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. 2015 Dec 29;10(12):e0145120. doi: 10.1371/journal.pone.0145120

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© 2015 Moll et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 3 — Strain-specific anti-DBL1α goat IgG (10 μg/ml) was used to detect surface expressed PfEMP1 in the parasite clones FCR3S1.2, PAvarO and R29 comparing cultures grown in blood group A and O. A: FACS blot overlays: For FCR3S1.2 and PAvarO remaining rosettes could mainly be observed after the staining procedure in blood group A, while in R29 rosettes were extinct. The cultures of FCR3S1.2 and PAvarO with remaining rosettes in blood group A showed a reduced surface reactivity with anti-DBL1α antibodies as compared to blood group O where rosettes were removed with a strain-specific anti-DBL1α antibody. In contrast, no difference in staining intensity was seen in R29 as comparing the two blood groups, where rosetting was abolished under both conditions. FACS blots show a representative example of one experiment out of five. B: Mean fluorescence intensity of PfEMP1-staining on the pRBC surface in blood group A versus O in presence or absence of rosettes: Cultures in blood group O and A of FCR3S1.2, PAvarO and R29 were compared for their MFI for PfEMP1 staining. FCR3S1.2 and PAvarO showed a significant higher MFI in blood group O with partly broken rosettes as compared to blood group A with intact rosettes (FCR3S1.2 p = 0,0214; PAVarO p = 0,0005) which disappeared after abolishment of rosetting (FCR3S1.2 p = 0,4874; PAVarO p = 0,0686). R29 with rosettes sensitive to anti-DBL1α antibodies displayed no difference in cultures in blood group A versus O when antibodies are added to trophozoite stage (p = 0,5561) and an even higher recognition in blood group A when antibodies are added to ring stages (p = 0,03). MFI was adjusted to arbitrary units for each experiment; bars show the mean of three experiments plus SD. Unpaired t test, two tailed was used to identify p values. C: Accessibility of PfEMP1 on the pRBC surface in rosetting versus non-rosetting pRBC of FCR3S1.2: Upper panel: FCR3S1.2 pRBC were grown in blood group A and O under rosetting and non-rosetting conditions and accessibility of PfEMP1 on the pRBC surface was assayed with a strain-specific anti-DBL1α goat IgG (50 μg/ml). Formation of rosettes in blood group A reduces antibody accessibility of PfEMP1; even in blood group O staining is less intense when partially rosetting as compared to complete abolishment of rosettes. After removal of rosettes, both blood groups show a similar staining intensity. FACS blot shows a representative example of one experiment out of three of generation three. Lower panel: Average MFI for PfEMP1 staining of cultures of generation one, two and three as described above was compared for rosetting and non-rosetting conditions in blood group A and O, statistical analysis did not show any significant difference. D: Impaired accessibility of the pRBC surface for anti-PfEMP1 antibodies within a rosette as seen by indirect immunofluorescence: FCR3S1.2 pRBC were labelled with anti-DBL1α antibodies visualized in confocal microscopy using 10 μg/ml anti-DBL1α goat IgG and anti-goat-Alexa488. PfEMP1 expressed on the pRBC surface is labelled, but antibodies are hampered in accessing PfEMP1 by uninfected RBC bound to the pRBC surface. pRBC in smaller rosettes as in blood group O (upper panel) are more accessible as compared to pRBC in larger rosettes with more RBC as in blood group A (middle panel). Staining over the whole pRBC surface is seen for non-rosetting pRBC (lower panel).