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Byline: Srija. Moulik, Ritika. Sengupta, Aishwarya. Dighal, Bikash. Sardar, Bibhuti. Saha, Nilay. Das, Mitali. Chatterjee This case report series alerts to the atypical manifestations of dermal leishmaniasis in an area [...]

Leishmania species of the subgenus Viannia and especially Leishmania braziliensis are responsible for a large proportion of New World leishmaniasis cases. The reproductive mode of Leishmania species has often been assumed to be predominantly clonal, but remains unsettled. We have investigated the genetic polymorphism at 12 microsatellite loci on 124 human strains of Leishmania braziliensis from 2 countries, Peru and Bolivia. There is substantial genetic diversity, with an average of 12.4 [+ or -] 4.4 alleles per locus. There is linkage disequilibrium at a genome-wide scale, as well as a substantial heterozygote deficit (more than 50% the expected value from Hardy-Weinberg equilibrium), which indicates high levels of inbreeding. These observations are inconsistent with a strictly clonal model of reproduction, which implies excess heterozygosity. Moreover, there is large genetic heterogeneity between populations within countries (Wahlund effect), which evinces a strong population structure at a microgeographic scale. Our findings are compatible with the existence of population foci at a microgeographic scale, where clonality alternates with sexuality of an endogamic nature, with possible occasional recombination events between individuals of different genotypes. These findings provide key clues on the ecology and transmission patterns of Leishmania parasites. clonality | microsatellites | population genetics | endogamyl | heterozygote defiency

Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key d biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal d or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence. inducible expression | FK506 | glycoconjugates | pathogen | trypanosomatid protozoa

Genetic exchange has not been shown to be a mechanism underlying the extensive diversity of Leishmania parasites. We report here evidence that the invertebrate stages of Leishmania are capable of having a sexual cycle consistent with a meiotic process like that described for African trypanosomes. Hybrid progeny were generated that bore fuji genomic complements from both parents, but kinetoplast DNA maxicircles from one parent. Mating occurred only in the sand fry vector, and hybrids were transmitted to the mammalian host by sand fly bite. Genetic exchange likely contributes to phenotypic diversity in natural populations, and analysis of hybrid progeny will be useful for positional cloning of the genes controlling traits such as virulence, tissue tropism, and drug resistance.

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, micro-satellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of [approximately equal to] 18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged [approximately equal to] 1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa. Leishmania infantum | Leishmaniasis | parasitic protozoa | phylogeny | population genetics

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II--directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression., Infection with pathogenic Leishmania results in a spectrum of human diseases, termed the leishmaniases, with an annual incidence of 2 million cases in 88 countries (1). Leishmania parasites are transmitted [...]

A comparison of gene content and genome architecture of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, revealed a conserved core proteome of about 6200 genes in large syntenic polycistronic gene clusters. Many species-specific genes, especially large surface antigen families, occur at nonsyntenic chromosome-internal and subtelomeric regions. Retroelements, structural RNAs, and gene family expansion are often associated with syntenic discontinuities that--along with gene divergence, acquisition and loss, and rearrangement within the syntenic regions--have shaped the genomes of each parasite. Contrary to recent reports, our analyses reveal no evidence that these species are descended from an ancestor that contained a photosynthetic endosymbiont., The protozoan pamogens Leishmania major, Trypanosoma cruzi, and Trypanosoma brucei (family Trypanosomatidae, order Kinetoplastida) collectively cause disease and death in millions of humans and countless infections in other mammals, primarily [...]

Leishmania are evolutionarily ancient protozoans (Kinetoplastidae) and important human pathogens that cause a spectrum of diseases ranging from the asymptomatic to the lethal. The Leishmania genome is relatively small [[approximately equal to] 34 megabases (Mb)], lacks substantial repetitive DNA, and is distributed among 36 chromosomes pairs ranging in size from 0.3 Mb to 2.5 Mb, making it a useful candidate for complete genome sequence determination. We report here the nucleotide sequence of the smallest chromosome, chr1. The sequence of chr1 has a 257-kilobase region that is densely packed with 79 protein.coding genes. This region is flanked by telomeric and subtelomeric repetitive elements that vary in number and content among the chr1 homologs, resulting in an [approximately equal to] 27.5-kilobase size difference. Strikingly, the first 29 genes are all encoded on one DNA strand, whereas the remaining 50 genes are encoded on the opposite strand. based on the gene density of chr1, we predict a total of [approximately equal to] 9,800 genes in Leishmania, of which 40% may encode unknown proteins.

Tumor necrosis factor [Alpha] (TNF[Alpha]) is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. For example, plasma levels of TNF[Alpha] are positively correlated with severity and mortality in malaria and leishmaniasis. We have previously described a polymorphism at -308 in the TNF[Alpha] promoter and shown that the rare allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3-DQ2, which is associated with autoimmunity and high TNF[Alpha] production. Homozygosity for TNF2 carries a sevenfold increased risk of death from cerebral malaria. Here we demonstrate, with reporter genes under the control of the two allelic TNF promoters, that TNF2 is a much stronger transcriptional activator than the common allele (TNF1) in a human B cell line. Footprint analysis using DNase I and B cell nuclear extract showed the generation of a hypersensitive site at -308 and an adjacent area of protection. There was no difference in affinity of the DNA-binding protein(s) between the two alleles. These results show that this polymorphism has direct effects on TNF[Alpha] gene regulation and may be responsible for the association of TNF2 with high TNF[Alpha] phenotype and more severe disease in infections such as malaria and leish-maniasis.

Leishmania are parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1[Beta] (IL-1[Beta]) and tumor necrosis factor [Alpha] are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid of Leishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1[Beta] steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1[Beta] mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor [Alpha] and Staphylococcus induction of IL-1[Beta] gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1[Beta] mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the -310 to -57 nucleotide region of the IL-1[Beta] promoter was found to mediate LPG's inhibitory activity. The requirement for the -310 to -57 promoter gene sequence for LPG's effect is demonstrated by the abrogation of LPG's inhibitory activity by truncation or deletion of the -310 to -57 promoter gene sequence. Furthermore, the minimal IL-1[Beta] promoter (positions -310 to +15) mediated LPG's inhibitory activity with dose and kinetic profiles that were similar to LPG's suppression of steady-state IL-1[Beta] mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a 'gene silencer,' a function, to our knowledge, not previously described. LPG's inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.

Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon [Gamma] to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon [Gamma] resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania parasites through their ability to regulate the expression of iNOS by macrophages.

Leishmania RNA virus 1 (LRV1) was electroporated in either an uninfected or an infected strain of Leishmania. PCR-based assays showed that the virus was successfully introduced, replicated but did not persist in Leishmania. These results indicate that LRV1 may be used as vector for the transfer and expression of foreign genes in Leishmania. However, futurestudies should address the question of why LRV1 does not persist in the host.

Experiments were conducted to develop homozygous knockouts of the Leishmania major dihydrofolate reductase-thymidylate synthese (dhfr-ts) gene by double gene replacement. The results showed that virulent Leishmania parasites were resistant to total inactivation of dhfr-ts, although dhfr-ts knockout could easily be achieved in attenuated strains. Further analysis showed that the virulent strains have the ability to undergo changes in chromosome number, which permits the retention of functional dhfr-ts genes despite double gene targeting.

A genetic characterization was carried out on several parasitic protozoa representing species of Trypanozoma, Leishmania and Plasmodium using the random amplified polymorphic DNA (RAPD) technique. The resulting relationships acquired by the RAPD method correlated well with previous results using multilocus enzyme electrophoresis. These results demonstrate the applicability of the RAPD technique in establishing genetic and evolutionary relationships among these organisms.

The transesterification model of RNA editing proposes that U residues are transferred directly from the oligo(U) 3' tail of small guide RNA (gRNA) to mRNA via a gRNA/mRNA chimeric intermediate. To further elucidate this model, an in vitro system for the chimeric formation of gND7-II gRNA and preedited ND7 mRNA was developed. The system utilized mitochondrial extracts from Leishmania tarentolae. Characterization of this in vitro system showed that it is similar to, but not identical to the in vivo system. Furthermore, chimera formation was dependent on the presence of an anchor sequence that allows fusion of the gRNA and the mRNA.

A study of the complete nucleotide sequence of the double-stranded RNA genome of the Leishmania RNA virus showed three open reading frames. Examination of the reading frames showed a plus one translational frameshift which produces a gag-pol type of fusion protein. It was further shown that the first open reading frame does not encode protein but initiates translation and or RNA stability.

Protein kinase C (PKC) dependent cell signaling was studied to investigate the defective stimulus response in mononuclear phagocytes. Results show altered kinetic properties and defective activation of PKC from leishmania infected mononuclear phagocytes. This was attributed to the decreased responsiveness to dioleoyl-rac-glycerol (DAG) which resulted to the covalent modification of PKC.

Leishmania are evolutionarily ancient protozoans of the order Kinetoplastidae that are linked with varied diseases affecting humans ranging from the asymptomatic to the lethal. The nucleotide sequence of the smallest chromosome of the Leishmania genome, chr1, has a 257-kilobase region that is densely packed with 79 protein-coding genes. It is possible that this region has a chromosomal centromere and an origin of replication. The higher order DNA structures and chromatin-associated protein complexes could define the centromeric functions and replication origin of chr1.

Researchers designed a transient transfection vector by designing a chimeric gene in which the firefly luciferase gene was inserted in frame into the coding site of the pgs 28 gene of plasmodium gallinaceum, with the aim of formulating a technique to functionally study malaria genes using DNA transfection. They effectively introduced and expressed a foreign gene in a malaria parasite.

The article examines the role of breed genetic background in susceptibility to canine leishmaniasis, particularly for dogs in the Mediterranean basin. The most frequently occurring haplotypes are identified; the boxer breed is used as illustration.

The genetic: determination of resistance to Leishmania major in mice is a topic of interest to many immunologists attempting to link the immunological mechanism with the responsible genes. In this [...]