Plasmodium alveolins possess distinct but structurally and functionally related multi-repeat domains

Abstract

The invasive and motile life stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive cortical structure termed the pellicle. The pellicle is characterised by a double-layered 'inner membrane complex' (IMC) located underneath the plasma membrane, which is supported by a cytoskeletal structure termed the subpellicular network (SPN). The SPN consists of intermediate filaments, whose major constituents include a family of proteins called alveolins. Here, we re-appraise the alveolins in the genus Plasmodium with respect to their repertoire, structure and interrelatedness. Amongst 13 family members identified, we distinguish two domain types that, albeit distinct at the primary structure level, are structurally related and contain tandem repeats with a consensus 12-amino acid periodicity. Analysis in Plasmodium berghei of the most divergent alveolin, PbIMC1d, reveals a zoite-specific expression in ookinetes and a subcellular localisation in the pellicle, consistent with its predicted role as a SPN component. Knockout of PbIMC1d gives rise to a wild-type phenotype with respect to ookinete morphogenesis, tensile strength, gliding motility and infectivity, presenting the first example of apparent functional redundancy amongst alveolin family members.

Fig. 1

Repertoire and domain structure of Plasmodium alveolins. a Phylogeny of conserved domains within the alveolin family members PbIMC1a to PbIMC1m (a–m). Numbers give amino acid coordinates of the conserved domains in the corresponding PbIMC1 protein. Type 1 (red) and type 2 (green) domains separate into distinct clades. b Schematic diagram depicting the 13 alveolin family members (a–m), showing relative positions of the type 1 (red) and type 2 (green) domains

Fig. 2

Generation and molecular analyses of genetically modified parasite lines. a General targeting strategy for the GFP tagging and gene disruption of pbimc1d via double crossover homologous recombination. Both wild-type (WT) GFP-tagged (IMC1d/GFP) and disrupted (IMC1d-KO) alleles are shown. The pbimc1d gene is indicated with coding sequence (wide bars) and non-coding sequence (narrow bars). Also indicated are the enhanced GFP module (gfp), the hDHFR selectable marker gene cassette (hdhfr) and primers used for diagnostic PCR amplification (P1–P3). b PCR diagnostic for the presence of the modified GFP-tagged pbimc1d alleles using primers P2 and P3 and the absence of the wild-type pbimc1d allele using primers P1 and P2, from clonal parasite populations of IMC1d/GFP and IMC1d-KO. WT parasites are included as positive controls for the wild-type alleles. c Confocal brightfield and GFP fluorescence image of a cultured, mature ookinete of parasite line IMC1d/GFP, showing cortical fluorescence. d Western blot analysis of purified, cultured ookinetes of parasite lines IMC1d/GFP using anti-GFP antibodies, showing the PbIMC1d::GFP fusion protein. e RT-PCR analysis of wild-type parasite samples enriched for asexual stages (ASX), gametocytes (GCT) and ookinetes (OOK) using primers specific for pbimc1d and pbtubulin1. Due to the primers flanking introns, for each gene, the larger PCR products are amplified from gDNA and the smaller from cDNA. f Confocal brightfield and GFP fluorescence image of a cultured, mature ookinete of parasite line IMC1d-KO, showing cytoplasmic fluorescence

Fig. 3

Tandem repeat identification in alveolins, articulins and plateins by the program HHrepID. a P. berghei IMC1e. b P. berghei IMC1b. c Euglena gracilis articulin (AAB23241.1). d Euplotes aediculatus alpha-2 platein precursor (AAM94463.1)