Ilia Bankov

Genetic diversity of liver flukes (Fasciola hepatica) from Eastern Europe

The genetic diversity of liver fluke populations in three different countries from Eastern Europe (Greece, Bulgaria, and Poland) was determined and compared with available data from other countries. Specifically, SNPs from regions of two nuclear genes, 28S rDNA, β-tubulin 3 and an informative region of the mitochondrial genome were examined. Two major lineages for the 28S rDNA gene based on the highly polymorphic 105th nucleotide position were found. These lineages were widely and almost equally spread not only through the countries studied but also in other investigated geographical areas. Two basic lineages and additional haplotypes were defined for the mtDNA gene region which consisted of the cytochrome c oxidase subunit III gene, transfer RNA histidine gene and cytochome b gene. The basic lineages were observed within Greek, Bulgarian, and Polish Fasciola hepatica populations but the distribution of additional haplotypes differed between the populations from the three countries. For the β-tubulin 3 gene multiple polymorphic sites were revealed but no explicit clades. The SNPs were spread unequally in all studied geographical regions with an evident distinction between the Greek and Polish specimens. Additional genotypes for the 28S rDNA region as well as haplotypes of the mtDNA region that were typical for the Greek or Polish populations were observed. Significant polymorphisms for β-tubulin 3 gene were displayed with decreasing percentage of presence within populations from Greece to Poland. There was an amino acid substitution in β-tubulin 3 protein found only among Polish specimens. It is hypothesized that genotypic differences between Greek, Bulgarian, and Polish liver fluke populations are due to territorial division and genetic drift in past epochs.

Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli

Ag‐NPA‐1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag‐NPA‐1 is a highly ordered, α‐helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag‐NPA‐1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr‐Trp dipole‐dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag‐NPA‐1 and the fluorescent fatty acid analogue 11‐[(5‐dimethylaminonaphthalene‐1‐ sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag‐NPA‐1 (Cys66 and Cys122) with the thiol reactive probes 5‐({[(2‐iodoacetyl)amino]ethyl}amino) naphthalene‐1‐sulfonic acid (IAEDANS) and N,N′‐dimethyl‐N‐(iodoacetyl)‐N′‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)ethylenediamine (IANBD), followed by MALDI‐TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag‐NPA‐1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag‐NPA‐1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag‐NPA‐1 plays an important role in the transport of fatty acids and retinoids.

The highly abundant protein Ag-Ibp55 from Ascaridia galli represents a novel type of lipid-binding proteins

Lipid-binding proteins exhibit important functions in lipid transport, cellular signaling, gene transcription, and cytoprotection. Their functional analogues in nematodes are nematode polyprotein allergens/antigens and fatty acid and retinoid-binding proteins. This work describes a novel 55-kDa protein, Ag-lbp55, purified from the parasitic nematode Ascaridia galli. By direct N-terminal sequencing, a partial amino acid sequence was obtained that allowed the design of oligonucleotide primers to obtain the full-length cDNA sequence. Sequence analysis revealed the presence of an N-terminal signal peptide of 25 amino acid residues and a FAR domain at the C terminus. Data base searches showed almost no significant homologies to other described proteins. The secondary structure of Ag-lbp55 was predominantly α-helical (65%) as shown by CD spectroscopy. It was found to bind with high affinity fatty acids (caprylic, oleic, and palmitic acid) and their fluorescent analogue dansylaminoundecanic acid. Immunolocalization showed that Ag-lbp55 is a highly abundant protein, mainly distributed in the inner hypodermis and extracellularly in the pseudocoelomatic fluid. A similar staining pattern was observed in other pathogenic nematodes, indicating the existence of similar proteins in these species.

Isolation and partial characterization of a fatty-acid-binding protein from Ascaris suum reproductive tissue

Abstract A 12-kDa fatty-acid-binding protein was purified to homogeneity from Ascaris suum reproductive tissue as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino-acid-sequence analysis of the protein revealed its identity with the ABA-1 allergen protein isolated from A. suum pseudocoelomic fluid. Fatty-acid binding by the protein from A. suum reproductive tissue was investigated using the Lipidex 1000 assay, which revealed the presence of a single class of fatty-acid-binding sites with an apparent dissociation constant for palmitate of about 0.8 μM.

A Novel Secretory Poly-Cysteine and Histidine-Tailed Metalloprotein (Ts-PCHTP) from Trichinella spiralis (Nematoda)

Background Trichinella spiralis is an unusual parasitic intracellular nematode causing dedifferentiation of the host myofiber. Trichinella proteomic analyses have identified proteins that act at the interface between the parasite and the host and are probably important for the infection and pathogenesis. Many parasitic proteins, including a number of metalloproteins are unique for the nematodes and trichinellids and therefore present good targets for future therapeutic developments. Furthermore, detailed information on such proteins and their function in the nematode organism would provide better understanding of the parasite - host interactions. Methodology/Principal Findings In this study we report the identification, biochemical characterization and localization of a novel poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP). The native Ts-PCHTP was purified from T. spiralis muscle larvae that were isolated from infected rats as a model system. The sequence analysis showed no homology with other proteins. Two unique poly-cysteine domains were found in the amino acid sequence of Ts-PCHTP. This protein is also the first reported natural histidine tailed protein. It was suggested that Ts-PCHTP has metal binding properties. Total Reflection X-ray Fluorescence (TXRF) assay revealed that it binds significant concentrations of iron, nickel and zinc at protein:metal ratio of about 1∶2. Immunohistochemical analysis showed that the Ts-PCHTP is localized in the cuticle and in all tissues of the larvae, but that it is not excreted outside the parasite. Conclusions/Significance Our data suggest that Ts-PCHTP is the first described member of a novel nematode poly-cysteine protein family and its function could be metal storage and/or transport. Since this protein family is unique for parasites from Superfamily Trichinelloidea its potential applications in diagnostics and treatment could be exploited in future.

Sphingomyelin synthesis in Fasciola hepatica

Whole worms and/or homogenates of F. hepatica incorporate label from cytidine-5-diphospho[methyl-14C]choline,[1-14C]palmitoylCoA,[U- 14C]serine,[2-14C]methionine, [U-14C]glycine, [U-14C]threonine and [U-14C]aspartate into the various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). This suggests that sphingomyelin synthesis in F. hepatica occurs by a pathway similar to that found in mammals. However, there is some evidence that in F. hepatica 3-ketosphinganine may be N-acylated prior to reduction and dehydrogenation.

Methionine and cysteine metabolism in Fasciola hepatica.

Radiolabel from the methyl groups of serine and methyltetrahydrofolate was readily incorporated into methionine in adult Fasciola hepatica, and a substantial proportion of the label from [35S]methionine appeared in cysteine. The data suggest that methionine synthesis is via methyltetrahydrofolate-homocysteine methyltransferase and that there is cysteine synthesis from methionine. Cystathionine-beta-synthase and gamma-cystathionase activities were demonstrated in homogenates.

Sphingomyelin synthesis in Ascaridia galli

Adult Ascaridia galli incorporate label from [U-14C] serine into various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). From the results it is concluded that A. galli possesses the five enzymes involved in sphingomyelin synthesis, namely: serine palmitoyltransferase, 3-ketosphinganine reductase, flavoprotein sphinganine reductase, sphingosine acyltransferase and ceramide choline phosphotransferase.