Exploring an uncommon host-parasite interaction to unveil the uniqueness of Perkinsus sp as a model organism

We report the exploration of some unique metabolic pathways in Perkinsus olseni a marine protist parasite, responsible to significant mortalities in mollusks, especially in bivalves all around the world. In Algarve, south of Portugal carpet shell clam Ruditapes decussatus mortalities can reach up to 70%, causing social and economic losses. The objective of studying those unique pathways, is finding new therapeutic strategies capable of controlling/eliminating P. olseni proliferation in clams. In that sense metabolic pathways, were explored, and drugs affecting these cycles were tested for activity. The first step involved the identification of the genes behind those pathways, the reconstitution of the main steps, and molecular characterization of those genes and later on, the identification of possible targets within the genes studied. Metabolic cycles were screened due to the fact of not being present in host or differ in a critical way, such as the following pathways: shikimate, MEP-­‐ isoprenoids, Leloir cycle for chitin production, purine biosynthesis (unique among protists), the de novo synthesis of folates (absent in metazoa) and some unique genes like, the alternative oxidase (a branch of respiratory chain) and the hypoxia sensor HPH. All those pathways were covered and possible chemical inhibition using therapeutic drugs was tested with positive results. The relation between the common host Ruditapes decussatus and P. olseni was also explored in a dimension not possible some years ago. With the accessibility to second generation sequencers and microarray analysis platforms, genes involved in host defense or parasite virulence and resistance to the host were deciphered, allowing aiming to new targets (mechanisms and pathways), offering new possibilities for the control of Perkinsus in close environments. The thousands of genes, generated by this work, sequenced and analyzed from this commercial valuable clam and for Perkinsus olseni will be an important and value tool for the scientific community, allowing a better understanding of host-­‐parasite interactions, promoting the usage of P. olseni as an emerging model for alveolata parasites.

Toxoplasma gondii: host-parasite interaction and behavior manipulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

Background The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. Results A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed. Conclusions This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

Host-parasite interaction between crustaceans of six fish species from the Brazilian Amazon.

Host-parasite interactions between crustaceans and six fish species (Psectrogaster falcata, Ageneiosus ucayalensis, Acestrorhynchus falcirostris, Hemiodus unimaculatus, Serrasalmus gibbus and Geophagus proximus) from a reservoir in eastern Amazon, northern Brazil, were investigated. Eight hundred and seventy-eight parasites belonging to three crustacean species, Excorallana berbicensis, Argulus chicomendesi and Ergasilus turucuyus, which parasitized the hosts? mouth, gills and tegument, were collected from 295 fish and examined. High infestation levels were caused by E. berbicensis on the body surface of the hosts. Excorallana berbicensis showed aggregate dispersion, except in S. gibbus, while E. turucuyus showed random dispersion in A. falcirostris. The host?s sex did not influence infestation by E. berbicensis, and high parasitism failed to affect the body conditions of the fish. In the case of some hosts, rainfall rates, temperature, dissolved oxygen levels and water pH affected the prevalence and abundance of E. berbicensis, the dominant parasite species. Results revealed that the environment and life-style of the hosts were determining factors in infestations by parasites. Current assay is the first report on E. berbicensis for the six hosts, as well as on A. chicomendesi for G. proximus and P. falcata.

Light and electron microscope studies of host-parasite relations in a mycoparasite

Light microscope studies of the mycoparasite Piptocephalis virginiana revealed that the cylindrical spores of the parasite became spherical upon germination and produced 1-4 germ tubes. Generally t"l.vO germ tubes were produced by each spore. When this parasite was inoculated on its potential hosts, Choanephora cucurbitarum and Phascolomyces articulosus, the germ tube nearest to the host hypha continued to grow and made contact with the host hypha. The tip of the parasite's germ tube became swollen to form a distinct appressorium. Up to this stage the behavior of the parasite was similar regardless of the nature of the host. In the compatible host-parasite combination, the parasite penetrated the host, established a nutritional relationship and continued to grow to cover the host completely with its buff colored spores in 3-4 days. In the incompatible host-parasite combination, the parasite penetrated the host but its further advance was arrested. As a result of failure to establish a nutritional relationship with the resistant host, the parasite made further attempts to penetrate the host at different sites producing multiple infections. In the absence of nutrition the parasite weakened and the host outgrew the parasite completely. In the presence of a non-host species, Linderina pennispora the parasite continued to grow across the non-host 1).yp_hae vlithout establishing an initial contact. Germination studies showed that the parasite germinated equally well in the presence of host and non-host species. Further electron microscope studies revealed that the host-parasite interaction between P. virginiana and its host, C. cucurbi tarum, was compatible when the host hyphae were young slender, with a thin cell wall of one layer. The parasite appeared to penetrate mechanically by pushing the host-cell wall inward. The host plasma membrane invaginated along the involuted cell wall. The older hyphae of C. cucurbitarum possessed two distinct layers of cell wall and-showed an incompatible interaction when challenged vlith the parasite. At the point of contact, the outer layer of the host-cell wall dissolved, probably by enzymatic digestion, and the inner layer became thickened and developed a papilla as a result of its response to the parasite. The haustoria of the parasite in the old hyphae were always surrounded by a thick, well developed sheath, whereas the haustoria of the same age in the young host mycelium were devoid of a sheath during early stages of infection. Instead, they were in direct contact with the host protoplast. The incompatible interaction between a resistant host, P. articulosus and the parasite showed similar results as with the old hyphae of C. cucurbitarum. The cell wall of P. articulosus appeared thick-with two or more layers even in the 18-22 h-old hyphae. No contact or interaction was established between the parasite and the non-host L. pennispora. The role of cell wall in the resistance mechanism is discussed.

Host-parasite relationships in paracoccidioidomycosis

A viewpoint of host-parasite relationships in paracoccidioidomycosis is presented. The characteristics of the fungus which are important to the host-parasite interaction are discussed. Aspects of inhibition of mycelium-to-yeast transformation by estrogens acting at receptors on the fungal wall and in the cytoplasm, and the role of polysaccharide components of the cell wall in virulence are reviewed. The natural mechanisms of host defense are also examined, including phagocytosis, complement system, natural-killer cells and genetic control of resistance and susceptibility. Finally, a discussion of granuloma morphogenesis and its relationship to the humoral and cellular anti-P. brasiliensis immune response is presented.

Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Characterization of the chitinolytic system during the mycoparasitic interaction between Trichoderma aggressivum f. aggressivum and different host strains of Agaricus bisporus /

Green mould is a serious disease of commercially grown mushrooms, the causal agent being attributed to the filamentous soil fungus Triclzodenna aggressivum f. aggressivu11l and T. aggressivum f. ellropaellm. Found worldwide, and capable of devastating crops, this disease has caused millions of dollars in lost revenue within the mushroom industry. One mechanism used by TricllOdenlla spp. in the antagonism of other fungi, is the secretion of lytic enzymes such as chitinases, which actively degrade a host's cell wall. Therefore, the intent of this study was to examine the production of chitinase enzymes during the host-parasite interaction of Agaricus bisporus (commercial mushroom) and Triclzodemza aggressivum, focusing specifically on chitinase involvement in the differential resistance of white, off-white, and brown commercial mushroom strains. Chitinases isolated from cultures of A. bisporus and T. aggressivu11l grown together and separately, were identified following native PAGE, and analysis of fluorescence based on specific enzymatic cleavage of 4-methylumbelliferyl glucoside substrates. Results indicate that the interaction between T. aggressivulll and A. bisporus involves a complex enzyme battle. It was determined that T. aggressivum produces a number of chitinases that appear to correlate to those isolated in previous studies using biocontrol strains of T. Izarziallilm. A 122 kDa N-acetylglucosaminidase of T. aggressivu11l revealed the highest and most variable activity, and is therefore believed to be an important predictor of antifungal activity. Furthermore, results indicate that brown strain resistance of mushrooms may be related to high levels of a 96 kDa N-acetylglucosaminidase, which showed elevated activity in both solitary and dual cultures with T. aggressivum. Overall, each host-parasite combination produced unique enzyme profiles, with the majority of the differences seen between day 0 and day 6 for the extracellular chitinases. Therefore, it was concluded that the antagonistic behaviour of T. aggressivli1ll does not involve a typical response, always producing the same types and levels of enzymes, but that mycoparasitism, specifically in the form of chitinase production, may be induced and regulated based on the host presented.

Pathogenesis II: Fungal responses to host responses: interaction of host cells with fungi

Most of our knowledge concerning the virulence determinants of pathogenic fungi comes from the infected host, mainly from animal models and more recently from in vitro studies with cell cultures. The fungi usually present intra- and/or extracellular host-parasite interfaces, with the parasitism phenomenon dependent on complementary surface molecules. Among living organisms, this has been characterized as a cohabitation event, where the fungus is able to recognize specific host tissues acting as an attractant, creating stable conditions for its survival. Several fungi pathogenic for humans and animals have evolved special strategies to deliver elements to their cellular targets that may be relevant to their pathogenicity. Most of these pathogens express surface factors that mediate binding to host cells either directly or indirectly, in the latter case binding to host adhesion components such as extracellular matrix (ECM) proteins, which act as 'interlinking' molecules. The entry of the pathogen into the host cell is initiated by fungal adherence to the cell surface, which generates an uptake signal that may induce its cytoplasmic internalization. Once this is accomplished, some fungi are able to alter the host cytoskeletal architecture, as manifested by a rearrangement of microtubule and microfilament proteins, and this can also induce epithelial host cells to become apoptotic. It is possible that fungal pathogens induce modulation of different host cell pathways in order to evade host defences and to foster their own proliferation. For a number of pathogens, the ability to bind ECM glycoproteins, the capability of internalization and the induction of apoptosis are considered important factors in virulence. Furthermore, specific recognition between fungal parasites and their host cell targets may be mediated by the interaction of carbohydrate-binding proteins, e.g., lectins on the surface of one type of cell, probably a parasite, that combine with complementary sugars on the surface of host-cell. These interactions supply precise models to study putative adhesins and receptor-containing molecules in the context of the fungus-host interface. The recognition of the host molecules by fungi such as Aspergillus fumigatus, Paracoccidioides brasiliensis and Histoplasma capsulatum, and their molecular mechanisms of adhesion and invasion, are reviewed in this paper.

Mistletoes Play Different Roles in a Modular Host-Parasite Network

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mistletoes Play Different Roles in a Modular Host-Parasite Network

Antagonistic interactions between host plants and mistletoes often form complex networks of interacting species. Adequate characterization of network organization requires a combination of qualitative and quantitative data. Therefore, we assessed the distribution of interactions between mistletoes and hosts in the Brazilian Pantanal and characterized the network structure in relation to nestedness and modularity. Interactions were highly asymmetric, with mistletoes presenting low host specificity (i.e., weak dependence) and with hosts being highly susceptible to mistletoe-specific infections. We found a non-nested and modular pattern of interactions, wherein each mistletoe species interacted with a particular set of host species. Psittacanthus spp. infected more species and individuals and also caused a high number of infections per individual, whereas the other mistletoes showed a more specialized pattern of infection. For this reason, Psittacanthus spp. were regarded as module hubs while the other mistletoe species showed a peripheral role. We hypothesize that this pattern is primarily the result of different seed dispersal systems. Although all mistletoe species in our study are bird dispersed, the frugivorous assemblage of Psittacanthus spp. is composed of a larger suite of birds, whereas Phoradendron are mainly dispersed by Euphonia species. The larger assemblage of bird species dispersing Psittacanthus seeds may also increase the number of hosts colonized and, consequently, its dominance in the study area. Nevertheless, other restrictions on the interactions among species, such as the differential capacity of mistletoe infections, defense strategies of hosts and habitat types, can also generate or enhance the observed pattern.

Characterization of the fetuin-binding fraction of Neospora caninum tachyzoites and its potential involvement in host-parasite interactions

Terminal sialic acid residues on surface-associated glycoconjugates mediate host cell interactions of many pathogens. Addition of sialic acid-rich fetuin enhanced, and the presence of the sialidiase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reduced, the physical interaction of Neospora caninum tachyzoites and bradyzoites with Vero cell monolayers. Thus, Neospora extracts were subjected to fetuin-agarose affinity chromatography in order to isolate components potentially interacting with sialic acid residues. SDS-PAGE and silver staining of the fetuin binding fraction revealed the presence of a single protein band of approximately 65 kDa, subsequently named NcFBP (Neospora caninum fetuin-binding protein), which was localized at the apical tip of the tachyzoites and was continuously released into the surrounding medium in a temperature-independent manner. NcFBP readily interacted with Vero cells and bound to chondroitin sulfate A and C, and anti-NcFBP antibodies interfered in tachyzoite adhesion to host cell monolayers. In additon, analysis of the fetuin binding fraction by gelatin substrate zymography was performed, and demonstrated the presence of two bands of 96 and 140 kDa exhibiting metalloprotease-activity. The metalloprotease activity readily degraded glycosylated proteins such as fetuin and bovine immunoglobulin G heavy chain, whereas non-glycosylated proteins such as bovine serum albumin and immunoglobulin G light chain were not affected. These findings suggest that the fetuin-binding fraction of Neospora caninum tachyzoites contains components that could be potentially involved in host-parasite interactions.

Proteins mediating the Neospora caninum-host cell interaction as targets for vaccination

Neospora caninum is an apicomplexan parasite that is capable of infecting, a wide range of tissues. The fact that Neospora represents an important abortion-causing parasite in cattle has transformed neosporosis research from an earlier, rather esoteric field, to a significant research topic, and considerable investments have been made in the last years to develop an efficacious vaccine or other means of intervention that would prevent infection and abortion due to N. caninum infection in cattle. Antigenic molecules associated with proteins involved in adhesion/invasion or other parasite-host-cell interaction processes can confer protection against Neospora caninum infection, and such proteins represent valuable targets for the development of a vaccine to limit economical losses due to neosporosis. Although not ideal, small laboratory animal models that mimic cerebral infection, acute disease and fetal loss upon infection during pregnancy have been used for the assessment of vaccine candidates, in parallel with studies on experimental infections in cattle. Herein, we review and critically assess these vaccination approaches and discuss potential options for improvements.

A blowfly strike vaccine requires an understanding of host-pathogen interactions

The phase-out of Mulesing by 2010 means the Australian wool industry requires immediate and viable alternatives for the control and prevention of blowfly strike, an economically important parasitic disease of sheep. In this review we have analysed previous research aimed toward the development of a vaccine against blowfly strike and the reasons why the approaches taken were unsuccessful at the time. Close scrutiny has provided new insight into this host-parasite interaction and identified new opportunities for the development of a vaccine. Here we propose that addressing immunosuppression together with the induction of cellular immunity is likely to result in an anti-blowfly strike vaccine, as opposed to the use of "standard" approaches aimed at inducing humoral immunity.