Synthesis, biological characterisation and structure activity relationships of aromatic bisamidines active against Plasmodium falciparum.

Malaria is one of the most significant tropical diseases and remains a major challenge due to the lack of a broadly effective vaccine and parasite resistance to current drugs. This means there is a need for new drug candidates with novel modes of action. Aromatic bisamidines, such as furamidine (DB75), were initially developed as anti-Trypanosoma agents however as clinical trials with furamidine highlighted potential side effects they were not pursued further in that setting. Despite apparent cytotoxicity liabilities the potency of furamidine against Plasmodium falciparum makes it a promising scaffold for the development of new anti-Plasmodium agents with improved selectivity. In this study a bisamidine compound series based on furamidine was synthesized by introducing modifications at the furan core structure and terminal amidine groups. The activity of the derived compounds was tested in vitro against drug sensitive and resistant P. falciparum lines and a human cell line (HEK293 cells) to generate anti-Plasmodium structure-activity relationships and to provide preliminary selectivity data.

The compatibiuty polymorphism in invertebrate host/trematodes interactions: research of molecular determinants.

The co-evolutionary dynamics that exist in many host-parasite interactions sometimes leads to compatibility polymorphism. This phenomenon is well documented in mollusc/trematodes interactions but its molecular base is unknown. In order to identify key molecules involved in this phenomenon, we developed several molecular approaches comparing compatible or incompatible strains of mollusc or parasite. These comparisons led to the identification of numerous candidate genes listed and discussed (some of them) in the present review.

Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: new candidate genes evidenced by a suppressive subtractive hybridization approach.

In order to elucidate mechanisms underlying snail/echinostome compatibility, numerous molecular studies comparing transcripts and proteins of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni were undertaken. These studies focused on plasma and haemocytes of the two strains and revealed that some transcripts and/or proteins were differentially expressed between strains. The aim of the present study was to develop a complementary transcriptomic approach by constructing subtractive libraries. This work revealed some candidate transcripts already identified in previous studies (calcium-binding proteins and glycolytic enzymes) as well as novel candidate transcripts that were differentially represented between strains of B. glabrata. Among these newly identified genes, we revealed several genes potentially involved in immune processes encoding proteases, protease inhibitors, a lectin, an aplysianin-like molecule, and cell adhesion molecules.

Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: Potential involvement of proteins from hemocytes revealed by a proteomic approach.

As an approach to investigate the suspected involvement of cellular factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns from hemocytes collected from susceptible and resistant snails. This proteomic approach revealed that twelve hemocytic proteins exhibited significant differences in their apparent abundance. The genes corresponding to five of them were characterized by a combination of mass spectrometry and molecular cloning. They encode an aldolase, an intermediate filament protein, a cytidine deaminase, the ribosomal protein P1 and the histone H4. Furthermore, we investigated their expression in parasite-exposed or -unexposed snails. These last experiments revealed changes in transcript levels corresponding to intermediate filament and histone H4 proteins post-infection.

Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: potential involvement of adhesion genes.

Because susceptibility or resistance of Biomphalaria glabrata to the trematode Echinostoma caproni correlates with differential hemocytic adhesive properties, we compared the expression of genes involved in adhesion processes between hemocytes from susceptible and resistant snails. Quantitative reverse transcriptase-PCR analysis revealed four genes whose transcripts were differentially represented between hemocytes from resistant and susceptible snails. These genes encode two dermatopontin-like, one matrilin-like and one cadherin-like proteins. Expression analyses performed following parasite exposure suggested that dermatopontins may be involved in the compatibility differences between these strains. We also investigated expression levels on whole snails of different genes potentially involved in extracellular matrix structure or coagulation. Our results support the hypothesis that susceptible snails possess a hemolymph coagulation-like system that is more potent than that of resistant snails. This system may prevent hemocyte migration towards the parasite larvae and therefore facilitate parasite settlement in susceptible snails.

Gene discovery and expression analysis of immune-relevant genes from Biomphalaria glabrata hemocytes.

The immune effector cells (hemocytes) of the snail host Biomphalaria glabrata are known to play a key role in recognition and elimination of larval helminths such as the human blood fluke Schistosoma mansoni. To identify novel immune-relevant genes, we undertook an expressed sequence tag program. A hemocyte cDNA library was constructed using snails that were not exposed to a particular pathogen or parasite but maintained in non-axenic conditions. Putative function could be assigned to 53% of the 1613 high quality cDNAs analysed. Based on sequence similarities, we identified 31 immune-relevant genes corresponding either to cellular defence effectors, proteases and protease inhibitors, pattern recognition receptors, cell adhesion molecules or immune regulators. In order to further investigate the potential involvement of these genes in snail-trematode immunobiological interactions, we analysed their expression in unchallenged and parasite-challenged snails, using the immunosuppressive trematode Echinostoma caproni and snail strains selected for resistance or susceptibility to this parasite. Real-time PCR analysis of expression ratios at 7 time-points post-exposure revealed both (i) genes displaying constitutive expression differences between the two strains; and (ii) genes differentially modulated after parasite exposure.

Characterisation of proteins differentially present in the plasma of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni.

Snail immune responses towards a trematode infection are known to rely on both plasmatic and cellular host factors. As an approach to further investigate the suspected involvement of plasmatic factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns of plasma collected from susceptible and resistant snails. This proteomic approach revealed that 13 plasmatic proteins exhibited significant differences in their apparent representativity. The genes corresponding to five of them were characterised by a combination of mass spectrometry and molecular cloning. They encode two isoforms of a glycolytic enzyme, two isoforms of a calcium binding protein and an inhibitor of cysteine protease. Furthermore, we investigated gene expression in parasite-exposed or -unexposed snails as well as in various tissues by quantitative PCR. This study showed that: (i) differential representation of plasma proteins between the snail strains was correlated with a differential level of transcripts; (ii) expression of these genes after parasite exposure was differentially regulated in the two strains; and (iii) these genes were expressed predominantly in the albumen gland.