QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02381

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Horrocks, P. Articles by Newbold, C. I. Search for Related Content PubMed PubMed Citation Articles by Horrocks, P. Articles by Newbold, C. I. Social Bookmarking                         What's this?

PfEMP1 expression is reduced on the surface of knobless Plasmodium falciparum infected erythrocytes

¹ Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK
² Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford, OX3 9DS, UK
³ Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, OX3 9DS, UK
⁴ Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK
⁵ FB Biologie, Philipps-Universität Marburg, 35032 Marburg, Germany

View larger version (67K): [in a new window]   Fig. 1. Molecular characterisation of isogenic knobby and knobless clones. (A) Schematic describing the preparation of isogenic clones used in this study. Rosette or mAbBC6 selected cultures were cloned by limiting dilution. Clones that share the same knobby phenotype and express the same PfEMP1 variant are boxed together. The K– clones expressing type 41 PfEMP1 variant were selected from clones originally cloned from rosetting parasites. See Table 1. (B) Schematic of the chromosome 2 breakpoints in the K– clones used in this study. The positions of the breakpoints upstream of the kahrp (PFB0100c) gene are indicated, with the distance from the telomere (located on left as a black semi-circle) indicated in kilobase pairs. All genes located to the left of the breakpoint are deleted. (C) Ethidium bromide staining of size-fractionated chromosomes shows that approximately 100 kilobase pairs is deleted from chromosome 2 in the K– clones. One from each pair of clones (name and knobby phenotype indicated) is shown here. (D) Northern blot analysis indicates specific transcription of var types 41 and 6 in the cloned parasites. Hybridisation with kahrp and EF-1 probes confirms the ethidium bromide (EtBr) staining indicating equal loading of RNA and that the kahrp gene is not transcribed in K– clones. Transcript sizes are indicated in kilobases (kb). (E) Scanning electron micrograph of a K+ IE adhering to a HDMEC monolayer. Note the numerous knobs (k) on the IE plasma membrane. Bar, 1 µm. (F) Scanning electron micrograph of a K– IE adhering to a HDMEC monolayer illustrating the irregular shape of the IE and the absence of knobs on the plasma membrane. Bar, 1 µm.

View larger version (31K): [in a new window]   Fig. 2. Static and flow adhesion is reduced in K– clones. (A) Results of static adhesion assays to CD36. The data are representative of four independent experiments (means±s.d.). The var variant transcribed and the knobby phenotye is indicated below each clone. (B) Results of static adhesion assays to ICAM1. The data are representative of three independent experiments (means±s.d.). (C) Flow adhesion of var type 41-expressing clones to recombinant ICAM1. Each spot represents a single flow adhesion experiment. Black dots represent data derived from 2B2 (K+) and 2F6B (K–); white dots represent 2E3 (K+) and 1C10B (K–). Data generated in the presence of ICAM-1 blocking antibodies are indicated by (block ab). Horizontal bars represent the mean. (D) Flow adhesion of var type 6-expressing clones to CD36 on HDMECs. Black dots represent data derived from 3C3 (K+) and 1C10 (K–); white dots represent 2H3 (K+) and 2A11 (K–). The presence of CD36 blocking antibodies is indicated by (block ab). Horizontal bars represent the mean.

View larger version (30K): [in a new window]   Fig. 3. Determination of the relative levels of surface PfEMP1 by flow cytometry. (A) Graph showing the proportion of type 41 PfEMP1-expressing IEs labelled with mAbBC6 as a function of the concentration of mAbBC6. MAbBC6 labelling of K+ clones (solid symbols) is indistinguishable from that of K– clones (open symbols). All analyses are representative of three independent experiments (mean±s.d.). (B) Graph showing the relative level of the mean fluorescence intensity (relative MFI; compared to the K+ clone 2B2) of type 41 PfEMP1-expressing IEs as a function of the concentration of mAbBC6. The key describes the symbols used, the var variant transcribed and the knobby phenotype for each clone in panels A and B. (C) Graph showing the proportion of IEs expressing var 6 PfEMP1 labelled with IgG from pooled adult hyperimmune serum (Adult IgG) as a function of the concentration of IgG. (D) Graph showing the relative MFI (compared to 2H3) of adult IgG-labelled IEs as a function of the concentration of IgG. (E) Graph showing the proportion of IEs expressing var 41 PfEMP1 labelled with adult IgG as a function of the concentration of IgG. (F) Graph showing the relative MFI (compared to 2B2) of adult IgG-labelled IEs as a function of the concentration of IgG.

View larger version (52K): [in a new window]   Fig. 4. Less PfEMP1 is expressed on the surface of K– IE. Western blot analysis of whole cell extracts isolated from K+ and K– trophozoite IEs expressing type 41 PfEMP1. Size-fractionated extracts were hybridised with antibodies to either the conserved C-terminus of type 41 PfEMP1 (-ATS), Exp1, aldolase or KAHRP. Fractions were also isolated from trypsin-treated (5 minutes in 100 µg ml–1) IEs. The mean and range of three densitometry measurements of the -ATS signal are indicated. Equal signal intensities to KAHRP, Exp-1 and aldolase confirm equal loading of protein extracts and the lack of KAHRP expression in K– IEs. The molecular weights of proteins labelled are indicated (kDa).

View larger version (88K): [in a new window]   Fig. 5. PfEMP1 on the surface of K+ and K– IEs appears to share a punctate and static distribution. (A) IFA of either unfixed (mAbBC6 and -CD59) or acetone-fixed (-KAHRP) type 41 PfEMP1-expressing K+ and K– IE. Binding of antibody reagents to IEs was detected using FITC-conjugated IgG (green) in the presence of the nuclear DAPI stain (blue). For each image the corresponding bright field image is shown. A Z-plane towards the centre of an IE is shown. Bar, 5 µm. (B) IFA of mAbBC6 stained type 41 PfEMP1-expressing K+ and K– IEs. The Z-plane has been selected to show the exterior face of the IE. Time-lapse photographs (0, 3 and 6 minutes) of the fluorescence signals are shown. Bar, 5 µm.

View larger version (62K): [in a new window]   Fig. 6. TEM analysis of PfEMP1 distribution on the surface of K+ and K– IEs. (A) Gold particle clusters decorate the exterior surface of K+ IE plasma membrane immediately adjacent to electron dense knobby protrusions (see arrows). Gold particle clusters are observed in the K– clone on the exterior face of IEs (see arrows), although no electron dense knobby protrusions are present. Bars, 200 nM. (B) The distribution of the numbers of gold particle clusters per IE was determined for both K+ and K– clones. The 25th-75th percentile of the distribution (box), the mean (line within box) and overall range of this distribution is indicated. (C) The distribution of gold particles within each cluster was determined for K+ and K– IEs. The 25th-75th percentile of the distribution (box), the mean (line within box) and overall range of this distribution is indicated.

View larger version (107K): [in a new window]   Fig. 7. TEM of the IE–endothelial-cell interaction in K+ and K– IEs. (A) Low-power micrograph of a K+ IE (P) that is adhering to the surface of an endothelial cell (En). Bar, 1 µm. (B) Detail of the interface between a K+ IE and an endotheial cell showing strands of connecting material specifically located at knobs (arrows). Note the presence of a Maurer's Cleft (M). Bar, 100 nm. (C) Interface between a K– IE and an endothelial cell in which individual clumps of electron dense material can be seen forming connections between the plasma membranes of the IE and endothelial cell (arrows). Bar, 100 nm.

View larger version (226K): [in a new window]   Fig. 8. Freeze-fracture examination of the distribution of intra-membraneous particles (IMP) on the plasma membrane P-face in K+ and K– IE. (A) Low power micrograph of a K+ IE in which the fracture plane passes through the erythrocyte plasma membrane and cytoplasm to the membrane of the parasitophorous vacuole (PV), which contains the parasite. Note that there is an uneven distribution of the intra-membraneous particle (IMP) within the erythrocyte plasma membrane (arrowheads). Bar, 1 µm. (B) Detail of a fracture through a K+ IE plasma membrane. Note the concentric ring formed from focal organisation of IMP and a zone free of IMP. IMP form foci at sites underlying knob (arrowheads). Bar, 100 nm. (C) Low power micrograph of a K– IE in which the fracture plane passes through the erythrocyte plasma membrane and cytoplasm to reveal the parasitophorous vacuole (PV). Bar, 1 µm. (D) Detail of the K– erythrocyte plasma membrane (boxed area in C) showing the even distribution of IMP in the absence of knob formation. Bar, 100 nm. (E) Detail of the plasma membrane of an uninfected erythrocyte for comparison to D to illustrate the lack of IMP rearrangement in the absence of knobs. Bar, 100 nm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter