Denis Sereno

Induction of a Peptide with Activity against a Broad Spectrum of Pathogens in the Aedes aegypti Salivary Gland, following Infection with Dengue Virus

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquitos salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vectors innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.

A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies

Background The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Methodology and Principal Findings Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? Conclusions and Significance We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

SIR2-Deficient Leishmania infantum Induces a Defined IFN-γ/IL-10 Pattern That Correlates with Protection

The ability to manipulate the Leishmania genome to create genetically modified parasites by introducing or eliminating genes is considered a powerful alternative for developing a new generation vaccine against leishmaniasis. Previously, we showed that the deletion of one allele of the Leishmania infantum silent information regulatory 2 (LiSIR2) locus was sufficient to dramatically affect amastigote axenic proliferation. Furthermore, LiSIR2 single knockout (LiSIR2+/−) amastigotes were unable to replicate in vitro inside macrophages. Because this L. infantum mutant persisted in BALB/c mice for up to 6 wk but failed to establish an infection, we tested its ability to provide protection toward a virulent L. infantum challenge. Strikingly, vaccination with a single i.p. injection of LiSIR2+/− single knockout elicits complete protection. Thus, vaccinated BALB/c mice showed a reversal of T cell anergy with specific anti-Leishmania cytotoxic activity and high levels of NO production. Moreover, vaccinated mice simultaneously generated specific anti-Leishmania IgG Ab subclasses suggestive of both type 1 and type 2 responses. A strong correlation was found between the elimination of the parasites and an increased Leishmania-specific IFN-γ/IL-10 ratio. Therefore, we propose that the polarization to a high IFN-γ/low IL-10 ratio after challenge is a clear indicator of vaccine success. Furthermore these mutants, which presented attenuated virulence, represent a good model to understand the correlatives of protection in visceral leishmaniasis.

Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death.

The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man. In previous studies, we reported the characterization of a Leishmania major gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2. Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm. In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmania infantum, revealed 93% overall amino acid identity with L. major SIR2 gene. Further, using L. infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions. This phenotype was also observed when L. infantum parasites were transfected with a cosmid vector (CLHyg), isolated from a L. infantum cosmid library, carrying the L. infantum SIR2 gene (CLHyg-LiSIR2). In contrast, no effect was observed on survival of the promastigote forms (insect stage) under similar culture conditions. However, when the glucose was used as a unique source of energy under starvation conditions, the viability of promastigotes was significantly enhanced. Moreover, we showed that amastigote forms in the stationary phase of culture died with a feature of apoptosis as revealed by the appearance of YOPRO-1 positive cells and that expression of LmSIR2 protein substantially delays this phenomenon. Taken together, these results demonstrate the existence of SIR2-related proteins encoding genes in different Leishmania species and suggest that LmSIR2p could participate among other factors in the control of cell death.

Use of an enzymatic micromethod to quantify amastigote stage of Leishmania amazonensis in vitro

Leishmaniasis is a significant cause of morbidity and mortality in tropical and subtropical areas. These protozoan parasites exist as a flagellate extracellular promastigote form in their sandfly vectors. In the mammalian hosts, only the nonflagellate amastigote form persists, surviving and dividing in the phagolysosome of macrophages. Leishmania promastigotes have often been quantified in vitro by an enzymatic method that involves the conversion of a tetrazolium salt, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), into a colored, insoluble formazan product, the amount of which depends on the number of viable parasites present (Mossman 1983; Cole 1986; Rabinovitch et al. 1986; Berg et al. 1994). The use of amastigote forms has been hampered by difficulties in obtaining sufficient amounts of the purified mammalian stage. Toward that end, we and other investigators have recently developed culture systems for the in vitro growth of large amounts of extracellular amastigote forms that are free of host-cell contamination (Bates 1993; Lemesre, patent PCT/FR 94/00577). In the present paper we report the use of an MTT-based microassay to quantify the proliferation and the viability of axenically grown amastigote forms of L. amazonensis. The efficiency of the method was compared with that of another conventional method, cell counting. A general procedure was used in the current study to generate large quantities of each of the parasite stages. Active and dividing populations of axenically grown amastigote forms of L. amazonensis (MHOM/BR/76/ LTB-012) were maintained at 32 ± 1 °C by weekly subpassage in a cell-free medium called MAA (medium for axenically grown amastigotes) in 25-cm flasks (Lemesre et al. 1994; Lemesre, patent PCT/FR 94/00577). From a starting inoculum of 5 × 10 amastigote forms/ ml a cell density of about 5 × 10 parasites/ml was obtained on day 7. Promastigote cultures were maintained at 25 ± 1 °C in RPMI 1640 medium (Gibco BRL) buffered with 25 mM HEPES and 2 mM NaHCO3 (pH 7.2) and supplemented with 20% heat-inactivated fetal calf serum (FCS). Initial parasite concentrations were 5 × 10 flagellates/ml of medium. For verification of the correlation between the MTT-based assay and the number of parasites, increasing concentrations of latelog-phase parasites, ranging from 2.5 to 40 × 10 cells/ well, were distributed in triplicate in 100 ll of media in 96-well flat-bottom microtrays. Plates were then incubated with 10 ll of MTT (10 mg/ml) for 4 h at 25° or 32 ± 1 °C, depending upon the parasite stage. The enzyme-substrate reaction was stopped by the addition of 100 ll of a solution of 50% isopropanol and 10% sodium dodecyl sulfate (SDS; pH 5.4). Microplates were further incubated for 30 min at room temperature. The optical density (OD) was determined at 570 nm with a Titertech 96-well scanner. To evaluate the possibility of using the MTT-based assay to achieve growth kinetics curves as is possible with cell-counting experiments, microorganisms were seeded at two inocula (1 and 2 × 10 parasites/well) and the dehydrogenase activities were measured daily as described above. Cell concentrations were determined by daily counting of parasites in a Thoma chamber at 400x magnification after adequate dilution in 0.01 M phosphate-buffered saline (PBS, pH 7.2). Morphological aspects of parasites were determined daily by microscope examination to detect the possible occurrence of differentiation phenomena in 96well plates. As shown in Fig. 1, for densities higher than or equal to 2.5 × 10 parasites/well a linear correlation between the OD signal and the parasite number was revealed when both amastigote and promastigote forms were used. Amastigote forms of L. amazonensis showed lower levels of dehydrogenase activities than did their corresponding promastigote forms. This difference was Parasitol Res (1997) 83: 401–403 uf6d9 Springer-Verlag 1997

Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica §

Temporins are small antimicrobial peptides isolated from North American and Eurasian ranid frogs that are particularly active against Gram-positive bacteria. To date, no temporins have been characterized from North African frog species. We isolated three novel members of the temporin family, named temporin-1Sa (FLSGIVGMLGKLF(amide)), -1Sb (FLPIVTNLLSGLL(amide)), and -1Sc (FLSHIAGFLSNLF(amide)), from the skin of the Sahara frog Pelophylax (Rana) saharica originating from Tunisia. These temporins were identified by a combined mass spectrometry/molecular cloning approach. Temporin-1Sa was found to be highly active against Gram-positive and Gram-negative bacteria, yeasts and fungi (MIC=2-30 microM). To our knowledge, this is the first 13-residue member of the temporin family with a net charge of +2 that shows such broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC=10 microM) and Pseudomonas aeruginosa (MIC=31 microM). Moreover, temporin-1Sa displays significant antiparasitic activity (IC50 approximately 20 microM) against the promastigote and amastigote stages of Leishmania infantum.

Leishmania major: Cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family

In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (eg logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti‐LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [35S]methionine‐labeled promastigote antigens after pulse chase experiments, using anti‐LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted‐secreted antigens (ESA). Moreover, immunoflurescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

Background The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011–2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. Methods/Principal Findings A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. Conclusions Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

Efficacy of second line drugs on antimonyl-resistant amastigotes of Leishmania infantum.

In a previous paper we have demonstrated that the induction, by direct drug pressure, of a resistance to Sb(III) antimony at physiological concentration in the amastigote stage of the parasite, led to a high cross-resistance to Sb(V) species in the form of Glucantime. In this paper, further chemoresistant clones were characterized. Axenic amastigotes of Leishmania infantum were adapted to survive in culture medium containing 4, 20, 30 and 120 microg/ml of potassium antimonyl tartrate Sb(II). These mutants were 12, 28, 35 and 44-fold more resistant to Sb(III) than the parental wild-type clone. They were able to resist at concentrations of Glucantime Sb(V) as high as 160 microg/ml when growing in THP-1 cells. We have investigated the efficacy of second line drugs in clinical use (pentamidine and amphotericin B) on the antimony-resistant mutants. Amphotericin B was toxic for both wild-type and chemoresistant mutants at concentrations ranging from 0.05 to 0.15 microM. Pentamidine which is extensively used when the first course of antimonial pentavalent compounds is unsuccessful, was more toxic for all the chemoresistant organisms than for the wild-type clone. In the same way, chemoresistant amastigotes growing within THP-1 cells were more susceptible to pentamidine than the wild-type clone. Our results showed that the resistance of the mutants was restricted to the antimony containing drugs and did not led to a cross-resistance against the other clinically relevant drugs. These results confirmed that these two drugs (pentamidine and amphotericin B) are good candidates to treat pentavalent antimonial unresponsiveness.

The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation

NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics‐based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown‐like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add‐back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target.