Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton

Abstract

The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture.

Figure 1. Erythrocyte ATP is required for merozoite invasion.

(a) Method for quantifying merozoite invasion by flow cytometry. Free GFP-expressing merozoites are added to fresh erythrocytes in a 96-well plate format under agitation for 40 min. Samples are stained with ethidium bromide (EtBr), which labels the parasite nucleus, washed and run on a flow cytometer using a high-throughput plate reader. After gating on erythrocytes, the proportion of red blood cells invaded by merozoites can be quantified. Erythrocytes alone are GFP and EtBr low, while red blood cells with associated parasites and free merozoites are GFP high. EtBr distinguishes between erythrocytes with bound extracellular merozoites, which stain high, and those infected with new rings, which stain low. Here, a new ring-stage parasitemia of 4.5% was achieved, a population that is absent when the invasion assay was performed in the presence of the invasion inhibitor heparin. (b) Invasion rate into cells depleted of ATP for different amounts of time, where invasion is expressed as a percentage relative to the new ring parasitemia of control cells that were incubated in PBS alone. **p < 0.05, ***p < 0.01, ****p < 0.0001, NS = non-significant difference, two-tailed unpaired t-test. Graph displays mean +/−SEM. n = 3 in triplicate. (c) When free D10-PHG merozoites are added to resealed erythrocytes and stained with EtBr, dextran-Alexa647 allows quantitation of invasion into resealed cells by flow cytometry. (d) Invasion rate into erythrocytes resealed in the presence of different concentrations of either ATP or AMP-PNP, where invasion is expressed as a percentage relative to the new ring parasitemia of control cells that were resealed in the presence of 1 mM ATP. ****p < 0.0001, two-tailed unpaired t-test. Graph displays mean +/−SEM. n = 3 in triplicate. (e) Live imaging of erythrocytes resealed under different conditions, where dextran-Alexa647 marks successfully resealed cells.

Figure 2. Imaging of the erythrocyte cytoskeleton throughout merozoite invasion.

Widefield deconvolution fluorescence imaging of fixed merozoite invasion samples labelled with anti-RON4, DAPI and markers of erythrocyte cytoskeletal proteins. (a) Schematic showing how RON4 labelling allows discernment of early, mid, late and complete invasion events. Early in invasion, during apical attachment, RON4 appears as a single point of fluorescence before expanding to form a ring around the invading merozoite as invasion progresses. When imaged in two dimensions, this ring appears as two single points of fluorescence on either side of the merozoite. The right junction marks the boundary between the erythrocyte plasma membrane and the parasitophorous vacuole membrane, and closes at the base of the parasite at the end of invasion. (b) Erythrocyte actin, labelled by phalloidin-Alexa594. (c,d) Band 3, ankyrin and adducin in invasion labelled with specific antibodies. PVM, parasitophorous vacuole membrane; RBC, red blood cell; TJ, tight junction.

Figure 3. Effect of actin and myosin inhibitors on merozoite invasion.

(a) Merozoite invasion when either erythrocytes or merozoites were pre-treated with cytochalasin D (CD), blebbistatin (bleb) or BDM. Invasion percentage is expressed relative to the new ring parasitemia of control cells that were incubated in either PBS alone (for DMSO, heparin and myosin inhibitor treatment) or DMSO (for cytochalasin D treatment). ****p < 0.0001, ***p <0.01, two-tailed unpaired t-test. Graph displays mean +/− SEM. n = 3 in triplicate for all samples except BDM at 10 mM and 50 mM, which was performed three times in either duplicate or triplicate. (b) Invasion rate into erythrocytes resealed in the presence of different concentrations of phalloidin, where invasion is expressed as a percentage relative to the new ring parasitemia of control cells that were resealed in the absence of phalloidin. Graph displays mean +/− SEM, n = 3 in triplicate.

Figure 4. Quantitative phospho-proteomics of merozoite invasion.

(a) Median fold-change values for high confidence phosphorylated and non-phosphorylated erythrocyte peptides were determined by comparing individual peptide abundances between test (containing added merozoites) and control (erythrocytes alone) samples. Data were transformed by median centering for the three conditions containing either heparin-based invasion inhibition, R1 mediated inhibition or invading merozoites. Bars represent the global median and inter-quartile range for each sample set. (b) Histograms from one invasion assay subjected to quantitative phospho-proteomics, displaying the transformed log₂ (median fold change) value for each phosphorylated and non-phosphorylated erythrocyte peptide detected. Outlier minimums, produced using the ROUT method, are marked with the dashed line. Histogram bin width is 0.2.

Figure 5. Phosphorylation of beta-spectrin and PIEZO1 in response to merozoites.

Median centered individual phospho-peptide fold-change values pooled across the four invasion proteomic experiments for two proteins that contain a shortlisted outlier peptide, (a) beta-spectrin and (b) PIEZO1. Graphs display box and whisker plots showing the median, maximum and minimum values and inter-quartile range. Schematic representations of (c) beta-spectrin and (d) PIEZO1. Beta-spectrin phospho-sites identified through quantitative phospho-proteomics of invasion and their locations within the protein relative to key domains and regions of protein-protein interactions. Predicted membrane topology of PIEZO1based on Uniprot-derived consensus features from multiple prediction algorithms. Location of identified outlier phospho-sites is marked in red.

Figure 6. Quantitative phospho-proteomics of erythrocyte response to microbeads.

(a) Median fold-change values for high confidence phosphorylated and non-phosphorylated erythrocyte peptides were determined by comparing individual peptide abundances between test (containing added microbeads either pre-incubated with WGA or in the presence of BlockAid) and control (erythrocytes alone) samples. Values were transformed by median centering for the two conditions. Bars represent the global median and inter-quartile range for each sample set. (b) Histograms of each erythrocyte bead assay subjected to quantitative phospho-proteomics, displaying the transformed log₂ (median fold change) values for each phosphorylated and non-phosphorylated erythrocyte peptide detected. Outlier boundaries, produced using the ROUT method, are marked with the dashed lines. Black dashed line represents upper boundary, where peptides with values above this line considered outliers, while the red dashed line marks the lower boundary, where peptides with values lower than this value identified as outliers. Histogram bin width is 0.05.