THE DIVALENT CATION TRANSPORTER NRAMP IN PARASITE PERKINSUS MARINUS: GENOMIC, MOLECULAR, STRUCTURAL, FUNCTIONAL AND EVOLUTIONARY ASPECTS

Abstract

Perkinsus marinus, the causative agent of Dermo disease in eastern oyster Crassostrea virginica has been a great hurdle for oyster population restoration. Iron was shown to be an essential element for P. marinus growth and virulence, but iron uptake pathways have not been elucidated. Natural Resistance-associated Macrophage Protein (Nramp), an iron transporter, was considered to be a potential virulence factor in intracellular pathogens. One Nramp homolog (PmNramp1) was reported in P. marinus previously. Two other PmNramp isotypes (PmNramp2 and PmNramp3) were identified through genome mining followed by molecular characterization. The three PmNramp isotypes with distinct gene structures were 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) were trans-spliced with a trans-splicing leader (SL) highly similar to dinoflagellate SL. No change in transcription level of those genes was detected by real-time quantitative reverse transcription PCR (qRT-PCR), under iron/manganese overload, iron depletion and host hemolymph exposure, indicating a constitutive polycistronic transcription in the parasite.

Functional study by yeast complementation assays suggested iron uptake activity by PmNramp1. Prediction of PmNramp1 topology by homologous modeling indicated that PmNramp1 was 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 the residues on the triplets in PmNramp1 caused the lost of complementation activity as iron transporter in yeast. A chimeric protein with PmNramp1 N- and C-termini but PmNramp3 core structure from TMS1 to TMS12 complemented yeast growth, suggesting PmNramp3 an iron transporter. Phylogeny data implied that all the three PmNramp isotypes were archetype Nramp. 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 are 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 the dissertation may serve as the reference for research in other protozoan Nramp and iron transporters.