Expression Profiling of <i>Plasmodium berghei HSP70</i> Genes for Generation of Bright Red Fluorescent Parasites

<div><p>Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite <i>Plasmodium berghei.</i> To allow constitutive and abundant expression of the mCherry protein we profiled expression of all members of the <i>P. berghei</i> heat shock protein 70 (HSP70) family. We identified <i>Pb</i>HSP70/1, an invariant ortholog of <i>Plasmodium falciparum</i> HSP70-1, as the protein with the highest expression levels during <i>Plasmodium</i> blood, mosquito, and liver infection. Stable allelic insertion of a mCherry expression cassette into the <i>PbHsp70/1</i> locus created constitutive red fluorescent <i>P. berghei</i> lines, termed <i>Pb</i>red. We show that these parasites can be used for live imaging of infected host cells and organs, including hepatocytes, erythrocytes, and whole <i>Anopheles</i> mosquitoes. Quantification of the fluorescence intensity of several <i>Pb</i>red parasite stages revealed significantly enhanced signal intensities in comparison to GFP expressed under the control of the constitutive EF1alpha promoter. We propose that systematic transcript profiling permits generation of reporter parasites, such as the <i>Pb</i>red lines described herein.</p></div

Generation of <i>Pb</i>red parasites.

<p>(A) Integration strategy to generate <i>Pb</i>red parasites. The <i>PbHSP70/1</i> genomic locus was targeted with an integration plasmid containing the 5′ region flanking the <i>HSP70/1</i> ORF (black box), the mCherry open reading frame (red box) and the 3′ region of the <i>PbDHFR</i>, followed by the <i>Tgdhfr/ts</i> cassette as positive selectable marker. Upon linearization of the plasmid with <i>Bst</i>BI, integration is expected to lead to allele duplication, resulting in mCherry expression under the <i>PbHSP70/1</i> promoter and an adjacent <i>PbHSP70/1</i> wild-type copy. Integration and wild type-specific test primer combinations and expected fragments are indicated. <b>(B</b>) Genotyping of two clonal <i>Pb</i>red parasite lines, <i>Pb</i>red c₅₀₇ and <i>Pb</i>red c_ANKA. Confirmation of the predicted integration is achieved by PCR analysis using primer combinations (<i>5′UTR</i> and <i>3′ UTR</i>), which only amplify a signal from the recombinant locus. A wild type-specific primer combination (WT) confirms the absence of residual wild type parasites in the clonal <i>Pb</i>red populations.</p

Live cell imaging of <i>Pb</i>red during mosquito infection.

<p>Representative live cell images of <i>Pb</i>red c₅₀₇ development inside the <i>Anopheles</i> vector. Presented are DIC images in combination with nuclear stain (Hoechst; left) and fluorescent images for mCherry (center) and GFP (right). Life cycle stages are indicated on the left. Scale bars, 5 µm for ookinete and sporozoite, and 10 µm for oocyst, respectively.</p

Quantitative analysis of mCherry and GFP fluorescence of <i>Pb</i>red c₅₀₇ parasites.

<p>(A) Distribution of fluorescence intensities in a representative trophozoite (upper left), ookinete (upper right), salivary gland sporozoite (lower left), and mature liver stage (lower right). Micrographs represent DIC, mCherry and GFP channels with indicated parasite “mask” (yellow), used for fluorescence determination. The point chart displays associated distribution of grey values (brightness) in numbers of pixels for each channel. (B) Quantification of Hsp70/1-mCherry and EF1a-GFP fluorescence in mixed blood stages (n = 12), ookinetes (ook; n = 13), salivary gland sporozoites (spz; n = 4) and extra-erythrocytic liver stages from 24 to 72 h post infection (eef; n = 7). Fluorescence intensities are presented as the mean of grey values for each fluorescent channel (± S.E.M.). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001 (unpaired students t-test).</p

Asexual blood-stage development is unaffected in <i>Pb</i>red parasites.

<p>Mice were infected intravenously with 1,000 infected erythrocytes. Parasitemia of recipient mice (n = 5 for WT and <i>Pb</i>red c₅₀₇; n = 3 for<i>Pb</i>red c_ANKA) was monitored daily by examination of Giemsa-stained blood smears. Shown are mean values (± S.E.M.).</p

Live imaging of <i>Pb</i>red-infected <i>Anopheles stephensi</i> mosquitoes.

<p>(A) Live imaging of salivary gland colonization of <i>Pb</i>red-infected <i>Anopheles stephensi</i> mosquitoes. Shown are representative live fluorescent images with the merge of fluorescence and white light illuminated images (left) and the mCherry signal only (right). (<b>B</b>) Live imaging of hemocoel sporozoites in <i>Pb</i>red-infected <i>Anopheles stephensi</i> mosquitoes. Shown are representative higher magnification live fluorescent images of the mosquito maxillary palps (top) and wings (bottom) with the merge of fluorescence and white light illuminated images (left), the mCherry signal (right), and the corresponding GFP signal (bottom right), exemplified in a wing vein.</p

Live cell imaging of <i>Pb</i>red parasites during infection of cultured hepatoma cells.

<p>Representative live cell images of <i>Pb</i>red c₅₀₇ -infected hepatoma cells at different stages of maturation. Presented are DIC images in combination with nuclear stain (Hoechst; left) and fluorescent images for mCherry (center) and GFP (right). Time points after sporozoite infection are indicated on the left. Scale bars, 10 µm for 24 h and 48 h time points and merosomes, and 20 µm for 72 h time points, respectively.</p

Live imaging and fluorescence quantification of <i>Pb</i>red during blood infection.

<p>(A) Live cell imaging of <i>Pb</i>red c₅₀₇ -infected erythrocytes during blood stage development. Representative differential interference contrast DIC (left) and live fluorescent images for mCherry (center) and the green fluorescent proteins GFP (right) of <i>Pb</i>red-infected erythrocytes at different stages of development are shown. Nuclear stains (Hoechst) are visualized as merge in the DIC images. Life cycle stages are indicated on the left. Scale bars, 5 µm. (B) Quantitative analysis of mCherry (red) and GFP (green) fluorescence in different blood stages. Fluorescence intensities are presented in mean of grey values (± S.E.M.) for rings, trophozoites and gametocytes. *, <i>P</i><0.05 (students t-test).</p

Coupling of Retrograde Flow to Force Production During Malaria Parasite Migration

Migration of malaria parasites is powered by a myosin motor that moves actin filaments, which in turn link to adhesive proteins spanning the plasma membrane. The retrograde flow of these adhesins appears to be coupled to forward locomotion. However, the contact dynamics between the parasite and the substrate as well as the generation of forces are complex and their relation to retrograde flow is unclear. Using optical tweezers we found retrograde flow rates up to 15 μm/s contrasting with parasite average speeds of 1–2 μm/s. We found that a surface protein, TLP, functions in reducing retrograde flow for the buildup of adhesive force and that actin dynamics appear optimized for the generation of force but not for maximizing the speed of retrograde flow. These data uncover that TLP acts by modulating actin dynamics or actin filament organization and couples retrograde flow to force production in malaria parasites

Discovery of <i>Plasmodium</i> (M)TRAP–Aldolase Interaction Stabilizers Interfering with Sporozoite Motility and Invasion

As obligate, intracellular parasites, <i>Plasmodium spp.</i> rely on invasion of host cells in order to replicate and continue their life cycle. The parasite needs to traverse the dermis and endothelium of blood vessels, invade hepatocytes and red blood cells, traverse the mosquito midgut, and enter the salivary glands to continue the cycle of infection and transmission. To traverse and invade cells, the parasite employs an actomyosin motor at the core of a larger invasion machinery complex known as the glideosome. The complex is comprised of multiple protein–protein interactions linking the motor to the internal cytoskeletal network of the parasite and to the extracellular adhesins, which directly contact the host tissue or cell surface. One key interaction is between the cytoplasmic tails of the thrombospondin related anonymous protein (TRAP) and aldolase, a bridging protein to the motor. Here, we present results from screening the Medicines for Malaria Venture (MMV) library of 400 compounds against this key protein–protein interaction. Using a surface plasmon resonance screen, we have identified several compounds that modulate the dynamics of the interaction between TRAP and aldolase. These compounds have been validated <i>in vitro</i> by studying their effects on sporozoite gliding motility and hepatocyte invasion. One of the MMV compounds identified reduced invasion levels by 89% at the lowest concentration tested (16 μM) and severely inhibited gliding at even lower concentrations (5 μM). By targeting protein–protein interactions, we investigated an under-explored area of parasite biology and general drug development, to identify potential antimalarial lead compounds