Transferrin polymorphism of common carp: link with disease resistance

P.M. Jurecka

Research output: Thesisinternal PhD, WU

Abstract

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.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Savelkoul, Huub, Promotor
  • Pilarczyk, A., Promotor, External person
  • Wiegertjes, Geert, Co-promotor
  • Irnazarow, I., Co-promotor, External person
Award date24 Nov 2008
Place of Publication[S.l.]
Print ISBNs9789085852438
Publication statusPublished - 2008

Keywords

  • carp
  • transferrin
  • polymorphism
  • disease resistance
  • genetic resistance
  • trypanoplasma borreli
  • experimental infection
  • gene expression
  • nitric oxide
  • macrophages
  • immunology

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