Functional Characterization of Actin Sequestering Proteins in Plasmodium berghei

Plasmodien spp. sind obligat intrazellulär lebende Parasiten, welche einen evolutionär konservierten aktinabhängigen molekularen Motor für die Fortbewegung und den Wirtszellein- und -austritt nutzen. In dieser Arbeit werden die Aktinregulatoren Adenylyl- Zyklase- assoziierte Protein (C-CAP), Profilin sowie die Aktin depolymerizierenden Faktoren 1 und 2 (ADF1, ADF2) in Plasmodium berghei charakterisiert. Die Geninaktivierung von C-CAP besitzt keinen Einfluss auf die Entwicklung von pathogenen Blutstadien. C-cap(-) Ookineten bewegen sich jedoch deutlich langsamer, sind aber in der Lage den invertebraten Wirt zu infizieren. Defekte treten während der extrazellulären Replikationsphase im Mosquito auf und führen zu Abbruch des Lebenszykluses. Die erfolgreiche Komplementierung der Defekte mit dem orthologen Gen aus Cryptosporidium parvum CpC-CAP bestätigt die funktionale Redundanz zwischen beiden Proteinen. Profilin, als ein weiteres G-Aktin bindendes Protein, ist hingegen nicht in der Lage die Defekte des c-cap(-) Parasiten auszugleichen. Mittels transgener Parasiten welche ein C-CAPmCherry Fusionsprotein exprimieren, wird das C-CAP Protein im Zytoplasma lokalisiert. Erstmals wird mit dieser Arbeit ein G-Aktin bindendes Protein, C-CAP beschrieben, welches eine essentielle Funktion während der Oozystenreifung in Plasmodium berghei besitzt. Die Transkription der Aktinregulatoren Profilin, ADF1 und ADF2 wird in Sporozoiten drastisch herunterreguliert und Profilin kann als Protein nicht mehr nachgewiesen werden. Um die Funktion von C-CAP und Profilin zu überprüfen, wurden beide Proteine spezifisch in Sporozoiten überexprimiert. Diese Parasiten sind nicht in der Lage die Speicheldrüsen des Wirtes zu besiedeln, was zum Abbruch des Lebenszykluses führt. Anhand dieser Ergebnisse entwickele ich ein „minimalistisches“ Model zur Beschreibung der Aktinregulation in Sporozoiten in welchem das ADF1 als regulatorisches Protein im Mittelpunkt steht.Plasmodium spp. are obligate intracellular parasites, which employ an conserved actin-dependent molecular motor machinery that facilitates their motility, host cell invasion and egress. In this work I report implications of the actin-regulators adenylyl cyclase-associated protein (C-CAP), profilin and actin depolymerization factor 1 and 2 (ADF1, ADF2) in distinct and previously unanticipated cellular processes during the life cycle of in the rodent malarial parasite Plasmodium berghei. Fluorescent tagging of the endogenous C-CAP genetic locus with mCherry revealed cytosolic distribution of the protein. Gene deletion demonstrates that the G-actin binding protein C-CAP is entirely dispensable for the pathogenic blood stages. Ookinetes show reduced motility, but are competent infecting the mosquito host. Defects emerging in the extracellular replication phase, leading to attenuation of oocyst maturation. Successful trans-species complementation with the C. parvum C-CAP ortholog, rescues the c-cap(-) phenotype and proves functional redundancy. The actin regulator profilin fails to rescue the defects of c-cap(-) parasites, despite sharing its actin sequestering activity with C-CAP. Taken together, C-CAP is the first G-actin sequestering protein of Plasmodium species that is not required for motility but performs essential functions during oocyst maturation. Characterization of the actin regulators profilin, ADF1 and ADF2 revealed dramatic transcriptional down-regulation and the absence of the profilin protein in sporozoites. To test whether G-actin binding proteins interfere with sporozoite functions, I ectopically overexpressed of profilin and C-CAP stage-specifically in sporozoites. This conducted to abolishment of salivary gland invasion and lifecycle arrest. Based on these unexpected findings and the available literature data, I developed a “minimalistic model” for actin regulation in sporozoites that predicts ADF1 as the main actin-turnover regulating factor

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

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

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 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

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