Comparative genomics: from genotype to disease phenotype in the leishmaniases

Recent progress in sequencing the genomes of several Leishmania species, causative agents of cutaneous, mucocutaneous and visceral leishmaniasis, is revealing unusual features of potential relevance to parasite virulence and pathogenesis in the host. While the genomes of Leishmania major, Leishmania braziliensis and Leishmania infantum are highly similar in content and organisation, species-specific genes and mechanisms distinguish one from another. In particular, the presence of retrotransposons and the components of a putative RNA interference machinery in L. braziliensis suggest the potential for both greater diversity and more tractable experimentation in this Leishmania Viannia species.     

Clonal diversity in the expression and stability of the metastatic capability of Leishmania guyanensis in the golden hamster

Metastatic disease is a major concern of dermal leishmaniasis caused by Leishmania of the Viannia subgenus. The golden hamster provides an experimental model of systemic dissemination and cutaneous metastasis of Leishmania Viannia. We have exploited this model to examine the expression of parasite virulence in cloned populations derived from a strain of L. guyanensis previously shown to be highly metastatic in the hamster. Metastatic capacity manifested as dissemination throughout the lymphoid organs; cachexia and secondary cutaneous lesions were found to differ among clones, yielding a spectrum of virulence. The metastatic phenotype of clonal populations was stable over 5 sequential passages in hamsters. In addition, the low or high propensity to disseminate and produce cutaneous metastatic lesions was reproduced. Capacity to disseminate from the inoculation site was conserved following subcloning of metastatic clones that had been passaged in culture for several generations; clinical manifestations, cachexia, and cutaneous metastatic lesions were variably expressed. Dissemination of parasites and cachexia were significantly related (P = 0.004). Overall, cachexia was an earlier manifestation of dissemination than cutaneous metastases (P < 0.001). The reproducible expression of virulence phenotypes by discrete populations of Leishmania in the golden hamster provides an experimental model for clinically relevant expression of virulence in human leishmaniasis.     

Mapping the proteome of Leishmania Viannia parasites using two-dimensional polyacrylamide gel electrophoresis and associated technologies

In this study we have demonstrated the potential of two-dimensional electrophoresis (2DE)-based technologies as tools for characterization of the Leishmania proteome (the expressed protein complement of the genome). Standardized neutral range (pH 5-7) proteome maps of Leishmania (Viannia) guyanensis and Leishmania (Viannia) panamensis promastigotes were reproducibly generated by 2DE of soluble parasite extracts, which were prepared using lysis buffer containing urea and nonidet P-40 detergent. The Coomassie blue and silver nitrate staining systems both yielded good resolution and representation of protein spots, enabling the detection of approximately 800 and 1,500 distinct proteins, respectively. Several reference protein spots common to the proteomes of all parasite species/strains studied were isolated and identified by peptide mass spectrometry (LC-ES-MS/MS), and bioinformatics approaches as members of the heat shock protein family, ribosomal protein S12, kinetoplast membrane protein 11 and a hypothetical Leishmania-specific 13 kDa protein of unknown function. Immunoblotting of Leishmania protein maps using a monoclonal antibody resulted in the specific detection of the 81.4 kDa and 77.5 kDa subunits of paraflagellar rod proteins 1 and 2, respectively. Moreover, differences in protein expression profiles between distinct parasite clones were reproducibly detected through comparative proteome analyses of paired maps using image analysis software. These data illustrate the resolving power of 2DE-based proteome analysis. The production and basic characterization of good quality Leishmania proteome maps provides an essential first step towards comparative protein expression studies aimed at identifying the molecular determinants of parasite drug resistance and virulence, as well as discovering new drug and vaccine targets.     

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5' extremity of each mRNA, supplying the 5'-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the virulence of the parasite in in vivo assays. The results presented here extend those findings to Viannia subgenus. Leishmania (Viannia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L. (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis.     

Leishmania (Viannia) lainsoni (Kinetoplastida: Trypanosomatidae), a divergent Leishmania of the Viannia subgenus--a mini review

Leishmania (Viannia) lainsoni is the Leishmania species that presents the most distinct biological (morphology, growth in axenic culture medium), biochemical (enzymatic electrophoresis profile), and molecular biology characteristics, when compared to other species of the Viannia subgenus. Development of promastigote forms of this parasite attached to the wall of the pyloric and hind gut regions of sand fly vectors is a solid characteristic that allows its positioning in the Viannia subgenus. However, taxonomic data from biochemical and molecular techniques on this Leishmania species are still not conclusive. It is evident the difficulty in taxonomically positioning this borderline Leishmania species. In this review we present the data accumulated since L. (Viannia) lainsoni has been described and we discuss its position in the Viannia subgenus.     

Reproductive strategies and population structure in Leishmania: substantial amount of sex in Leishmania Viannia guyanensis

Leishmania species of the subgenus Viannia and especially Leishmania Viannia guyanensis are responsible for a large proportion of New World leishmaniasis cases. Since a recent publication on Leishmania Viannia braziliensis, the debate on the mode of reproduction of Leishmania parasites has been reopened. A predominant endogamic reproductive mode (mating with relatives), together with strong Wahlund effects (sampling of strains from heterogeneous subpopulations), was indeed evidenced. To determine whether this hypothesis can be generalized to other Leishmania Viannia species, we performed a population genetic study on 153 human strains of L. (V.) guyanensis from French Guiana based on 12 microsatellite loci. The results revealed important homozygosity and very modest linkage disequilibrium, which is in agreement with a high level of sexual recombination and substantial endogamy. These results also revealed a significant isolation by distance with relatively small neighbourhoods and hence substantial viscosity of Leishmania populations in French Guiana. These results are of epidemiological relevance and suggest a major role for natural hosts and/or vectors in parasite strain diffusion across the country as compared to human hosts.     

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.     

Is lipophosphoglycan a virulence factor? A surprising diversity between Leishmania species

Lipophosphoglycan is a prominent member of the phosphoglycan-containing surface glycoconjugates of Leishmania. Genetic tests enable confirmation of its role in parasite virulence and permit discrimination between the roles of lipophosphoglycan and related glycoconjugates. When two different lipophosphoglycan biosynthetic genes from Leishmania major were knocked out, there was a clear loss of virulence in several steps of the infectious cycle but, with Leishmania mexicana, no effect on virulence was found. This points to an unexpected diversity in the reliance of Leishmania species on virulence factors, a finding underscored by recent studies showing great diversity in the host response to Leishmania species.     

Leishmania (Viannia) utingensis n. sp., a parasite from the sandfly Lutzomyia (Viannamyia) tuberculata in Amazonian Brazil

A leishmanial parasite isolated in 1977 from a specimen of the sandfly Lutzomyia tuberculata from Pará State, Amazonian Brazil, has been characterized following its comparison with other species of Leishmania from the same region, using isoenzyme profiles, monoclonal antibodies and characterization of the miniexon gene repeat, using the polymerase chain reaction technique (PCR). It is described here under the name of Leishmania (Viannia) utingensis n. sp.     

Inhibition of macrophage invasion by monoclonal antibodies specific to Leishmania (Viannia) braziliensis promastigotes and characterisation of their antigens

Monoclonal antibodies that specifically recognise Leishmania (Viannia) braziliensis promastigotes were produced and termed SST-2, SST-3 and SST-4. SST-2 recognises a conformational epitope present in a 24-28 kDa doublet and in a 72 kDa component, as verified by Western blotting. Indirect immunofluorescence showed that the antigen recognised by SST-2 is distributed homogeneously on the parasite surface. SST-3 recognises a flagellar glycoprotein of approximately 180 kDa. The reactivity of this mAb was abolished by sodium m-periodate treatment, indicating that SST-3 reacts with a carbohydrate epitope of the 180 kDa antigen. SST-4 recognises a conformational epitope of a 98 kDa antigen. SST-2, SST-3 and SST-4 were specific to L. (V.) braziliensis promastigote forms. Indirect immunofluorescence did not show reactivity of SST-2 or SST-3 with amastigotes of L. (V.) braziliensis, or with promastigotes of Leishmania (Viannia) panamensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Viannia) lainsoni, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major, or Leishmania (Leishmania) chagasi. We also evaluated the involvement of SST-2, SST-3 and SST-4 antigens in parasite-macrophage interaction. Fab fragments of SST-3 and SST-4 significantly inhibited the infectivity of L. (V.) braziliensis promastigotes to mouse peritoneal macrophages.     

Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania

Species of the subgenus Leishmania (Leishmania) cause the debilitating disease leishmaniasis on four continents. Species grouped within the Leishmania donovani complex cause visceral leishmaniasis, a life-threatening disease, often associated with poverty, and affecting some 0.5 million people each year. The Leishmania glycoprotein GP63, or major surface protease, is a metalloprotease involved in parasite survival, infectivity and virulence. Here, we show that evolution of the gp63 multigene family is influenced by mosaic or fragmental gene conversion. This is a major evolutionary force for both homogenisation and for generating diversity, even in the absence of sexual reproduction. We propose here that the high GC content at the third codon position in the gp63 family of Old World Leishmania may be higher in multicopy regions, under the biased gene conversion model, because increased copy numbers may lead to increased rates of recombination. We confirm that one class of gp63 genes with an extended 3'end signal, gp63(EXT), reveals genetic groups within the complex and gives insights into evolution and host associations. Gp63(EXT) genes can also provide the basis for rapid and reliable genotyping of strains in the L. donovani complex. Our results confirmed that a more stringent definition of Leishmania infantum is required and that the species Leishmania archibaldi should be suppressed.     

The in vitro leishmanicidal activity of hexadecylphosphocholine (miltefosine) against four medically relevant Leishmania species of Brazil

The in vitro leishmanicidal activity of miltefosine? (Zentaris GmbH) was assessed against four medically relevant Leishmania species of Brazil: Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis and Leishmania (Leishmania) chagasi. The activity of miltefosine against these New World species was compared to its activity against the Old World strain, Leishmania (Leishmania) donovani, which is known to be sensitive to the effects of miltefosine. The IC50 and IC90 results suggested the New World species harboured similar in vitro susceptibilities to miltefosine; however, miltefosine was approximately 20 times more active against the Old World L. (L.) donovani than against the New World L. (L.) chagasi species. The selectivity index varied from 17.2-28.9 for the New World Leishmania species and up to 420.0 for L. (L.) donovani. The differences in susceptibility to miltefosine suggest that future clinical trials with this drug should include a laboratory pre-evaluation and a dose-defining step.     

Minicircle variable region probes for characterization of Leishmania (Viannia) species.

The minicircle molecules present in the kinetoplast DNA (kDNA) network constitute a particularly useful molecular tool because they are a multicopy target and present a variable region that differs among minicircle classes in the same network. Using the polymerase chain reaction (PCR) and a set of primers directed outwardly from the minicircle conserved region, it is possible to prepare molecular probes representing the pool of variable regions from the different minicircle classes in the kDNA. In order to examine the specificity of the minicircle variable region as hybridization probes in Leishmania (Viannia) species, such fragments were amplified from reference strains and from a panel of isolates representing the zymodeme diversity of Leishmania (Viannia) in Colombia. The size of the amplified products was conserved in Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, and Leishmania (Viannia) panamensis (650 bp) and diverged in Leishmania (Viannia) equatorensis and Leishmania (Viannia) colombiensis (850 bp). The amplified products were further hybridized to variable region pools of Leishmania braziliensis, Leishmania panamensis, Leishmania guyanensis, and Leishmania equatorensis reference strains. The results obtained from the hybridization experiments support this approach as a means of defining relationships among strains. Hybridization allowed homologies to be perceived, whereas restriction fragment length analysis of the amplified products yielded strain-specific profiles. Apparently, L. (V.) equatorensis and L. (V.) colombiensis minicircle variable regions have no or only low homology with those of other Leishmania (Viannia) species, showing the divergence of those species within the subgenus.