Transferrin polymorphism of common carp: link with disease resistance

Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many metabolic functions. Iron concentrations are tightly regulated, for example by ferritin, because excessive iron leads to tissue damage. Iron cannot cross cellular membranes directly and most cells acquire iron from the iron transporting protein transferrin (Tf), via transferrin receptors. During nutritional immunity the body reacts with a metabolic adjustment in order to render important nutrients unavailable to invading microorganisms. However, pathogens also have evolved a range of mechanisms to acquire iron from the host (chapter 1). In the study described in this thesis, we used a natural host-parasite model of common carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan kinetoplastid, extracellular blood parasite of carp to get more insight in the competition for iron between host and parasite. Transferrin of common carp is highly polymorphic with several alleles identified according to differences in electrophoretic mobility. We studied the implications of Tf polymorphisms for iron binding and modulation of immune function. We performed a series of challenge experiments infecting five genetically different, commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype may influence the susceptibility to pathogens. We observed a significant association of the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’ and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We also showed that variation in resistance to T. borreli could be controlled by sex-related genetic factors. Examination of parasite growth in vitro, in culture media supplemented with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in number of parasites in media supplemented with serum from DD-typed animals (chapter 2). In general, pathogens also have mechanisms to acquire iron from the host. We developed a method for Tf depletion of carp serum using specific antibodies to carp Tf, and compared T. borreli multiplication and survival in the presence or absence of Tf in vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly showed that Tf is essential for parasite growth and multiplication (chapter 3). We isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography (chapter 5). We showed that parasite growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in culture media containing different sera typed differently for Tf genotypes (chapter 3). We identified four complete coding sequences for common carp Tf alleles C, D, F and G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs of other species. We could show that carp Tf differs significantly in critical iron-binding sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species (chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe indeed seems to affect the ability to bind iron, which may be compensated for by higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene expression of two Tf alleles D and G showed a comparably high gene expression level in liver and small but consistent differences in gene expression for allele D over allele G in other immunologically important organs (chapter 4). Our data suggest that the allelic polymorphism is not related to differences in iron binding and/or binding to the host Tf receptor but could be linked with other factors, such as competition for iron with pathogens (chapter 4). Transferrin itself may also exert effects that are not directly linked with maintaining iron levels and Tf cleavage products have been shown to stimulate macrophages to produce large amounts of nitric oxide (NO). To study the induction of NO in carp head kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G) to purity and showed that the level of activation of macrophages by Tf was different for the D and G allele (chapter 5). Differences in NO levels induced could be related to different cleavage forms of the two alleles D and G, as shown by Western blot, confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products induced significantly higher nitric oxide (NO) production than cleavage products of Gtype Tf. (chapter 5). Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The TfR-Tf complex then is internalised and transported to lysosomes, where Tf is proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like cysteine proteinase from T. borreli and production of a recombinant and biologically active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is able to digest host transferrin. Likely, Tf cleavage fragments are released from the trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages (chapter 7). Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in immunity of common carp and the influence of allelic polymorphism on competition for iron between host and pathogen (chapter 7). Further investigations should shed more light on the selective advantage of particular alleles to provide a basis for incorporating Tf as a genetic marker in marker-assisted selection programmes for increased resistance to diseases. This could contribute to improved survival of carp kept under semiintensive farming systems in ponds.