Fig 1.
SURFIN4.2 gene and its encoding protein.
Polyclonal antibody specificity tested on a peptide array. (A) Schematic representation of the SURFIN4.2 protein and its encoding gene (PfIT_0422600). CRD: Cysteine-rich domain; Var1 and 2: Variable region 1 and 2; TM: Transmembrane domain; WR: Tryptophan-rich domain. (B) Anti-SURFIN4.2 polyclonal antibodies reactivity on a peptide array. Reactivity profile of each anti-SURFIN4.2 polyclonal antibody against the whole sequence of the SURFIN4.2 protein. Sequence is depicted from N- to C terminus. (C) Number of reactive peptides for each polyclonal antibody discriminated by protein family. For the SURFIN family each member is depicted separately.
Fig 2.
SURFIN4.2 protein expression during the parasite asexual cycle.
All panels depict PAGE followed by immunoblot with αSURFIN4.2. (A) SDS-PAGE on a parasite time-course during the asexual cycle inside RBCs, pellet (P) and supernatant (S) after saponin pellet extraction with the detergent SB3-10. (B) SDS-PAGE on cellular pellet (lane 1) and culture supernatant (lanes 2 and 3, before and after ultracentrifugation respectively) upon schizont rupture. (C) BN-PAGE on a parasite time-course, only supernatants after extraction with SB3-10 were tested.
Fig 3.
SURFIN4.2 protein forms a complex with GLURP and RON4.
IP with αSURFIN4.2 specifically pulls down SURFIN4.2 from schizont (A) and merozoite (B) protein extract as compared with control IgGs. (C) IP with αSURFIN4.2 also pulls down GLURP and RON4. (D) IP with αGLURP R2 also pulls down RON4 but not SURFIN4.2; IP with αRON4 also pulls down GLURP and SURFIN4.2. Input corresponds to the supernatant fraction after SB3-10 extraction followed by detergent removal and pre-clearing on control beads; FT: Flow-through corresponds to unbound material; E1 and 2: Eluted fraction corresponds to bound material to the antibody-coupled beads.
Table 1.
Proteins identified by Mass Spectrometry (MS) after immunoprecipitation (IP) with anti-SURFIN4.2 antibodies both from schizont and merozoite protein extract.
Fig 4.
SURFIN4.2 localizes at the apical end and surface of the merozoites.
(A) IFA on a time course, showing SURFIN4.2 labeling throughout the asexual cycle inside the RBCs and after schizont rupture. (B) IFA on double-labeled schizonts. (C) IFA on double-labeled invading merozoites upon apical attachments (upper panel) and during active invasion into the nascent PV (lower panel). SURFIN4.2 is shown in green, surface markers (MSP-1 and MSP-3) and microneme markers (EBA-175 and AMA-1) are shown in red. Scale bar represents 5μm.
Fig 5.
SURFIN4.2, GLURP and RON4 localization in schizonts and free merozoites.
(A) IFA on single or double-labeled schizonts. (B) IFA on single or double-labeled free purified merozoites, SURFIN4.2 and GLURP are shown in green. RON4 is shown in red. Scale bar represents 5μm.
Fig 6.
SURFIN4.2 localizes at the rhoptry neck.
(A) Whole merozoites where the electrodense club-shaped rhoptries are clearly observed. (B) Image enlargements of the corresponding pictures shown in A. showing the rhoptries in more detail, SURFIN4.2 is clearly present in the most terminal part of the organelle: the neck. Scale bar represent 200nm.
Fig 7.
(A) Bright-field images of group O RBCs binding to CHO cells expressing SURFIN4.2 CRD, RIFIN-A, untransfected (Control) or transfected with empty pDisplay plasmid (PD). (B) Number of RBCs binding to 1000 CHO cells, a positive control (RIF-A) known to bind RBCs was included. The values correspond to the mean±standard deviation (SD) of three independent experiments. Stars indicate significant difference.
Fig 8.
Antibodies against SURFIN4.2 partially inhibit invasion into RBCs by the parasite FCR3S1.2.
Parasitaemia as percentage of a control (without antibody) is shown. The values correspond to the mean±standard deviation (SD) of three independent experiments. Stars indicate significant difference when compared with the rabbit IgG control.