Application of magnetically induced hyperthermia in the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections

Background: Magnetic hyperthermia is currently a clinical therapy approved in the European Union for treatment of tumor cells, and uses magnetic nanoparticles (MNPs) under time-varying magnetic fields (TVMFs). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, given that the therapeutic drugs available have severe side effects and that there are drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe3O4 MNPs in order to provoke cell death remotely using TVMFs. Methods: Iron oxide MNPs with average diameters of approximately 30 nm were synthesized by precipitation of FeSO4 in basic medium. The MNPs were added to C. fasciculata choanomastigotes in the exponential phase and incubated overnight, removing excess MNPs using a DEAE-cellulose resin column. The amount of MNPs uploaded per cell was determined by magnetic measurement. The cells bearing MNPs were submitted to TVMFs using a homemade AC field applicator (f = 249 kHz, H = 13 kA/m), and the temperature variation during the experiments was measured. Scanning electron microscopy was used to assess morphological changes after the TVMF experiments. Cell viability was analyzed using an MTT colorimetric assay and flow cytometry. Results: MNPs were incorporated into the cells, with no noticeable cytotoxicity. When a TVMF was applied to cells bearing MNPs, massive cell death was induced via a nonapoptotic mechanism. No effects were observed by applying TVMF to control cells not loaded with MNPs. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. Conclusion: As a proof of principle, these data indicate that intracellular hyperthermia is a suitable technology to induce death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies combating parasitic infection.

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

Abstract Background In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.

Highlights of the XXI annual meeting of the Brazilian Society of Protozoology, the XXXII annual meeting on Basic Research in Chagas' disease & an international symposium on vesicle trafficking in parasitic Protozoa – 7 to 9 November 2005, Caxambu, Minas Gerais, Brazil

This report focuses on the 2005 Annual meeting held in Caxambu, Minas Gerais, Brazil that was convened and organized by the Brazilian Society of Protozoology http://www.sbpz.org.br/. This is an annual event and details of these meetings can be found on the Society's website. Within the space available it has been impossible to cover all the important and fascinating contributions and what is presented are our personal views of the meetings scientific highlights and new developments. The contents undoubtedly reflect each author's scientific interests and expertise. Fuller details of the round tables, seminars and posters can be consulted on line at http://www.sbpz.org.br/livroderesumos2005.php.

Nematicidal effect of volatile organic compounds (VOCs) on the plant-parasitic nematode Meloidogyne javanica

Previous studies have demonstrated that volatile organic compounds (VOCs), produced by the yeast Saccharomyces cerevisiae, were able to inhibit the development of phytopathogenic fungi. In this context, the nematicidal potential of the synthetic mixture of VOCs, constituted of alcohols and esters, was evaluated for the control of the root-knot nematode Meloidogyne javanica, which causes losses to crops of high economic value. The fumigation of substrate containing second-stage juveniles with VOCs exhibited nematicidal effect higher than 30% for the lowest concentration tested (33.3 µL g-1 substrate), whereas at 66.6 and 133.3 µL g-1 substrate, the nematode mortality was 100%. The present results stimulate other studies on VOCs for nematode management.

Innate Immunity in Insects, Function and Regulation of Hemolin from Hyalophora cecropia

Insects are useful models for the study of innate immune reactions and development. The distinction between recognition mechanisms preceding the breakdown of apoptotic cells during metamorphosis, and the breakdown of cells in response to infections, is unclear. Hemolin, a Lepidopteran member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and hemolin gene regulation at a molecular level. We investigated the binding and cell adhesion properties of hemolin from H. cecropia and demonstrated that the proteins could homodimerize in presence of calcium. Moreover, a higher molecular weight membrane form of hemolin was present on hemocytes. These results, taken together with an earlier finding that soluble hemolin inhibits hemocyte adhesion, indicated that the secreted hemolin could modulate hemocyte aggregation in a competitive manner in the blood. In addition, hemolin was expressed in different tissues and at different developmental stages. Since hemolin is expressed both during development and during the immune response, its different regulatory factors must act in concert. We found that the third intron contains an enhancer, through which Dif, C/EBP and HMGI synergistically activate a reporter construct in vitro. We concluded that the enhancer is used during infection, since the κB-site is crucial for an immune response. Interestingly, we also found that the active form of the steroid hormone, ecdysone, induces the hemolin gene transcription in vivo, and in addition, acts synergistically during bacterial infection. Preliminary in vivo results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of hemolin. To explore the use of Drosophila as a genetic tool for understanding hemolin function and regulation, we sought to isolate the functional homologue in this species. A fly cDNA library in yeast was screened using H. cecropia hemolin as bait. The screen was not successful. However, it did lead to the discovery of a Drosophila protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named yippee. Yippee is distantly related to zinc finger proteins and represents a novel family of proteins present in numerous eukaryotes, including fungi, plants and humans. Notably, when the Drosophila genome sequence was revealed, no hemolin orthologue could be detected. Finally, an extensive Drosophila genome chip analysis was initiated. The goal was to investigate the Drosophila immune response, and, in contrast to earlier studies of artificially injected flies, to examine a set of natural microbes, orally and externally applied. In parallel experiments viruses, bacteria, fungi and parasites were compared to unchallenged controls. We obtained a unique set of genes that were up-regulated in the response to the parasite Octosporea muscadomesticae and to the fungus Beauveria bassiana. We expect both down-regulated and up-regulated genes to serve as a source for the discovery of new effector molecules, in particular those that are active against parasites and fungi.

New associations between native parasitoids and exotic insects introduced in Italy and in Europe

The introduction of exotic species is one of the most important threats to biodiversity.This phenomenon may cause economic and environmental damage. To prevent these invasions there are institutions like EPPO. Nevertheless, the introduction of exotic pests is an increasing issue, difficult to control. Classic biological control, based on importation of natural enemies from the country of origin, has been successfully used for over 120 years, but it has also raised some criticism. My research work has focused on the study of the new associations occurring between indigenous parasitoids and three exotic pests introduced in Italy and Europe. The three target insects considered were: Cacyreus marshalli Butler (Lepidoptera: Lycaenidae), a pest of Geranium plants; Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), a plague of Castanea sp. and Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). This ladybug has been introduced as a biological control agent, but since some years it considered as an invasive species. For C. marshalli I performed laboratory tests on acceptance and suitability of immature stages of this butterfly by Exorista larvarum (Diptera: Tachinidae) and Brachymeria tibialis (Hymenoptera: Chalcidicae). The experiments showed that these two parasitoids could be used to contain this pest. For D. kuriphilus I performed field samplings in an infested chestnut area, the samples were maintained in rearing chamber until gall wasp or parasitoids emergence. In the 3-year research many parasitoids of gall wasps were found; one of these, Torymus flavipes (Walker), was found in large number. For H. axyridis the research work included a first phase of field sampling, during which I searched indigenous parasitoids which had adapted to this new host; the only species found was Dinocampus coccinellae (Schrank) (Hymenoptera: Braconidae). Laboratory tests were performed on the wasp rearing, biology and capacity to contain H. axyridis.

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

Background Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences. Results About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein. An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins. Conclusions The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of C. inanitus appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.

Comparative genomics of metabolic networks of free-living and parasitic eukaryotes

Background Obligate endoparasites often lack particular metabolic pathways as compared to free-living organisms. This phenomenon comprises anabolic as well as catabolic reactions. Presumably, the corresponding enzymes were lost in adaptation to parasitism. Here we compare the predicted core metabolic graphs of obligate endoparasites and non-parasites (free living organisms and facultative parasites) in order to analyze how the parasites' metabolic networks shrunk in the course of evolution. Results Core metabolic graphs comprising biochemical reactions present in the presumed ancestor of parasites and non-parasites were reconstructed from the Kyoto Encyclopedia of Genes and Genomes. While the parasites' networks had fewer nodes (metabolites) and edges (reactions), other parameters such as average connectivity, network diameter and number of isolated edges were similar in parasites and non-parasites. The parasites' networks contained a higher percentage of ATP-consuming reactions and a lower percentage of NAD-requiring reactions. Control networks, shrunk to the size of the parasites' by random deletion of edges, were scale-free but exhibited smaller diameters and more isolated edges. Conclusions The parasites' networks were smaller than those of the non-parasites regarding number of nodes or edges, but not regarding network diameters. Network integrity but not scale-freeness has acted as a selective principle during the evolutionary reduction of parasite metabolism. ATP-requiring reactions in particular have been retained in the parasites' core metabolism while NADH- or NADPH-requiring reactions were lost preferentially.

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-d- and all-l-cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of l-cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

Identification of a host cell target for the thiazolide class of broad-spectrum anti-parasitic drugs

The thiazolide nitazoxanide (NTZ) and some derivatives exhibit considerable in vitro activities against a broad range of parasites, including the apicomplexans Neospora caninum and Toxoplasma gondii tachyzoites. In order to identify potential molecular targets for this compound in both parasites, RM4847 was coupled to epoxy-agarose and affinity chromatography was performed. A protein of approximately 35 kDa was eluted upon RM4847-affinity-chromatography from extracts of N. caninum-infected human foreskin fibroblasts (HFF) and non-infected HFF, but no protein was eluted when affinity chromatography was performed with T. gondii or N. caninum tachyzoite extracts. Mass spectrometry analysis identified the 35 kDa protein as human quinone reductase NQO1 (P15559; QR). Within 8h after infection of HFF with N. caninum tachyzoites, QR transcript expression levels were notably increased, but no such increase was observed upon infection with T. gondii tachyzoites. Treatment of non-infected HFF with RM4847 did also lead to an increase of QR transcript levels. The enzymatic activity of 6-histidine-tagged recombinant QR (recQR) was assayed using menadione as a substrate. The thiazolides NTZ, tizoxanide and RM4847 inhibited recQR activity on menadione in a concentration-dependent manner. Moreover, a small residual reducing activity was observed when these thiazolides were offered as substrates.

The use of light- and electron microscopy for studies on the cell- and molecular biology of parasites and parasitic diseases

Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host.

Travel medicine of parasitic diseases in the dog

Companion animals are increasingly brought along by their owners to foreign countries. Thus, small animal travel medicine is becoming more important. The field includes both prophylaxis and metaphylaxis against various infectious diseases, as well as their diagnosis and treatment. Dogs returning from Southern Europe, but also from more tropical regions, may be infected with exotic pathogens. In addition, imported pedigree or working dogs, and especially stray dogs imported through welfare organisations, are at high risk.The present overview summarises the clinical and practical aspects of exotic parasitic diseases that may affect such dogs, and the risk of such diseases becoming autochthonously transmitted in Switzerland. Furthermore, the zoonotic potential of these infections will be considered.