Caractérisation moléculaire des souches de leishmania infantum et évaluation de nouveaux médicaments pour soigner la leishmaniose viscérale

La leishmaniose viscérale (LV) est la forme la plus sévère de la leishmaniose humaine. Elle est transmise par la piqûre d'un phlébme. La leishmaniose viscérale est mortelle en l'absence de traitement. Les options thérapeutiques courantes contre la leishmaniose viscérale sont limitées. En région méditerranéenne, la LV est due à Leishmania infantum, zoonose dont le chien est le principal réservoir. A côté de quelques cas d'infection systémique, un nombre important d'humains porteurs asymptomatiques a été mis en evidence. En première partie, nous avons étudié l'intérêt du MultiLocus Microsatellite Genotyping (MLMT) pour l'identification des souches de Leishmania du sud de la France. Par MLMT nous avons étudié la variabilité génétique de différentes souches et recherché une association avec les différentes formes cliniques, la résistance aux médicaments et les phénomènes de rechutes. Nous avons observé une hétérogénéité génétique entre les différentes souches de L. infantum MON-1. Si l'association de certains génotypes avec les différentes expressions cliniques de la leishmaniose n'a pu être démontrée, nous avons par contre observé une répartition préférentielle géographique de certains génotypes. En deuxième partie, nous avons mis au point un protocole expérimental destiné au criblage de nouveaux agents anti-Leishmania infantum ayant pour cible la machinerie cellulaire mise en route par la cellule hôte pour l'élimination du parasite intracellulaire. Nos résultats ont montré que les altérations du système de trafic intracellulaire de la cellule-hôte induites par certains composés étaient corrélées à la mort du parasite et à son élimination.Visceral leishmaniasis (VL) is the most severe form of Human Leishmaniases, which occurs when protozoan parasites Leishmania donovani or L. infantum, given by phlebotomine sandfly bites. The disease is fatal when untreated. Current treatment options against VL are very limited with few drug molecules, often expensive, not always safe and able to induce resistance phenomenon.In this report, we have characterized on one hand, different genetic variants of Leishmania infantum strains isolated in different geographical areas from southern France and on the other hand have identified new potential anti-Leishmania infantum compounds and characterized their molecular mechanism of action.In the first part, we studied the interest of Multilocus Microsatellite Genotyping (MLMT) for the identification of Leishmania strains from southern France. By genotyping technique MLMT, we studied the genetic variability of different strains and sought an association with different clinical forms of leishmaniasis, resistance to drugs and relapse. We observed genetic heterogeneity among different strains of L. infantum-MON-1. we observed a preferential geographic distribution of certain genotypes.In the second part, we have developed an experimental protocol for the screening of new anti-Leishmania infantum compounds that target the host cell machinery responsible for the intracellular parasite killing, we studied the different steps of endocytic pathways potentially targeted by these compounds. Our results showed that with some compounds, modifications of the intracellular trafficking of the host cell were correlated with parasite death and its elimination

Multilocus microsatellite typing of Leishmania and clinical applications: a review

Microsatellite markers have been used for Leishmania genetic studies worldwide, giving useful insight into leishmaniasis epidemiology. Understanding the geographic distribution, dynamics of Leishmania populations, and disease epidemiology improved markedly with this tool. In endemic foci, the origins of antimony-resistant strains and multidrug treatment failures were explored with multilocus microsatellite typing (MLMT). High genetic variability was detected but no association between parasite genotypes and drug resistance was established. An association between MLMT profiles and clinical disease manifestations was highlighted in only three studies and this data needs further confirmation. At the individual level, MLMT provided information on relapse and reinfection when multiple leishmaniasis episodes occurred. This information could improve knowledge of epidemiology and guide therapeutic choices for active chronic visceral leishmaniasis, the disease form in some HIV-positive patients

Geographical distribution of isolates from Southeastern France.

<p>Isolates clustered into four endemic areas. The geographic areas where samples were isolated from Cévennes (blue), Provence (green), Alpes-Maritimes (red) and Corsica (violet) are indicated. The Alpes-Maritimes and Provence endemic areas are located in the PACA region.</p

A. Localization of the genotypes.

<p>Eleven repeated genotypes, which were observed in four or more isolates, are represented with colored squares on the map of the endemic areas of Alpes-Maritimes and Provence. The two endemic areas are separated by a blue line. The remaining genotypes are represented by light brown squares. The area in the black rectangle is shown in further detail in Fig 3B. B. Detailed localization of isolates in the Alpes-Maritimes endemic area and more precisely the surrounding region of Nice. Isolates were localized at the city level. The black line represents the delimitation of Nice.</p

Estimated population structure for <i>L</i>. <i>infantum</i> from Southeastern France assessed using STRUCTURE software based on the analysis of the 270 <i>L</i>. <i>infantum</i> DNA samples at 12 microsatellite markers.

<p><b>A</b>: The plots show the estimated membership coefficient (Q) of each isolates. Each isolate is represented by a single vertical line divided into K colors, in which K is the number of populations assumed. Each color represents one population, and the length of the colored segment shows the estimated proportion of isolates membership in that population. The derived graph for delta K shows K = 2, thereby indicating the presence of two populations in the investigated sample set. <b>B</b>: Isolates of the sub-populations A and B with delta K values for each sub-population. Two close values were observed for K in sub-population A: K = 4 and K = 6. For sub-population B, K = 2.</p

Repeated genotypes.

<p>Arbitrary numbers assigned to the 30 repeated genotypes in (x-axis) and number of isolates belonging to each genotype (y-axis). The origin of each isolates is indicated by the different colors.</p

Unrooted neighbor-joining tree inferred from genetic distances derived from the proportion of alleles shared among the 270 isolates of <i>L</i>. <i>infantum</i> based on 12 microsatellite markers.

<p>The two populations defined using STRUCTURE are highlighted. The blue stars correspond to the non-MON-1 strains. At the end of each branche of the network, the first three characters correspond to the arbitrarily assigned number for each genotype, the following two characters (AM, P, Ce, Co) correspond to the endemic area of sample collection, and then the last two characters correspond to the number of isolates with the given genotype. The orange rectangles represent the isolates with mixed genotypes. The sub populations are represented with colored ovals at K = 6 for A sub-populations and K = 2 for B sub-populations. The isolates with no color are those with mixed genotypes within A sub-populations at K = 6. MEGA 4 software was used to visualize the neighbor-joining tree.</p

Temporal distribution of the 11 genotypes represented by more than four isolates.

<p>Temporal distribution of the 11 genotypes represented by more than four isolates.</p