Isabel L. Mauricio

Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy

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, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of ≈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 ≈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.

The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania: looking back and to the future

Trypanosoma cruzi is the protozoan agent of Chagas disease, and the most important parasitic disease in Latin America. Protozoa of the genus Leishmania are global agents of visceral and cutaneous leishmaniasis, fatal and disfiguring diseases. In the 1970s multilocus enzyme electrophoresis demonstrated that T. cruzi is a heterogeneous complex. Six zymodemes were described, corresponding with currently recognized lineages, TcI and TcIIa-e--now defined by multiple genetic markers. Molecular epidemiology has substantially resolved the phylogeography and ecological niches of the T. cruzi lineages. Genetic hybridization has fundamentally influenced T. cruzi evolution and epidemiology of Chagas disease. Genetic exchange of T. cruzi in vitro involves fusion of diploids and genome erosion, producing aneuploid hybrids. Transgenic fluorescent clones are new tools to elucidate molecular genetics and phenotypic variation. We speculate that pericardial sequestration plays a role in pathogenesis. Multilocus sequence typing, microsatellites and, ultimately, comparative genomics are improving understanding of T. cruzi population genetics. Similarly, in Leishmania, genetic groups have been defined, including epidemiologically important hybrids; genetic exchange can occur in the sand fly vector. We describe the profound impact of this parallel research on genetic diversity of T. cruzi and Leishmania, in the context of epidemiology, taxonomy and disease control.

Genomic diversity in the Leishmania donovani complex

The Leishmania donovani complex is considered to be composed of 3 species; L. donovani , L. infantum and L. chagasi , although this classification has been challenged. Genotypic relationships within the complex were evaluated at different levels by: binding of the probe Lmet9, specific for L. chagasi and Old World Leishmania spp.; partial sequencing of a constitutive major surface protease single gene ( mspC ) and random amplification of polymorphic DNA (RAPD). The Old World Leishmania spp. and the L. donovani complex have a monophyletic origin. Leishmania chagasi clearly belongs to the L. donovani complex but it is indistinguishable from L. infantum , which suggests introduction of L. chagasi into the New World in recent history. Leishmania infantum / L. chagasi was identified as a monophyletic group within the L. donovani complex but L. donovani may be paraphyletic. Diversity within L. donovani is substantial and phylogeographical patterns of association were found.

Comparative microsatellite typing of new world leishmania infantum reveals low heterogeneity among populations and its recent old world origin.

Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis (VL) in the New World (NW) with endemic regions extending from southern USA to northern Argentina. The two hypotheses about the origin of VL in the NW suggest (1) recent importation of L. infantum from the Old World (OW), or (2) an indigenous origin and a distinct taxonomic rank for the NW parasite. Multilocus microsatellite typing was applied in a survey of 98 L. infantum isolates from different NW foci. The microsatellite profiles obtained were compared to those of 308 L. infantum and 20 L. donovani strains from OW countries previously assigned to well-defined populations. Two main populations were identified for both NW and OW L. infantum. Most of the NW strains belonged to population 1, which corresponded to the OW MON-1 population. However, the NW population was much more homogeneous. A second, more heterogeneous, population comprised most Caribbean strains and corresponded to the OW non-MON-1 population. All Brazilian L. infantum strains belonged to population 1, although they represented 61% of the sample and originated from 9 states. Population analysis including the OW L. infantum populations indicated that the NW strains were more similar to MON-1 and non-MON-1 sub-populations of L. infantum from southwest Europe, than to any other OW sub-population. Moreover, similarity between NW and Southwest European L. infantum was higher than between OW L. infantum from distinct parts of the Mediterranean region, Middle East and Central Asia. No correlation was found between NW L. infantum genotypes and clinical picture or host background. This study represents the first continent-wide analysis of NW L. infantum population structure. It confirmed that the agent of VL in the NW is L. infantum and that the parasite has been recently imported multiple times to the NW from southwest Europe.

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

In most aerobic organisms hemoperoxidases play a major role in H2O2-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis–Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H2O2. To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.

Molecular approaches for a better understanding of the epidemiology and population genetics of Leishmania

Molecular approaches are being used increasingly for epidemiological studies of visceral and cutaneous leishmaniases. Several molecular markers resolving genetic differences between Leishmania parasites at species and strain levels have been developed to address key epidemiological and population genetic questions. The current gold standard, multilocus enzyme typing (MLEE), needs cultured parasites and lacks discriminatory power. PCR assays identifying species directly with clinical samples have proven useful in numerous field studies. Multilocus sequence typing (MLST) is potentially the most powerful phylogenetic approach and will, most probably, replace MLEE in the future. Multilocus microsatellite typing (MLMT) is able to discriminate below the zymodeme level and seems to be the best candidate for becoming the gold standard for distinction of strains. Population genetic studies by MLMT revealed geographical and hierarchic population structure in L. tropica, L. major and the L. donovani complex. The existence of hybrids and gene flow between Leishmania populations suggests that sexual recombination is more frequent than previously thought. However, typing and analytical tools need to be further improved. Accessible databases should be created and sustained for integrating data obtained by different researchers. This would allow for global analyses and help to avoid biases in analyses due to small sample sizes.

Genetic typing and phylogeny of the Leishmania donovani complex by restriction analysis of PCR amplified gp63 intergenic regions

Protozoan parasites of the Leishmania donovani complex (L. donovani, L. infantum/L. chagasi) are causative agents of visceral leishmaniasis. To understand phylogeny and taxonomy within this group better we have developed 2 new polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) analyses of the major surface protease (msp or gp63) intergenic (ITG) regions. We have named this approach msp intergenic region RFLP typing (MIRT). One intergenic region lies between the constitutive msp (mspC) and stationary phase msp (mspS4) genes (ITG/CS) and the other between multicopy logarithmic phase msp (mspL) genes (ITG/L). The markers generated robust and congruent phylogenies, identifying 5 genetic clusters within L. donovani. One cluster was synonymous with L. infantum (L. chagasi); clusters strongly correlated with isoenzyme typing and some with geographical origin. These genetic groups may be important for epidemiological and clinical studies. The congruence of the groups identified indicates suitability of these genomic targets for taxonomic studies. Furthermore, subgroups of L. donovani were of equivalent phylogenetic status to L. infantum. No evidence was found to support the existence of L. archibaldi. It is likely to be necessary in future to re-evaluate the taxonomic status of L. donovani or of L. infantum, as discrete species.

TcGPXII, a glutathione-dependent Trypanosoma cruzi peroxidase with substrate specificity restricted to fatty acid and phospholipid hydroperoxides, is localized to the endoplasmic reticulum.

Until recently, it had been thought that trypanosomes lack glutathione peroxidase activity. Here we report the subcellular localization and biochemical properties of a second glutathione-dependent peroxidase from Trypanosoma cruzi (TcGPXII). TcGPXII is a single-copy gene which encodes a 16 kDa protein that appears to be specifically dependent on glutathione as the source of reducing equivalents. Recombinant TcGPXII was purified and shown to have peroxidase activity towards a narrow substrate range, restricted to hydroperoxides of fatty acids and phospholipids. Analysis of the pathway revealed that TcGPXII activity could be readily saturated by glutathione and that the peroxidase functioned by a Ping Pong mechanism. Enzyme reduction was shown to be the rate-limiting step in this pathway. Using immunofluorescence, TcGPXII was shown to co-localize with a homologue of immunoglobulin heavy-chain binding protein (BiP), a protein restricted to the endoplasmic reticulum and Golgi. As the smooth endoplasmic reticulum is the site of phospholipid and fatty acid biosynthesis, this suggests that TcGPXII may play a specific role in the T. cruzi oxidative defence system by protecting newly synthesized lipids from peroxidation.

Leishmaniases in the Mediterranean in the era of molecular epidemiology

Molecular tools are used increasingly for descriptive epidemiological studies in different Mediterranean foci of visceral and cutaneous leishmaniases. Several molecular markers with different resolution levels have been developed to address key epidemiological questions related to the (re-)emergence and spread of leishmaniases, as well as its risk factors: environmental changes, immunosuppression and treatment failure. Typing and analytical tools are improving but are not yet addressing all epidemiological issues satisfactorily. There is an urgent need for better cooperation between laboratory scientists and epidemiologists and for regional epidemiological surveillance of these infectious diseases that affect all Mediterranean countries.

Is it time to revise the nomenclature of Leishmania

Leishmania is notable for a large number of described species. Molecular phylogenies of Leishmania have increasingly suggested that the number of species could be too large. A recent phylogenetic analysis of the heat-shock protein 70 gene (hsp70) by Fraga and colleagues supports the existence of only eight medically relevant species compared to 17 species as defined by multilocus enzyme electrophoresis. Whether a revised classification system should be introduced for Leishmania is discussed.