The divalent cation transporter Nramp in parasite Perkinsus marinus: Genomic, molecular, structural, functional and evolutionary aspects

Perkinsus marinus, the causative agent of Dermo disease in eastern oyster Crassostrea virginica has been a great hurdle for oyster population restoration along the atlantic and gulf coasts of USA. Iron was shown to be an essential element for P. marinus growth and virulence, but iron uptake pathways have not been elucidated.;The N&barbelow;atural R&barbelow;esistance-A&barbelow;ssociated M&barbelow;acrophage P&barbelow;rotein (Nramp), an iron transporter initially identified in the mouse as a marker for resistance to intracellular pathogens, was also proposed as a potential virulence factor for intracellular pathogens. An Nramp homologue was identified by our laboratory in P. marinus (PmNramp1). In this study, two other PmNramp isotypes (PmNramp2 and PmNramp3) were identified through genome mining followed by molecular characterization. The three PmNramp isotypes are encoded by genes of distinct organization, and are all transcribed in parasite trophozoites cultured in defined medium. Transcripts of a number of P. marinus genes, including PmNramp isotypes, superoxide dismutases (PmSOD), ascorbate peroxidase (PmAPX) and heat shock proteins (PmHSP70 and PmHSP90) display a trans-splicing leader (SL) highly similar to that from dinoflagellates. No changes in transcription levels of those genes were detected by real-time quantitative reverse transcription PCR (qRT-PCR), under iron/manganese overload, iron depletion, and host hemolymph exposure, suggesting a constitutive polycistronic transcription in the parasite.;Functional studies by yeast complementation assays suggested iron uptake activity by PmNramp1, but not for PmNramp2 and PmNramp3. Prediction of PmNramp1 topology by homologous modeling indicated that PmNramp1 is an integral protein with 12 transmembrane segments (TMS). The central position of the Nramp-specific triplets Asp-Pro-Gly (TMS1) and Met-Pro-His (TMS6) in a three-dimensional (3D) arrangement formed with TMS3 and TMS8 provided the mechanistic basis for iron acquisition via PmNramp1. Site-directed mutagenesis of selected residues in the TMS6 triplets in PmNramp1 resulted in loss of complementation of the mutant in yeast. A chimeric protein with PmNramp1 N- and C-termini and a PmNramp3 core structure from TMS1 to TMS12 actively complemented yeast growth, suggesting that PmNramp3 can function as an iron transporter. A phylogenetic analysis indicated that all the three PmNramp isotypes are of the archetype Nramp cluster. Protein sequence divergence among PmNramp isotypes was not related to diversification of critical functional elements, which remained constrained by purifying selection. This result was consistent with the function of both PmNramp1 and PmNramp3 as iron transporters in yeast, despite their different evolutionary rate and substitution patterns. Subcellular localization of PmNramp isotypes in P. marinus trophozoites is in progress. PmNramp3 was shown to localize on cell peripheral when the parasite proliferates by binary fission. The data were consistent with the previous observation that iron is important for P. marinus growth.;As the first functional study of Nramp homolog in protozoan parasites, the work in this dissertation may serve as a platform for research in other protozoan Nramp and iron transporters.