Mehdi Chenik

Identification of a Disulfide Isomerase Protein of Leishmania major as a Putative Virulence Factor

ABSTRACT Several approaches have been previously used to elucidate the genetic basis of Leishmania virulence. In general, they were based on laboratory Leishmania clones genetically modified or grown in the presence of selecting agents. In a previous study, we demonstrated that Leishmania major freshly isolated from human cutaneous lesions showed significant differences in the severity of the experimental disease induced in BALB/c mice. Here, using the mRNA differential display technique, we analyzed gene expression in L. major promastigotes showing different levels of virulence. We have identified a novel Leishmania gene encoding a 477-amino-acid protein exhibiting two distinct regions that are identical to the putative active-site sequence (CGHC) of the eukaryotic protein disulfide isomerase (PDI). The recombinant protein displayed a specific PDI enzymatic activity. This L. major disulfide isomerase protein (LmPDI) is predominantly expressed, at both the mRNA and protein levels, in highly virulent strains. Specific PDI inhibitors abolished the enzymatic activity of the recombinant protein and profoundly affected parasite growth. These findings suggest that LmPDI may play an important role in Leishmania natural pathogenicity and may constitute a new target for anti-Leishmania chemotherapy.

Heterogeneity of Wild Leishmania major Isolates in Experimental Murine Pathogenicity and Specific Immune Response

ABSTRACT Virulence variability was investigated by analyzing the experimental pathogenicity of 19 Leishmania major strains in susceptible BALB/c mice. Twelve strains were isolated from Tunisian patients with zoonotic cutaneous leishmaniasis; seven strains were isolated in Syria (n = 1), Saudi Arabia (n = 2), Jordan (n = 2), or Israel (n = 2). BALB/c mice were injected in the hind footpad with 2 × 106 amastigotes of the various isolates, and lesion progression was recorded weekly for 9 weeks. Interleukin-4 (IL-4) and gamma interferon (IFN-γ) production of lymph node mononuclear cells activated in vitro with parasite antigens were evaluated 5 weeks after infection. We show that disease progression induced by different L. major isolates was largely heterogeneous although reproducible results were obtained when using the same isolate. Interestingly, isolates from the Middle East induced a more severe disease than did the majority of Tunisian isolates. Strains with the highest virulence tend to generate more IL-4 and less IFN-γ in vitro at week 5 postinfection as well as higher levels of early IL-4 mRNA in the lymph node draining the inoculation site at 16 h postinfection. These results suggest that L. major isolates from the field may differ in virulence, which influences the course of the disease induced in mice and the type of immune response elicited by the infected host.

Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system

We report the cloning and sequence analysis of Cerastes vipera and Macrovipera lebetina transmediterranea cDNAs coding for short non-RGD (Arg-Gly-Asp) disintegrins and for dimeric disintegrin subunits. The mRNAs belong to the short-coding class, suggesting that these disintegrin mRNAs may be more widely distributed than previously thought. Our data also argue for a common ancestry of the mRNAs of short disintegrins and those coding for precursors of dimeric disintegrin chains. The Macrovipera lebetina transmediterranea dimeric disintegrin reported to inhibit the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1 was analysed using a proteomic approach and was shown to bear MLD (Met-Leu-Asp) and VGD (Val-Gly-Asp) motifs. The results highlight the fact that disintegrins have evolved a restricted panel of integrin-blocking sequences that segregate with defined branches of the phylogenetic tree of the integrin alpha-chains, providing novel insights into the evolutionary adaptation of the snake venom antagonists to the ligand-binding sites of their target integrin receptors.

Approaches for the identification of potential excreted/secreted proteins of Leishmania major parasites

Leishmania parasites are able to survive in host macrophages despite the harsh phagolysosomal vacuoles conditions. This could reflect, in part, their capacity to secrete proteins that may play an essential role in the establishment of infection and serve as targets for cellular immune responses. To characterize Leishmania major proteins excreted/secreted early after promastigote entry into the host macrophage, we have generated antibodies against culture supernatants of stationary-phase promastigotes collected 6 h after incubation in conditions that partially reproduce those prevailing in the parasitophorous vacuole. The screening of an L. major cDNA library with these antibodies led us to isolate 33 different cDNA clones that we report here. Sequence analysis revealed that the corresponding proteins could be classified in 3 groups: 9 proteins have been previously described as excreted/secreted in Leishmania and/or other species; 11 correspond to known proteins already characterized in Leishmania and/or other species although it is unknown whether they are excreted/secreted and 13 code for unknown proteins. Interestingly, the latter are transcribed as shown by RT-PCR and some of them are stage regulated. The L. major excreted/secreted proteins may constitute putative virulence factors, vaccine candidates and/or new drug targets.

An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis

Protozoa of the Leishmania genus are transmitted to humans by the bite of infected sandflies, and are the causative agents of leishmaniasis which ranges from cutaneous to visceral clinical forms. The definitive diagnosis of leishmaniasis has relied traditionally on parasite demonstration, either by microscopy or culture; in the last years, diagnosis based on PCR methods has overcome some drawbacks of traditional methods, increasing sensitivity and allowing using less invasive sampling for diagnosis. However, there are not defined protocols and almost each laboratory applies its own in-house method. Although there are several studies comparing the performance of different methods within the same laboratory, those addressing interlaboratory comparison are scarce, in spite of the growing number of collaborative projects between partners from different leishmaniasis endemic and non-endemic countries. In this work we propose a protocol for interlaboratory comparison of conventional and real-time PCR methods involving four participant laboratories from four different endemic regions in four continents; the protocol includes a quality control step and reduces the variability among the samples tested by each participant. A panel of 77 samples from human origin and 9 from different parasite strains was blindly tested by the participants, aiming to assess the sensitivity of the different methods as well as their usefulness for species identification. Real-time PCR methods targeting the kDNA minicircles returned the highest sensitivity, while both PCR targeting ITS-1 and further HaeIII digestion and a combined algorithm including hsp70 PCR and restriction fragment length polymorphism analysis were the most appropriate approaches for species identification.

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.

Selection of endogenous reference genes for gene expression analysis in Leishmania major developmental stages.

At the era of post-genomics, gene expression analysis constitutes an important step for understanding the biological functions of genes. For this, reverse transcription and real-time polymerase chain reaction (RT-PCR) is one of the most accurate techniques available to date. Normalization with a proper internal control is critical for the generation of reliable results with biological significance. This is particularly true for pathogens, like Leishmania (L.) parasites, that alternate between different stages during their life cycle. In this study, we evaluate six different sequences for their potential as suitable internal control for the study of gene expression in three different developmental stages (procyclic and metacyclic promastigotes and amastigotes) of the parasite Leishmania major. Experiments were performed on RNA purified from three L. major isolates using the RT-PCR technique. Data analysis was performed using GeNorm and NormFinder programs. We could determine that a sequence encoding rRNA45 is the most stable in the three developmental stages of the parasite and can thus be used as a reference gene in gene expression studies in L. major.

Comparative Evaluation of Two Vaccine Candidates against Experimental Leishmaniasis Due to Leishmania major Infection in Four Inbred Mouse Strains

ABSATRCT Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.

Gene expression analysis of wild Leishmania major isolates: identification of genes preferentially expressed in amastigotes.

Trying to identify virulence genes of wild Leishmania (L.) major parasites, the species responsible for zoonotic cutaneous leishmaniasis, we compared, using differential display technique, gene expression in two L. major isolates obtained from human lesions and characterized by their contrasting pathogenicity in the BALB/c mouse model. The analysis was performed on amastigotes derived from BALB/c mice lesions. A total of 13 different clones were identified, but the use of reverse transcription and real-time polymerase chain reaction technique did not allow us to confirm any of these clones as differentially expressed. However, the fact that we used the amastigote stage of the parasite led us the identification of amastigote-specific genes, essentially (8 among 13). They are overexpressed, two to seven times, in amastigotes relative to promastigotes. Sequence analysis revealed that two of them namely LPG3 and the ATP dependent RNA helicase correspond to previously described amastigote-specific genes. The others correspond to genes involved in important biological process. Their better characterization could help the development of new drugs targeting the processes in which these molecules are involved.

Characterization of two different mucolipin-like genes from Leishmania major

Here, we report the existence of two different mucolipin-like genes in Leishmania parasites. The Leishmania major mucolipin-like A and B genes (lmmlA and lmmlB) encode two proteins of 776 and 590 amino acids, respectively, and may be classified among the mucolipins family [transient receptors potential mucolipin (TRPML)] because (1) they include a large region that exhibits significant similarities with specific domains of ion transport proteins and transient receptors potential (TRP) channels, (2) they contain at least 173 residues that display significant homologies with conserved regions of different mucolipins from several species, and (3) as TRPMLs, they include six predicted transmembrane domains. Gene expression analysis reveals that lmmlB is upregulated in metacyclics and amastigotes relative to procyclics, while lmmlA is constitutively expressed in the three Leishmania developmental stages. These genes could constitute potential drug targets.