An attenuated total reflection (ATR) and Raman spectroscopic investigation into the effects of chloroquine on Plasmodium falciparum-infected red blood cells

Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) and Raman spectroscopy were used to compare chloroquine (CQ)-treated and untreated cultured Plasmodium falciparum-infected human red blood cells (iRBCs). The studies were carried out in parallel from the same starting cultures using both spectroscopic techniques, in duplicate. ATR FTIR spectra showed modifications in the heme vibrational bands as well as increases in the CH2/CH3 stretching bands in the 3100–2800 cm−1 region of CQ-treated iRBCs consistent with an increase in lipid content. Other changes consisted of secondary structural variations including shifts in the amide I and II modes, along with changes in RNA and carbohydrate bands. Raman microspectroscopy of single red blood cells using 532 nm revealed subtle changes in the positions and intensity of ν37 of the core size region marker band and ν4 in the pyrrole ring-stretching region between untreated and CQ-treated iRBCs. Similar patterns in the corresponding relations were also observed in the non-fundamental (overtone region) between the control and treated cells. These differences were consistent with higher levels of oxygenated hemoglobin (oxyHb) in the treated cells as shown in a Principle Component Analysis (PCA) loadings plot. The results obtained demonstrate that vibrational spectroscopic techniques can provide insight into the effect of quinolines on iRBCs and thus may assist understanding the sensitivity and resistance of new and existing anti-malarial drugs

Human Aurora kinase inhibitor Hesperadin reveals epistatic interaction between Plasmodium falciparum PfArk1 and PfNek1 kinases

Mitosis has been validated by numerous anti-cancer drugs as being a druggable process, and selective inhibition of parasite proliferation provides an obvious opportunity for therapeutic intervention against malaria. Mitosis is controlled through the interplay between several protein kinases and phosphatases. We show here that inhibitors of human mitotic kinases belonging to the Aurora family inhibit P. falciparum proliferation in vitro with various potencies, and that a genetic selection for mutant parasites resistant to one of the drugs, Hesperadin, identifies a resistance mechanism mediated by a member of a different kinase family, PfNek1 (PF3D7_1228300). Intriguingly, loss of PfNek1 catalytic activity provides protection against drug action. This points to an undescribed functional interaction between Ark and Nek kinases and shows that existing inhibitors can be used to validate additional essential and druggable kinase functions in the parasite.Larkins Fellowship, NIH, MMV, Bill and Melinda Gates Foundation, Medical Research Council and South African Research Chairs Initiative of the Department of Science and Technology.https://www.nature.com/commsbiopm2021BiochemistryGeneticsMicrobiology and Plant Patholog

Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development.

Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria

Expression profile of the <i>Pfge</i> genes through gametocytogenesis.

<p>MACS purified late stage asexual parasite cultures were set up at 6% hematocrit and sorbitol synchronized 2 hours later to remove all but the newly invaded ring stage parasites. Parasitemia was monitored by daily Giemsa-stained smear. Ring (blue), schizont stage parasitemias (red), and stage II gametocytemia (green) are plotted (A & B). The relative abundance, (log2) of the indicated gene in relation to the expression level on day 2 was calculated using the 2^{−ΔΔ<i>C</i>}_T method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Schmittgen1" target="_blank">[43]</a> with seryl tRNA synthetase as the reference and plotted in C and D): <i>Pfaldolase</i> (blue), <i>Pfkahrp</i> (brown), <i>Pfmsp7-5</i> (purple), <i>Pfs16 (</i>green), <i>Pfgdv1</i> (bright red); while <i>Pfge1</i> (orange), <i>Pfge2</i> (pale pink), <i>Pfge3</i> (beige), <i>Pfg27</i> (light blue), <i>Pfge7</i> (dark pink), <i>Pfge8</i> (light green), <i>Pfs47</i>(turquoise) and <i>Pfs16</i> (green) are shown in (E and F). The 3 asexual cycles are indicated by numbers as well as gray dotted lines, and NAG treatment is indicated by the gray box. Representative data from one of three independent experiments is shown. The samples from the different time points were tested in triplicate and the average relative expression is plotted with the error bars representing the range.</p

Identification of <i>Plasmodium falciparum</i> gametocyte development 1 gene (<i>Pfgdv1</i>).

<p>A) Schematic of chromosome 9 (1–1,541,723 bp) showing the segment deleted (arrows) in the 3D7.G_def clone. For orientation, the putative centromere is indicated by an O and the 1,500,000 bp position is marked (1500 k). Colored boxes indicate the location of annotated genes: blue, genes transcribed toward the telomere; red, transcribed toward the centromere; horizontal stripes, <i>Pfgdv1;</i> diagonal stripes, <i>var</i> and <i>rifins</i>. The 70-mer oligonucleotide that identified the chromosome 9 deletion in 3D7.G_def parasites is indicated by an asterisk (*). The numbered gray bars below the line indicate the positions of the eight PCR products used to map the chromosome 9 deletion. B) Amplification products from chromosome 9 using Indochina, 3D7.G_def, FCR3.G_def, or HB3.G_def gDNA as a template. Peak gametocytemias attained in two independent experiments are indicated on the right of the ethidium bromide-stained agarose gel of the PCR products generated using the eight primer pairs (1–8). C) Southern blot of <i>Bsa</i>BI-digested gDNA from 3D7.G+ (+), 3D7.G_def (d), 3D7.G_def +<i>Pfgdv1</i> (a), 3D7.G_def +HA.<i>Pfgdv1</i> (b) and the parental 3D7 parasites (WT). Digested gDNA was probed with <i>Pfgdv1</i> (bp 1423–1800) or <i>Pfg27</i> (bp 1–654). D–E) Gametocyte production in WT (black square), 3D7.G_def (black triangle), complemented line a, 3D7.G_def +<i>Pfgdv1</i> (Panel D, black inverted triangle) and line b, 3D7.G_def +HA.<i>Pfgdv1</i> (Panel E, unfilled inverted triangle). Cultures set up at 0.1% asexual parasitemia on day 0 were followed for gametocyte production by Giemsa-stained smears for the next 16 days. Mean gametocytemia and standard deviation of two (D) or three (E) independent experiments are shown (<i>P</i><0.003 by linear regression analysis). F) Giemsa-stained smear of parasitized erythrocytes purified on a 16% Nycodenz cushion from day 16 gametocyte cultures of WT, 3D7.G_def +HA.<i>Pfgdv1</i> (HA.<i>Pfgdv1</i>), and 3D7.G_def (G_def) lines. G) Northern blots of RNA harvested from WT 3D7 (w), 3D7.G_def (d), 3D7.G_def<i>+Pfgdv1</i> (a), and 3D7.G_def +HA.<i>Pfgdv1</i> (b) complemented lines were hybridized with probes corresponding to <i>Pfgdv1</i> (<i>gdv1</i>), <i>Pfge</i> genes (<i>ge1–3, 5–11</i>), and merozoite surface protein-1 (<i>msp1</i>) as an asexual parasite control. Autoradiographs are shown with the corresponding ethidium bromide-stained gel. H) Expression of gametocyte specific antigen Pfs48/45. Methanol-fixed WT, 3D7.G_def+<i>Pfgdv1</i> (<i>a</i>) and 3D7.G_def+HA.<i>Pfgdv1</i> (b) mature gametocyte cultures were incubated with Pfs48/45 mAb IIC5B10 (1∶250 dilution) and labeled secondary antibodies (1∶500 dilution). I) The average gametocytemia of 4 independent cultures of WT 3D7 (WT), WT 3D7 transformed with a <i>Pfgdv1</i> episomal expression plasmid (WT+HA.<i>Pfgdv1</i>) and the G_def (G_def) line is graphed. The error bars represent the SEM and a significant difference from WT and G_def is indicated by an asterisk (p<0.05 ANOVA followed by Tukey multiple comparison test).</p

Model for continuous gametocytogenesis.

<p>During each round of the asexual cycle, a proportion of the schizonts (S_c, light blue) produced are committed to producing merozoites (M_c, light blue) that will differentiate into gametocytes (G_c, G_I–V) after invading a RBC. The expression profile for <i>Pfgdv1</i> is indicated by a line under the corresponding stage.</p

Subcellular localization of PfGDV1.

<p>Parasites transformed with GFP- or HA-tagged PfGDV1 were stained with DAPI (DNA stain) and the indicated anti-sera, and then examined by fluorescence microscopy (Zeiss Axiovert 200, 1000× magnification). Images are shown of the DAPI stain (DNA), GFP-tagged PfGDV1 epifluorescence (GDV1), and antibodies specific for HA (αHA), Pfs16 (αPfs16), PfGE3 (αPfGE3), PfMCM2 (αMCM2), and PfSir2 (αSir2). The corresponding merged and bright field (BF) images are included on the right. PfGDV1 expression is indicated with an arrow; locations of parasites in the BF image are indicated with a P for parasite or S for schizont. A) A schizont (S) (<i>Upper</i>) expressing GDV1 and a doubly infected erythrocyte (<i>Lower</i>) with one parasite (P1) expressing HA-tagged PfGDV1 (αHA) and another negative (P2) for anti-HA antibodies. B) Co-staining of parasites expressing GDV1 with early gametocytogenesis markers. A doubly infected erythrocyte (<i>Upper</i>) with one parasite (P1) positive for GDV1 and αPfs16 and the other (P2) negative for both. An erythrocyte (<i>Lower</i>) infected with a parasite (P) positive for GDV1 and αPfGE3. C) Co-localization of PfGDV1 with nuclear proteins. A doubly infected erythrocyte (<i>Upper</i>) with one parasite in the plane of the image (P1) and the other below (P2). Both P1 and P2 are positive for GDV1 and αMCM2. A schizont (S) (<i>Lower</i>) expressing GDV1 stained with αSir2.</p

<i>In vivo Pfge</i> gene expression profiling.

<p>RNA from 20 gametocytemic patients was analyzed using a <i>P. falciparum</i> whole genome microarray. The color coded cluster analysis of the quantile normalized expression data for the <i>Pfge</i> genes with a G+/G_def ratio>10 is shown with mature gametocyte specific genes <i>Pfs25</i> and <i>Pfs28</i> (Underlined in gray). The gene name and standardized intensity of the color code is indicated below and the patient cluster associated with high gametocytemia is indicated on the right with a vertical green bar. The gametocytemia (G'cyte) and asexual parasitemia (Asex) of the patient samples are represented both by color code and numerically on the right.</p

Expression analysis of the 3D7.G+ and 3D7.G_def clones identifies <i>P. falciparum</i>gametocytogenesis early genes (<i>Pfge</i>).

<p>Mean ratios of microarray signals (mean±SEM) obtained from the 3D7.G+ and 3D7.G_def clones at 1.4/0.9% parasitemia (dark gray bar) and at 5.2/5.5% parasitemia (light gray bar), respectively, for the 11 <i>Pfge</i> genes with a ratio of 3D7.G+/3D7.G_def>10 are plotted in descending order. <i>Pfgdv1</i> (PFI1710w) had the fourth highest ratio and is indicated by an ^o. Previously described <i>Pfge</i> genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Young1" target="_blank">[17]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Bruce2" target="_blank">[20]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-vanSchaijk1" target="_blank">[22]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Silvestrini3" target="_blank">[82]</a>, are denoted with an asterisk (*) and a vertical line indicates a gene represented by two oligonucleotides. <i>Pfge</i> number, PlasmoDB ID, and common name are listed on the right. The central panel is a summary of the characteristics of the <i>Pfgdv1</i>-dependent genes. The first row (L) indicates whether the gene is subtelomeric (<150 kb from the telomere) (gray square) or within a region of the chromosome that has synteny with other species (black square) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002964#ppat.1002964-Kooij1" target="_blank">[83]</a>. The panel on the left indicates whether the gene encodes a secretory signal sequence (S, black square), PEXEL/HTS export domain (E, black square) or transmembrane domain (T, black square).</p