Host Cell Phosphatidylcholine Is a Key Mediator of Malaria Parasite Survival during Liver Stage Infection

During invasion, Plasmodium, the causative agent of malaria, wraps itself in a parasitophorous vacuole membrane (PVM), which constitutes a critical interface between the parasite and its host cell. Within hepatocytes, each Plasmodium sporozoite generates thousands of new parasites, creating high demand for lipids to support this replication and enlarge the PVM. Here, a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes reveals an enrichment of neutral lipids and the major membrane phospholipid, phosphatidylcholine (PC). While infection is unaffected in mice deficient in key enzymes involved in neutral lipid synthesis and lipolysis, ablation of rate-limiting enzymes in hepatic PC biosynthetic pathways significantly decreases parasite numbers. Host PC is taken up by both P. berghei and P. falciparum and is necessary for correct localization of parasite proteins to the PVM, which is essential for parasite survival. Thus, Plasmodium relies on the abundance of these lipids within hepatocytes to support infection.Seventh Framework Programme (European Commission) (Grant Agreement 311502)Fundacao para a Ciencia e a Tecnologia (Grant EXCL/IMI-MIC/0056/2012)Fundacao para a Ciencia e a Tecnologia (Grant PTDC/IMI-MIC/1568/2012

Rethinking human resources and capacity building needs for malaria control and elimination in Africa.

Despite considerable success in controlling malaria worldwide, progress toward achieving malaria elimination has largely stalled. In particular, strategies to overcome roadblocks in malaria control and elimination in Africa are critical to achieving worldwide malaria elimination goals-this continent carries 94% of the global malaria case burden. To identify key areas for targeted efforts, we combined a comprehensive review of current literature with direct feedback gathered from frontline malaria workers, leaders, and scholars from Africa. Our analysis identified deficiencies in human resources, training, and capacity building at all levels, from research and development to community involvement. Addressing these needs will require active and coordinated engagement of stakeholders as well as implementation of effective strategies, with malaria-endemic countries owning the relevant processes. This paper reports those valuable identified needs and their concomitant opportunities to accelerate progress toward the goals of the World Health Organization's Global Technical Strategy for Malaria 2016-2030. Ultimately, we underscore the critical need to re-think current approaches and expand concerted efforts toward increasing relevant human resources for health and capacity building at all levels if we are to develop the relevant competencies necessary to maintain current gains while accelerating momentum toward malaria control and elimination

Precise coordination between nutrient transporters ensures fertility in the malaria mosquito Anopheles gambiae.

Females from many mosquito species feed on blood to acquire nutrients for egg development. The oogenetic cycle has been characterized in the arboviral vector Aedes aegypti, where after a bloodmeal, the lipid transporter lipophorin (Lp) shuttles lipids from the midgut and fat body to the ovaries, and a yolk precursor protein, vitellogenin (Vg), is deposited into the oocyte by receptor-mediated endocytosis. Our understanding of how the roles of these two nutrient transporters are mutually coordinated is however limited in this and other mosquito species. Here, we demonstrate that in the malaria mosquito Anopheles gambiae, Lp and Vg are reciprocally regulated in a timely manner to optimize egg development and ensure fertility. Defective lipid transport via Lp knockdown triggers abortive ovarian follicle development, leading to misregulation of Vg and aberrant yolk granules. Conversely, depletion of Vg causes an upregulation of Lp in the fat body in a manner that appears to be at least partially dependent on target of rapamycin (TOR) signaling, resulting in excess lipid accumulation in the developing follicles. Embryos deposited by Vg-depleted mothers are completely inviable, and are arrested early during development, likely due to severely reduced amino acid levels and protein synthesis. Our findings demonstrate that the mutual regulation of these two nutrient transporters is essential to safeguard fertility by ensuring correct nutrient balance in the developing oocyte, and validate Vg and Lp as two potential candidates for mosquito control

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential.

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite's extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R0, and find the average R0 is higher (range: 10.1%-12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions

Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes.

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver

Hepatocyte cytoskeleton reorganization depends on the presence of <i>P. berghei</i>.

<p>Percentage of parasites (<i>P. berghei</i> and <i>T. gondii</i>) and beads associated with hepatocyte actin reorganization between 10 and 16 hours p.i. or bead internalization. Numbers of parasites or beads associated with actin reorganization/total numbers of parasites or beads analysed are indicated above the bars. Error bars represent Standard Errors * p<0.05 *** p≤0.001.</p

Gelsolin is important for actin reorganization around developing <i>P. berghei</i> and parasite disappearance.

<p>(<b>A</b>) GSN mRNA (top) and protein (bottom) expression after knockdown with shRNA. GSN and α-tubulin bands corresponds to 93 kDa and 55 kDa molecular weights, respectively. (<b>B</b>) Phalloidin staining of Huh7 cells transduced with Scramble and GSN shRNA (grey: F-actin). (<b>C</b>) Effect of GSN knockdown on the percentage of <i>P. berghei</i> parasites associated with hepatocyte actin reorganization between 10 and 16 hours p.i.. Numbers of parasites associated with actin reorganization/total numbers of parasites analysed are indicated above the bars. Error bars represent Standard Errors * p<0.05 ** p<0.01; scale bars represent 10 µm.</p

Gelsolin expression and distribution around developing <i>P. berghei</i>.

<p>(<b>A</b>) Microarray data of GSN expression levels in <i>P. berghei</i>- infected and non infected Hepa1-6 cells at different time points of infection, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029408#pone.0029408-Albuquerque1" target="_blank">[18]</a> (<b>B</b>) qRT-PCR data of GSN expression in <i>P. berghei</i> infected and non-infected Huh7 cells at 12 hours p.i.. (<b>C</b>) Huh7 cells showing an accumulation of GSN around GFP-Pb in a pattern similar to an actin cloud and tail-like actin cloud (red and grey: anti-GSN antibody, green: GFP-Pb; blue: nuclei). Scale bars represent 10 µm.</p

Hepatocyte cytoskeleton reorganization around developing P. berghei <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Actin but not tubulin is reorganized in Huh7 cell lines infected with GFP-Pb parasites (red and grey: mCherry::α-tubulin or mCherry::β-actin; green: GFP-Pb). Plot profiles represent the pixel intensity (gray values) of the regions indicated in the pictures (lines); <b>o</b> represents the parasite. Pie plots represent the percentages and absolute numbers of parasites associated (▪) or not (□) with host cytoskeleton reorganization. (<b>B</b>) Distribution of the percentage of <i>P. berghei</i> parasites associated with host cell actin reorganization in different infection periods. Numbers of parasites associated with actin reorganization/total numbers of parasites analysed are indicated above the bars. (<b>C</b>) Hepatocyte F-actin around <i>P. berghei</i> EEFs at 24 hours p.i. in BALB/c mice livers (red and grey: Phalloidin AlexaFluor 594; green: GFP-Pb; blue: nuclei). The pie plot represents the percentage and absolute numbers of parasites associated (▪) or not (□) with host actin reorganization. Plot profile represents the pixel intensity (gray values) along the white line; <b>o</b> represents the parasite. Error bars represent Standard Errors ** p<0.01, scale bars represent 10 µm.</p

Gametocytemia and COI for <i>P</i>. <i>falciparum</i> field isolates.

Gametocytemia and COI for P. falciparum field isolates.</p