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