Ghada Mkannez

MicroRNA Expression Profile in Human Macrophages in Response to Leishmania major Infection

Background Leishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts. Methodology/Principal Findings We report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h). We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation. Conclusions/Significance Our data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the dynamics of gene expression in host cells during leishmaniasis.

An in silico immunological approach for prediction of CD8+ T cell epitopes of Leishmania major proteins in susceptible BALB/c and resistant C57BL/6 murine models of infection

It is well established that MHC class II restricted-CD4 T cells are dominant during the development of immunity against Leishmania (L) in the C57BL/6-resistant mouse strain. However and in agreement with a number of previous observations indicating that specific CD8 T cells are primed during natural infection or vaccination in humans, a great deal of evidence obtained recently with the susceptible BALB/c murine model of infection by Leishmania major indicates that CD8 T cells participate in both pathogenesis and immunity to cutaneous leishmaniasis. Our goal herein was to identify in silico all parasitic peptides present in the whole L. major predicted proteome, using several public computational systems for the prediction of peptide binding to all MHC (histocompatibility complex-2) molecules in BALB/c and C57BL/6 mice (Syfpeithi, Rankpep, PRED(BALB/c) and Bimas). Peptides that were predicted to bind to different H2 molecules were then analysed for their homology with any of the murine proteins annotated so far, using the BLAST algorithm. Sets of selected peptides for each H2 molecule were defined by different prediction systems and compared to each other. Surprisingly, the results showed that a higher number of L. major peptides were predicted to bind H2 BALB/c molecules and very few or none to bind H2 C57BL/6 molecules. Our finding illustrates how a hybrid immuno-computational approach may be useful for biologists to target an in silico set of selected proteins to define potential candidate antigens for experimental vaccination with greater accuracy as well as a reduced number of T cell antigens.

Genetic micro-heterogeneity of Leishmania major in emerging foci of zoonotic cutaneous leishmaniasis in Tunisia.

Tunisia is endemic for zoonotic cutaneous leishmaniasis (ZCL), a parasitic disease caused by Leishmania (L.) major. ZCL displays a wide clinical polymorphism, with severe forms present more frequently in emerging foci where naive populations are dominant. In this study, we applied the multi-locus microsatellite typing (MLMT) using ten highly informative and discriminative markers to investigate the genetic structure of 35 Tunisian Leishmania (L.) major isolates collected from patients living in five different foci of Central Tunisia (two old and three emerging foci). Phylogenetic reconstructions based on genetic distances showed that nine of the ten tested loci were homogeneous in all isolates with homozygous alleles, whereas one locus (71AT) had a 58/64-bp bi-allelic profile with an allele linked to emerging foci. Promastigote-stage parasites with the 58-bp allele tend to be more resistant to in vitro complement lysis. These results, which stress the geographical dependence of the genetic micro-heterogeneity, may improve our understanding of the ZCL epidemiology and clinical outcome.

Comparative genomics of Tunisian Leishmania major isolates causing human cutaneous leishmaniasis with contrasting clinical severity.

Zoonotic cutaneous leishmaniasis caused by Leishmania (L.) major parasites affects urban and suburban areas in the center and south of Tunisia where the disease is endemo-epidemic. Several cases were reported in human patients for which infection due to L. major induced lesions with a broad range of severity. However, very little is known about the mechanisms underlying this diversity. Our hypothesis is that parasite genomic variability could, in addition to the host immunological background, contribute to the intra-species clinical variability observed in patients and explain the lesion size differences observed in the experimental model. Based on several epidemiological, in vivo and in vitro experiments, we focused on two clinical isolates showing contrasted severity in patients and BALB/c experimental mice model. We used DNA-seq as a high-throughput technology to facilitate the identification of genetic variants with discriminating potential between both isolates. Our results demonstrate that various levels of heterogeneity could be found between both L. major isolates in terms of chromosome or gene copy number variation (CNV), and that the intra-species divergence could surprisingly be related to single nucleotide polymorphisms (SNPs) and Insertion/Deletion (InDels) events. Interestingly, we particularly focused here on genes affected by both types of variants and correlated them with the observed gene CNV. Whether these differences are sufficient to explain the severity in patients is obviously still open to debate, but we do believe that additional layers of -omic information is needed to complement the genomic screen in order to draw a more complete map of severity determinants.

Do scars caused by past history of Leishmania major Ainfection may harbor persistent parasites

Based on the knowledge that a cured infection protects the individual from re-infection, the development of a vaccine to prevent leishmaniasis has been a goal for nearly a century. Indeed, it is generally believed that after healing of leishmaniasis, sterile cure is never achieved and that few residual living parasites will remain sequestered within some host cells that offer them a safe shelter and hence maintain anti-parasite immune memory. This statement is mainly supported by data from experimental leishmaniasis in mice of susceptible or resistant phenotype, in which, live parasites could be recovered from lesions even after healing, and in which disease reactivation can be obtained by immune manipulation even after apparent complete cure. Whether maintenance of a long-term immune effector memory in humans will also require persistence of live parasites is presently unknown but is very important in the perspective of a vaccine development. Our aim herein was to address the issue of Leishmania major parasite persistence vs. sterile healing in zoonotic cutaneous leishmaniasis (ZCL) by analyzing biopsies of scars from healed volunteers. Skin-punch scars’ biopsies (n=59, range of scar age: 1– 5 years) have been obtained from volunteers (18–55 years old) living in two ZCL endemic foci, who had a confirmed past history, are clinically cured of ZCL and who gave their written consent. The whole protocol was approved by the local IRB. The specimens were taken under sterile conditions and local anaesthesia. Each specimen was divided into three parts: (i) the first sample was processed for quantitative real time PCR, (ii) the second was cultured in vitro in enriched medium and (iii) the third was inoculated into the footpad of susceptible BALB/c mice which were kept under observation for five months. For in vitro isolation and after microscopic observation for at least 8 weeks, all cultures were found negative. For in vivo isolation, biopsy-inoculated mice were killed five months later, and skin fragments, draining lymph nodes and spleen were inoculated into culture medium, observed carefully for at least 8 weeks before being also designated as negative. PCR results were found negative for the majority of biopsies. These results indicate that any potential persistent living parasites after cure are unlikely to be sequestered in ZCL scars. This issue is of importance in strategies for control of leishmaniasis and requires further discussion.