The Natural Resistance-Associated Macrophage Protein from the Protozoan Parasite Perkinsus marinus Mediates Iron Uptake

Abstract

Microbial pathogens succeed in acquiring essential metals such as iron and manganese despite their limited availability because of the host's immune response. The eukaryotic natural resistance-associated macrophage proteins mediate uptake of divalent metals and, during infection, may compete directly for metal acquisition with the pathogens' transporters. In this study, we characterize the Nramp gene family of Perkinsus marinus, an intracellular parasite of the eastern oyster, and through yeast complementation, we demonstrate for the first time for a protozoan parasite that Nramp imports environmental Fe. Three PmNramp isogenes differ in their exon-intron structures and encode transcripts that display a trans splicing leader at the 5' end. The protein sequences share conserved properties predicted for the Nramp/Solute carrier 11 (Slc11) family, such as 12-transmembrane segment (TMS) topology (N- and C-termini cytoplasmic) and preferential conservation of four TMS predicted to form a pseudosymmetric proton/metal symport pathway. Yeast fet3fet4 mutant complementation assays showed iron transport activity for PmNramp1 and a fusion chimera of the PmNramp3 hydrophobic core and PmNramp1 N- and C-termini. PmNramp1 site-directed mutagenesis demonstrated that Slc11 invariant and predicted pseudosymmetric motifs (TMS1 Asp-Pro-Gly and TMS6 Met-Pro-His) are key for transport function. PmNramp1 TMS1 mutants D76E, G78A, and D76E/G78A prevented membrane protein expression, while TMS6 M250A, H252Y, and M250A/H252Y specifically abrogated Fe uptake; the TMS6 H252Y mutation also correlates with divergence from Nramp specificity for divalent metals.

Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species

“…Thus, it is likely that as we observed for CvGal1 (22), the CvGal2 transcripts identified in these tissues actually originate in the infiltrating hemocytes. The P. marinus trophozoites enter the oyster through filter-feeding and initially become in contact with the gills, palps, gut or mantle, where they are phagocytosed by resident hemocytes (65) and survive intracellular oxidative stress by their effective anti-oxidative mechanisms (53, 54, 66, 67). The trophozoites proliferate in phagosome-like structures, eventually lysing the host hemocytes, and causing systemic infection and death of the oyster, which by releasing large numbers of parasites into the water column spreads the infection to neighboring oysters (20, 35).…”

The Galectin CvGal1 from the Eastern Oyster (Crassostrea virginica) Binds to Blood Group A Oligosaccharides on the Hemocyte Surface*

“…Dominin is of particular interest because it is a major plasma protein in the oyster hemolymph that exhibits a Cu,Zn-superoxide-like domain and has high similarity to cavortin, an iron-binding protein from the Pacific oyster (C. gigas) (47). We have characterized the role of iron for P. marinus virulence (48 -50) and intracellular survival in oyster hemocytes (51) and identified the iron uptake mechanisms via the Nramp transporter (52,53), responsible for a "tug of war" for available iron between the parasite and its oyster host (54). A rigorous glycomic analysis of dominin associated with hemocytes and from plasma revealed that it also displays biantennary glycans carrying blood group A moieties (61), thus opening the possibility that CvGal1 cross-links dominin to the hemocyte surface as a mechanism to sequester plasma iron into the intracellular pools, a defense mechanism against infectious challenge that has been conserved from invertebrates to mammals (55).…”

Biochemical Characterization of Oyster and Clam Galectins: Selective Recognition of Carbohydrate Ligands on Host Hemocytes and Perkinsus Parasites

“…Dominin is a major plasma protein that houses a Cu/Zn superoxide-like domain, and is highly similar to cavortin, an iron binding-protein from the Pacific oyster (C. gigas) (Itoh et al, 2011). Iron is critical for P. marinus intracellular survival in oyster hemocytes (Schott and Vasta, 2003;Schott et al, 2003a;Fernández-Robledo et al, 2008;Alavi et al, 2009), and the iron transporters (Nramp; Robledo et al, 2004;Lin et al, 2011) in both the parasite and the oyster host are involved in their competition for available iron (Cellier et al, 2007). Another interesting ligand for CvGal1 identified on the hemocyte surface is β-integrin (Zhuo et al, 2008;Feng et al, 2013), and this was of particular interest as this transmembrane signaling glycoprotein is a key receptor in cell activation processes (Mayadas and Cullere, 2005;Lim and Hotchin, 2012).…”