Plasmodium Development Research Articles

Effects of insecticide resistance and exposure on Plasmodium development in Anopheles mosquitoes.

The spread of insecticide resistance in anopheline mosquitoes is a serious threat to the success of malaria control and prospects of elimination, but the potential impact(s) of insecticide resistance or sublethal insecticide exposure on Plasmodium-Anopheles interactions are poorly understood. Only a few studies have attempted to investigate such interactions, despite their clear epidemiological significance for malaria transmission. This short review provides an update on our understanding of the interactions between insecticide resistance and exposure and Plasmodium development, focusing on the mechanisms which might underpin any interactions, and identifying some key knowledge gaps.

G-strand binding protein 2 is involved in asexual and sexual development of Plasmodium berghei

G-strand binding protein 2 (GBP2) is a Ser/Arg-rich (SR) protein involved in mRNA surveillance and nuclear mRNA quality control in yeast. However, the roles of GBP2 in virulence and sexual development in Plasmodium parasites are unclear, although GBP2 is involved in the asexual development of Plasmodium berghei, the rodent malaria parasite. In this study, we investigated the role of GBP2 in virulence and sexual development of P. berghei using gbp2-deleted P. berghei (Δgbp2 parasites). Then, to identify factors affected by gbp2 deletion, we performed a comparative proteomic analysis of the Δgbp2 parasites. We found that GBP2 was not associated with the development of experimental cerebral malaria during infection with P. berghei, but asexual development of the parasite was delayed with deletion of gbp2. However, the development of P. berghei gametocytes was significantly reduced with deletion of gbp2. Comparative proteomic analysis revealed that the levels of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in Δgbp2 parasites were significantly higher than those in wild-type (WT) parasites, suggesting that biosynthesis of purine nucleotides may be involved in function of GBP2. Therefore, we investigated the effect of purine starvation on the sexual development and proteome. In nt1-deleted P. berghei (Δnt1 parasites), the production of male and female gametocytes was significantly reduced compared to that in WT parasites. Moreover, we found that protein levels of GBP2 in Δnt1 parasites were markedly lower than in WT parasites. These findings suggest that GBP2 is primarily involved in the sexual development of malaria parasites, and its function may be suppressed by purine starvation.

Association of Plasmodium berghei With the Apical Domain of Hepatocytes Is Necessary for the Parasite's Liver Stage Development.

Plasmodium parasites undergo a dramatic transformation during the liver stage of their life cycle, amplifying over 10,000-fold inside infected hepatocytes within a few days. Such a rapid growth requires large-scale interactions with, and manipulations of, host cell functions. Whereas hepatocyte polarity is well-known to be critical for liver function, little is presently known about its involvement during the liver stage of Plasmodium development. Apical domains of hepatocytes are critical components of their polarity machinery and constitute the bile canalicular network, which is central to liver function. Here, we employed high resolution 3-D imaging and advanced image analysis of Plasmodium-infected liver tissues to show that the parasite associates preferentially with the apical domain of hepatocytes and induces alterations in the organization of these regions, resulting in localized changes in the bile canalicular architecture in the liver tissue. Pharmacological perturbation of the bile canalicular network by modulation of AMPK activity reduces the parasite's association with bile canaliculi and arrests the parasite development. Our findings using Plasmodium-infected liver tissues reveal a host-Plasmodium interaction at the level of liver tissue organization. We demonstrate for the first time a role for bile canaliculi, a central component of the hepatocyte polarity machinery, during the liver stage of Plasmodium development.

Gene copy number and function of the APL1 immune factor changed during Anopheles evolution

BackgroundThe recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined.MethodsThe APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis.ResultsAPL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus.ConclusionsAPL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa.

Hierarchical transcriptional control regulates Plasmodium falciparum sexual differentiation

BackgroundMalaria pathogenesis relies on sexual gametocyte forms of the malaria parasite to be transmitted between the infected human and the mosquito host but the molecular mechanisms controlling gametocytogenesis remains poorly understood. Here we provide a high-resolution transcriptome of Plasmodium falciparum as it commits to and develops through gametocytogenesis.ResultsThe gametocyte-associated transcriptome is significantly different from that of the asexual parasites, with dynamic gene expression shifts characterizing early, intermediate and late-stage gametocyte development and results in differential timing for sex-specific transcripts. The transcriptional dynamics suggest strict transcriptional control during gametocytogenesis in P. falciparum, which we propose is mediated by putative regulators including epigenetic mechanisms (driving active repression of proliferation-associated processes) and a cascade-like expression of ApiAP2 transcription factors.ConclusionsThe gametocyte transcriptome serves as the blueprint for sexual differentiation and will be a rich resource for future functional studies on this critical stage of Plasmodium development, as the intraerythrocytic transcriptome has been for our understanding of the asexual cycle.

Plasmodium falciparum R2TP complex: driver of parasite Hsp90 function.

Heat shock protein 90 (Hsp90) is essential for the development of the main malaria agent, Plasmodium falciparum. Inhibitors that target Hsp90 function are known to not only kill the parasite, but also reverse resistance of the parasite to traditional antimalarials such as chloroquine. For this reason, Hsp90 has been tagged as a promising antimalarial drug target. As a molecular chaperone, Hsp90 facilitates folding of proteins such as steroid hormone receptors and kinases implicated in cell cycle and development. Central to Hsp90 function is its regulation by several co-chaperones. Various co-chaperones interact with Hsp90 to modulate its co-operation with other molecular chaperones such as Hsp70 and to regulate its interaction with substrates. The role of Hsp90 in the development of malaria parasites continues to receive research attention, and several Hsp90 co-chaperones have been mapped out. Recently, focus has shifted to P. falciparum R2TP proteins, which are thought to couple Hsp90 to a diverse set of client proteins. R2TP proteins are generally known to form a complex with Hsp90, and this complex drives multiple cellular processes central to signal transduction and cell division. Given the central role that the R2TP complex may play, the current review highlights the structure-function features of Hsp90 relative to R2TPs of P. falciparum.

Identification of antimalarial leads with dual falcipain-2 and falcipain-3 inhibitory activity

Falcipains (FPs), cysteine proteases in the malarial parasite, are emerging as the promising antimalarial drug targets. In order to identify novel FP inhibitors, we generated a pharmacophore derived from the reported co-crystal structures of inhibitors of Plasmodium falciparum Falcipain-3 to screen the ZINC library. Further, the filters were applied for dock score, drug-like characters, and clustering of similar structures. Sixteen molecules were purchased and subject to in vitro enzyme (FP-2 and FP-3) inhibition assays. Two compounds showed in vitro inhibition of FP-2 and FP-3 at low µM concentration. The selectivity of the inhibitors can be explained based on the predicted interactions of the molecule in the active site. Further, the inhibitors were evaluated in a functional assay and were found to induce morphological changes in line with their mode of action arresting Plasmodium development. Compound 15 was most potent inhibitor identified in this study.

Quantitative Proteomic Analysis of Simian Primary Hepatocytes Reveals Candidate Molecular Markers for PermissivenesstoRelapsingMalariaPlasmodiumcynomolgi.

A major obstacle impeding malaria research is the lack of an in vitro system capable of supporting infection through the entire liver stage cycle of the parasite, including that of the dormant forms known as hypnozoites. Primary hepatocytes lose their liver specific functions in long-term in vitro culture. The malaria parasite Plasmodium initiates infection in hepatocyte. This corresponds to the first step of clinically silent infection and development of malaria parasite Plasmodium in the liver. Thus, the liver stage is an ideal target for development of novel antimalarial interventions and vaccines. However, drug discovery against Plasmodium liver stage is severely hampered by the poor understanding of host-parasite interactions during the liver stage infection and development. In this study, tandem mass tag labeling based quantitative proteomic analysis is performed in simian primary hepatocytes cultured in three different systems of susceptibility to Plasmodium infection. The results display potential candidate molecular markers, including asialoglycoprotein receptor, apolipoproteins, squalene synthase, and scavenger receptor B1 (SR-BI) that facilitate productive infection and full development in relapsing Plasmodium species. The identification of these candidate proteins required for constructive infection and development of hepatic malaria liver stages paves the way to explore them as therapeutic targets.

Asaia Activates Immune Genes in Mosquito Eliciting an Anti-Plasmodium Response: Implications in Malaria Control.

In mosquitoes, the discovery of the numerous interactions between components of the microbiota and the host immune response opens up the attractive possibility of the development of novel control strategies against mosquito borne diseases. We have focused our attention to Asaia, a symbiont of several mosquito vectors who has been proposed as one of the most potential tool for paratransgenic applications; although being extensively characterized, its interactions with the mosquito immune system has never been investigated. Here we report a study aimed at describing the interactions between Asaia and the immune system of two vectors of malaria, Anophelesstephensi and An. gambiae. The introduction of Asaia isolates induced the activation of the basal level of mosquito immunity and lower the development of malaria parasite in An. stephensi. These findings confirm and expand the potential of Asaia in mosquito borne diseases control, not only through paratransgenesis, but also as a natural effector for mosquito immune priming.

Inhibitors of Eicosanoid Biosynthesis Reveal that Multiple Lipid Signaling Pathways Influence Malaria Parasite Survival in Anopheles gambiae.

Eicosanoids are bioactive signaling lipids derived from the oxidation of fatty acids that act as important regulators of immune homeostasis and inflammation. As a result, effective anti-inflammatory drugs have been widely used to reduce pain and inflammation which target key eicosanoid biosynthesis enzymes. Conserved from vertebrates to insects, the use of these eicosanoid pathway inhibitors offer opportunities to evaluate the roles of eicosanoids in less-characterized insect systems. In this study, we examine the potential roles of eicosanoids on malaria parasite survival in the mosquito Anopheles gambiae. Using Plasmodium oocyst numbers to evaluate parasite infection, general or specific inhibitors of eicosanoid biosynthesis pathways were evaluated. Following the administration of dexamethasone and indomethacin, respective inhibitors of phospholipid A2 (PLA2) and cyclooxygenase (COX), oocyst numbers were unaffected. However, inhibition of lipoxygenase (LOX) activity through the use of esculetin significantly increased oocyst survival. In contrast, 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid (AUDA), an inhibitor of epoxide hydroxylase (EH), decreased oocyst numbers. These experiments were further validated through RNAi experiments to silence candidate genes homologous to EH in An. gambiae to confirm their contributions to Plasmodium development. Similar to the results of AUDA treatment, the silencing of EH significantly reduced oocyst numbers. These results imply that specific eicosanoids in An. gambiae can have either agonist or antagonistic roles on malaria parasite survival in the mosquito host.

Life Cycles of Myxogastria Stemonitopsis typhina and Stemonitis fusca on Agar Culture.

Myxogastria is a group of protozoa characterized by cellular uninucleate amoeboflagellates (myxamoebae and flagellated swarm cell), acellular multinucleate plasmodia, and stationary spore‐bearing sporocarps. The Stemonitales is a large order in the Myxogastria and contains approximately 230 species, but only 13 species have their completed life cycles observed so far. Here, we described the life cycles of two species in Stemonitales, Stemonitopsis typhina and Stemonitis fusca by culturing in water agar medium and observing the morphogenesis of their spore germination, plasmodium, and sporocarp development. The spore‐to‐spore life cycles of Ste. typhina and S. fusca were completed in approximately 67 and 12 d, respectively. Both species possessed an aphanoplasmodium. However, the spores of Ste. typhina and S. fusca germinated by the V‐shape split and pore methods, respectively. Unlike S. fusca with an evanescent peridium, Ste. typhina produced a shiny persistent peridium which was continuous with the membrane surrounding its stalk. The information will contribute to a better understanding of their taxonomy and phylogeny.

Host Cytoskeleton Remodeling throughout the Blood Stages of Plasmodium falciparum.

The asexual intraerythrocytic development of Plasmodium falciparum, causing the most severe form of human malaria, is marked by extensive host cell remodeling. Throughout the processes of invasion, intracellular development, and egress, the erythrocyte membrane skeleton is remodeled by the parasite as required for each specific developmental stage. The remodeling is facilitated by a plethora of exported parasite proteins, and the erythrocyte membrane skeleton is the interface of most of the observed interactions between the parasite and host cell proteins. Host cell remodeling has been extensively described and there is a vast body of information on protein export or the description of parasite-induced structures such as Maurer's clefts or knobs on the host cell surface. Here we specifically review the molecular level of each host cell-remodeling step at each stage of the intraerythrocytic development of P. falciparum We describe key events, such as invasion, knob formation, and egress, and identify the interactions between exported parasite proteins and the host cell cytoskeleton. We discuss each remodeling step with respect to time and specific requirement of the developing parasite to explain host cell remodeling in a stage-specific manner. Thus, we highlight the interaction with the host membrane skeleton as a key event in parasite survival.

A Gut Symbiotic Bacterium Serratia marcescens Renders Mosquito Resistance to Plasmodium Infection Through Activation of Mosquito Immune Responses.

The malaria development in the mosquito midgut is a complex process that results in considerable parasite losses. The mosquito gut microbiota influences the outcome of pathogen infection in mosquitoes, but the underlying mechanisms through which gut symbiotic bacteria affect vector competence remain elusive. Here, we identified two Serratia strains (Y1 and J1) isolated from field-caught female Anopheles sinensis from China and assessed their effect on Plasmodium development in An. stephensi. Colonization of An. stephensi midgut by Serratia Y1 significantly renders the mosquito resistant to Plasmodium berghei infection, while Serratia J1 has no impact on parasite development. Parasite inhibition by Serratia Y1 is induced by the activation of the mosquito immune system. Genome-wide transcriptomic analysis by RNA-seq shows a similar pattern of midgut gene expression in response to Serratia Y1 and J1 in sugar-fed mosquitoes. However, 24 h after blood ingestion, Serratia Y1 modulates more midgut genes than Serratia J1 including the c-type lectins (CTLs), CLIP serine proteases and other immune effectors. Furthermore, silencing of several Serratia Y1-induced anti-Plasmodium factors like the thioester-containing protein 1 (TEP1), fibrinogen immunolectin 9 (FBN9) or leucine-rich repeat protein LRRD7 can rescue parasite oocyst development in the presence of Serratia Y1, suggesting that these factors modulate the Serratia Y1-mediated anti-Plasmodium effect. This study enhances our understanding of how gut bacteria influence mosquito-Plasmodium interactions.

Parasitism: Anopheles Mosquitoes and Plasmodium Parasites Share Resources

Female Anopheles mosquitoes are the definitive hosts of Plasmodium parasites. A new study has found that successful establishment and development of Plasmodium in the Anopheles midgut requires mosquito oogenesis, without affecting egg production.

Blocking Plasmodium Development in Mosquitoes: A Powerful New Approach for Expanding Malaria Control Efforts

Blocking Plasmodium Development in Mosquitoes: A Powerful New Approach for Expanding Malaria Control Efforts

Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

CXCR4 regulates Plasmodium development in mouse and human hepatocytes.

The liver stage of the etiological agent of malaria, Plasmodium, is obligatory for successful infection of its various mammalian hosts. Differentiation of the rod-shaped sporozoites of Plasmodium into spherical exoerythrocytic forms (EEFs) via bulbous expansion is essential for parasite development in the liver. However, little is known about the host factors regulating the morphological transformation of Plasmodium sporozoites in this organ. Here, we show that sporozoite differentiation into EEFs in the liver involves protein kinase C ζ-mediated NF-κB activation, which robustly induces the expression of C-X-C chemokine receptor type 4 (CXCR4) in hepatocytes and subsequently elevates intracellular Ca2+ levels, thereby triggering sporozoite transformation into EEFs. Blocking CXCR4 expression by genetic or pharmacological intervention profoundly inhibited the liver-stage development of the Plasmodium berghei rodent malaria parasite and the human Plasmodium falciparum parasite. Collectively, our experiments show that CXCR4 is a key host factor for Plasmodium development in the liver, and CXCR4 warrants further investigation for malaria prophylaxis.

Modeling the Potential Role of Engineered Symbiotic Bacteria in Malaria Control.

Recent experimental study suggests that the engineered symbiotic bacteria Serratia AS1 may provide a novel, effective and sustainable biocontrol of malaria. These recombinant bacteria have been shown to be able to rapidly disseminate throughout mosquito populations and to efficiently inhibit development of malaria parasites in mosquitoes in controlled laboratory experiments. In this paper, we develop a climate-based malaria model which involves both vertical and horizontal transmissions of the engineered Serratia AS1 bacteria in mosquito population. We show that the dynamics of the model system is totally determined by the vector reproduction ratio [Formula: see text], and the basic reproduction ratio [Formula: see text]. If [Formula: see text], then the mosquito-free state is globally attractive. If [Formula: see text] and [Formula: see text], then the disease-free periodic solution is globally attractive. If [Formula: see text] and [Formula: see text], then the positive periodic solution is globally attractive. Numerically, we verify the obtained analytic result and evaluate the effects of releasing the engineered Serratia AS1 bacteria in field by conducting a case study for Douala, Cameroon. We find that ideally, by using Serratia AS1 alone, it takes at least 25 years to eliminate malaria from Douala. This implies that continued long-term investment is needed in the fight against malaria and confirms the necessity of integrating multiple control measures.

Reduced mosquito survival in metal-roof houses may contribute to a decline in malaria transmission in sub-Saharan Africa

In The Gambia, metal-roof houses were hotter during the day than thatched-roof houses. After 24 h, the mortality of Anopheles gambiae, the principal African malaria vector, was 38% higher in metal-roof houses than thatched ones. During the day, mosquitoes in metal-roof houses moved from the hot roof to cooler places near the floor, where the temperature was still high, reaching 35 °C. In laboratory studies, at 35 °C few mosquitoes survived 10 days, the minimum period required for malaria parasite development. Analysis of epidemiological data showed there was less malaria and lower vector survival rates in Gambian villages with a higher proportion of metal roofs. Our findings are consistent with the hypothesis that the indoor climate of metal-roof houses, with higher temperatures and lower humidity, reduces survivorship of indoor-resting mosquitoes and may have contributed to the observed reduction in malaria burden in parts of sub-Saharan Africa.

Steroid Hormone Function Controls Non-competitive Plasmodium Development in Anopheles

Transmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20-hydroxyecdysone (20E), remains largely unknown. Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P.falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.