluation of the activity of these kinase inhibitors against mammalian stage Leishmania parasites in vitro and in animal models of infection, as well as studies to genetically validate CK1 as a therapeutic target in trypanosomatid parasites.     

Ploidy changes associated with disruption of two adjacent genes on Leishmania major chromosome 1

Leishmania major Friedlin (LmjF) is a kinetoplastid protozoan whose genomic sequence has been recently elucidated. About 60% of the identified genes do not have a known function, and many are trypanosomatid-specific. Here we characterise two adjacent genes from LmjF chromosome 1 (chr1): LmjF01.0750, which encodes a predicted protein with a serine/threonine protein kinase motif and LmjF01.0760, which encodes a product with no similarity to other known proteins. Orthologues of both genes are present in Trypanosoma cruzi, but neither occur in Trypanosoma brucei. We have mapped polyadenylation and spliced-leader acceptor sites for both genes, and show that they differ between Leishmania species. Attempts to generate null mutants of LmjF01.0750 by homologous recombination were unsuccessful and led to the apparent triploidy of the entire genome, suggesting that it is an essential gene. Interestingly, at least two copies of LmjF01.0750 are required for cell survival. Further evidence of genome plasticity in Leishmania was provided by changes in chr1 copy number that occurred during in vitro growth of wild-type LmjF promastigotes and following replacement of a single copy of LmjF01.0760.     

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran CL and VL strains using Mediterranean L. infantum strains as a reference. Macrophage infectivity and serum sensitivity, properties thought to be associated with virulence, were similar between CL and VL strains from both regions. Attention focused on the genome organisation of genes for two candidate virulence factors: Leishmania mitogen activated protein kinase (LMPK) and cysteine proteinase b (Cpb). Interestingly, the Mediterranean strains exhibited restriction enzyme polymorphisms associated with tropism for both LMPK and Cpb genes whereas no differences were observed for the Honduran strains. We also report relative genetic homogeneity of the Honduran strains as compared to the Mediterranean strains and discuss it in terms of the probable origin for the Central American L. chagasi/infantum.     

Leishmania major: clathrin and adaptin complexes of an intra-cellular parasite

To investigate the role of clathrin-mediated trafficking during the Leishmania lifecycle, open reading frames encoding clathrin heavy chain and the beta-adaptins, major components of the adaptor complexes, have been analysed both in silico and experimentally. The Leishmania genome encodes three beta-adaptins, which arose at a time predating speciation of these divergent trypanosomatids. Unlike Trypanosoma brucei, both clathrin heavy chain and beta-adaptin1 are constitutively expressed throughout the Leishmania life cycle. Clathrin relocalises in amastigotes relative to promastigotes, consistent with developmental alterations to the morphology of the endo-membrane system.     

Identification of novel Leishmania major antigens that elicit IgG2a response in resistant and susceptible mice

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.     

Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism

We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress. The attenuated line was found to be significantly more susceptible to hydrogen peroxide, a change which may explain the loss of virulence. In a direct assay of trypanothione-dependent peroxidase activity, hydrogen peroxide metabolism in the H-line was significantly lower than in wild type. Furthermore, trypanothione reductase activity was significantly lower in the H-line, suggesting that gentamicin selection may result in pleiotropic affects on thiol metabolism in Leishmania. A putative RNA-binding protein was very strongly up-regulated in the attenuated line, suggesting a possible target for gentamicin in Leishmania.     

Protein turnover and differentiation in Leishmania

Leishmania occurs in several developmental forms and thus undergoes complex cell differentiation events during its life-cycle. Those are required to allow the parasite to adapt to the different environmental conditions. The sequencing of the genome of L. major has facilitated the identification of the parasite's vast arsenal of proteolytic enzymes, a few of which have already been carefully studied and found to be important for the development and virulence of the parasite. This review focuses on these peptidases and their role in the cellular differentiation of Leishmania through their key involvement in a variety of degradative pathways in the lysosomal and autophagy networks.     

Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana

Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail. 3'-Nucleotidase/nuclease gene (mRNA) and protein (enzyme activity) expression were examined in three different L. mexicana developmental forms: procyclic promastigotes, metacyclic promastigotes and amastigotes. Results of both approaches demonstrated that the 3'-nucleotidase/nuclease was a stage-specific enzyme, being expressed by promastigote forms (stages restricted to the insect vector), but not by amastigotes (which produce disease in mammalian hosts). Starvation of these parasites for purines resulted in the significant up-regulation of both 3'-nucleotidase/nuclease mRNA and enzyme activity in promastigotes, but not in amastigotes. These results underscore the critical role that the 3'-nucleotidase/nuclease must play in purine salvage during the rapid multiplicative expansion of the parasite population within its insect vector. To our knowledge, the L. mexicana 3'-nucleotidase/nuclease is the first example of a nutrient-induced and developmentally regulated enzyme in any parasitic protozoan.     

A suspected new species of Leishmania, the causative agent of visceral leishmaniasis in a Thai patient

A suspected new species of Leishmania is described as the causative agent of the third reported case of autochthonous visceral leishmaniasis in a Thai man living in Southern Thailand. The results of PCR-restriction fragment length polymorphism and sequence analysis of the internal transcribed spacer 1 of ssrRNA and the mini-exon genes were different from those of previously reported Leishmania species. A direct agglutination test (DAT) revealed that antibody against Leishmania infection was detected in nine domestic cats. No potential vectors could be identified. A large-scale epidemiological survey of leishmaniasis should be urgently conducted since visceral leishmaniasis is considered an emerging disease of public health concern in Thailand.