KSAYMRFAMIDE - A NOVEL FMRFAMIDE-RELATED HEPTAPEPTIDE FROM THE FREE-LIVING NEMATODE, PANAGRELLUS-REDIVIVUS, WHICH IS MYOACTIVE IN THE PARASITIC NEMATODE, ASCARIS-SUUM

In nematodes, FMRFamide-related peptides (FaRPs) have been structurally characterised from the parasite, Ascaris suum, and from two free-living species, Panagrellus redivivus and Caenorhabditis elegans. While both FaRPs isolated from P. redivivus (PF1 and PF2) have been identified in C. elegans the two heptapeptides isolated from A. suum (AF1 and AF2) have until recently been considered unique to this parasitic species. We have recently isolated AF2 from P. redivivus and, during this study, an additional novel heptapeptide amide, Lys-Ser-Ala-Tyr-Met-Arg-Phe amide (KSAYMRFamide), was structurally characterised. A synthetic replicate of this peptide induced a rapid concentration-dependent muscle tension increase in an isolated A. suum somatic muscle preparation, with a threshold of approximately 0.1 mu M. These data suggest that the complement of FaRPs in parasitic and free-living nematodes may not be as radically different as preliminary studies would suggest, and that the absence of AF1, AF2 and KSAYMRFamide on the C. elegans FMRFamide-related peptide gene (flp-1) may imply the presence of at least two different FaRP genes in nematodes. (C) 1994 Academic Press, Inc.

Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

Serological correlate of protection against norovirus-induced gastroenteritis

Norovirus infection is the leading cause of acute nonbacterial gastroenteritis. Histoblood group antigens (HBGAs) are host susceptibility determinants for Norwalk virus (NV) infection. We hypothesized that antibodies that block NV-HBGA binding are associated with protection from clinical illness following NV exposure.

Transcriptome analysis of a parasitic clade V nematode:comparative analysis of potential molecular anthelmintic targets in Cylicostephanus goldi

Clade V nematodes comprise several parasitic species that include the cyathostomins, primary helminth pathogens of horses. Next generation transcriptome datasets are available for eight parasitic clade V nematodes, although no equine parasites are included in this group. Here, we report next generation transcriptome sequencing analysis for the common cyathostomin species, Cylicostephanus goldi. A cDNA library was generated from RNA extracted from 17 C. goldi male and female adult parasites. Following sequencing using a 454 GS FLX pyrosequencer, a total of 475,215 sequencing reads were generated, which were assembled into 26,910 contigs. Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, 27% of the transcriptome was annotated. Further in-depth analysis was carried out by comparing the C. goldi dataset with the next generation transcriptomes and genomes of other clade V nematodes, with the Oesophagostomum dentatum transcriptome and the Haemonchus contortus genome showing the highest levels of sequence identity with the cyathostomin dataset (45%). The C. goldi transcriptome was mined for genes associated with anthelmintic mode of action and/or resistance. Sequences encoding proteins previously associated with the three major anthelmintic classes used in horses were identified, with the exception of the P-glycoprotein group. Targeted resequencing of the glutamate gated chloride channel α4 subunit (glc-3), one of the primary targets of the macrocyclic lactone anthelmintics, was performed for several cyathostomin species. We believe this study reports the first transcriptome dataset for an equine helminth parasite, providing the opportunity for in-depth analysis of these important parasites at the molecular level. Sequences encoding enzymes involved in key processes and genes associated with levamisole/pyrantel and macrocyclic lactone resistance, in particular the glutamate gated chloride channels, were identified. This novel data will inform cyathostomin biology and anthelmintic resistance studies in future.

New insights into the FLPergic complements of parasitic nematodes: Informing deorphanisation approaches

FMRFamide-like peptide (FLP) receptors are appealing as putative anthelmintic targets. To date, 31 flp-encoding genes have been identified in Caenorhabditis elegans and thirteen FLP-activated G-protein coupled receptors (FLP-GPCRs) have been reported. The lack of knowledge on FLPs and FLP-GPCRs in parasites impedes their functional characterisation and chemotherapeutic exploitation. Using homology-based BLAST searches and phylogenetic analyses this study describes the identification of putative flp and flp-GPCR gene homologues in 17 nematode parasites providing the first pan-phylum genome-based overview of the FLPergic complement. These data will facilitate FLP-receptor deorphanisation efforts in the quest for novel control targets for nematode parasites.

POPULATION DYNAMICS OF PLANT PARASITIC NEMATODES AND THEIR RELATIONSHIPS WITH SOIL PHYSICO-CHEMICAL PARAMETERS DURING THE FALLOW PERIOD AFTER SUGARBEET AND LEADING INTO TOMATO CULTIVATION

The main objective of this research was the study of the soil nematode community, and in particular plant parasitic nematodes (PPN), from a field located in Portugal’s southern region, used for sugarbeet production. The study was performed from February to July 2003, covering part of the fallow period previous to tomato cultivation, the alternative crop in the rotation. The end of the fallow period in March and the soil preparation period in May were marked by a significant reduction in the numbers of PPN, whereas their numbers increased on the following tomato crop. The genus Helicotylenchus stood out as the most representative group, forming 90% of all PPN counted each month. The genus Heterodera was relatively abundant in the months following the previous sugarbeet crop, and numbers of the genus Meloidogyne increased during the tomato crop. The correlations between these group and environmental parameters show that, apart from the direct influence of the host, pH, organic matter, temperature and soil moisture significantly influenced nematode abundance and community composition.

Peroxide-based hybrid and prodrug antimalarials to deliver cysteine protease inhibitors into the parasitic food vacuole

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Does the generalist parasitic plant Cuscuta campestris selectively forage in heterogeneous plant communities?

Cuscuta spp. are holoparasitic plants that can simultaneously parasitise several host plants. It has been suggested that Cuscuta has evolved a foraging strategy based on a positive relationship between preuptake investment and subsequent reward on different host species. Here we establish reliable parasite size measures and show that parasitism on individuals of different host species alters the biomass of C. campestris but that within host species size and age also contributes to the heterogeneous resource landscape. We then performed two additional experiments to test whether C. campestris achieves greater resource acquisition by parasitising two host species rather than one and whether C. campestris forages in communities of hosts offering different rewards (a choice experiment). There was no evidence in either experiment for direct benefits of a mixed host diet. Cuscuta campestris foraged by parasitising the most rewarding hosts the fastest and then investing the most on them. We conclude that our data present strong evidence for foraging in the parasitic plant C. campestris.

Variabilidad de electroferotipos y serotipos G y P de rotavirus en casos de gastroenteritis infantil [por] Juan Fco. Contreras Cordero

Tesis (Maestría en Ciencias con Especialidad en Inmunobiología) U.A.N.L.