Pathogenic role of mitogen activated protein kinases in protozoan parasites.

Protozoan parasites with complex life cycles have high mortality rates affecting billions of human lives. Available anti-parasitic drugs are inadequate due to variable efficacy, toxicity, poor patient compliance and drug-resistance. Hence, there is an urgent need for the development of safer and better chemotherapeutics. Mitogen Activated Protein Kinases (MAPKs) have drawn much attention as potential drug targets. This review summarizes unique structural and functional features of MAP kinases and their possible role in pathogenesis of obligate intracellular protozoan parasites namely, Leishmania, Trypanosoma, Plasmodium and Toxoplasma. It also provides an overview of available knowledge concerning the target proteins of parasite MAPKs and the need to understand and unravel unknown interaction network(s) of MAPK(s).

Susceptibility of Field-Collected Nyssorhynchus darlingi to Plasmodium spp. in Western Amazonian Brazil.

Mosquito susceptibility to Plasmodium spp. infection is of paramount importance for malaria occurrence and sustainable transmission. Therefore, understanding the genetic features underlying the mechanisms of susceptibility traits is pivotal to assessing malaria transmission dynamics in endemic areas. The aim of this study was to investigate the susceptibility of Nyssorhynchus darlingi-the dominant malaria vector in Brazil-to Plasmodium spp. using a reduced representation genome-sequencing protocol. The investigation was performed using a genome-wide association study (GWAS) to identify mosquito genes that are predicted to modulate the susceptibility of natural populations of the mosquito to Plasmodium infection. After applying the sequence alignment protocol, we generated the variant panel and filtered variants; leading to the detection of 202,837 SNPs in all specimens analyzed. The resulting panel was used to perform GWAS by comparing the pool of SNP variants present in Ny. darlingi infected with Plasmodium spp. with the pool obtained in field-collected mosquitoes with no evidence of infection by the parasite (all mosquitoes were tested separately using RT-PCR). The GWAS results for infection status showed two statistically significant variants adjacent to important genes that can be associated with susceptibility to Plasmodium infection: Cytochrome P450 (cyp450) and chitinase. This study provides relevant knowledge on malaria transmission dynamics by using a genomic approach to identify mosquito genes associated with susceptibility to Plasmodium infection in Ny. darlingi in western Amazonian Brazil.

Immunopotentiation by Lymph-Node Targeting of a Malaria Transmission-Blocking Nanovaccine.

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs.

Lipid transport proteins in malaria, from Plasmodium parasites to their hosts.

Eukaryotic unicellular pathogens from the genus Plasmodium are the etiological agents of malaria, a disease that persists over a wide range of vertebrate species, including humans. During its dynamic lifecycle, survival in the different hosts depends on the parasite's ability to establish a suitable environmental milieu. To achieve this, specific host processes are exploited to support optimal growth, including extensive modifications to the infected host cell. These modifications include the formation of novel membranous structures, which are induced by the parasite. Consequently, to maintain a finely tuned and dynamic lipid environment, the organisation and distribution of lipids to different cell sites likely requires specialised lipid transfer proteins (LTPs). Indeed, several parasite and host-derived LTPs have been identified and shown to be essential at specific stages. Here we describe the roles of LTPs in parasite development and adaptation to its host including how the latest studies are profiting from the improved genetic, lipidomic and imaging toolkits available to study Plasmodium parasites. Lastly, a list of predicted Plasmodium LTPs is provided to encourage research in this field.

Plasmodium chitinases: revisiting a target of transmission-blockade against malaria.

Malaria is a life-threatening disease caused by one of the five species of Plasmodium, among which Plasmodium falciparum cause the deadliest form of the disease. Plasmodium species are dependent on a vertebrate host and a blood-sucking insect vector to complete their life cycle. Plasmodium chitinases belonging to the GH18 family are secreted inside the mosquito midgut, during the ookinete stage of the parasite. Chitinases mediate the penetration of parasite through the peritrophic membrane, facilitating access to the gut epithelial layer. In this review, we describe Plasmodium chitinases with special emphasis on chitinases from P. falciparum and P. vivax, the representative examples of the short and long forms of this protein. In addition to the chitinase domain, chitinases belonging to the long form contain a pro-domain and chitin-binding domain. Amino acid sequence alignment of long and short form chitinase domains reveals multiple positions containing variant residues. A subset of these positions was found to be conserved or invariant within long or short forms, indicating the role of these positions in attributing form-specific activity. The reported differences in affinities to allosamidin for P. vivax and P. falciparum were predicted to be due to different residues at two amino acid positions, resulting in altered interactions with the inhibitor. Understanding the role of these amino acids in Plasmodium chitinases will help us elucidate the mechanism of catalysis and the mode of inhibition, which will be the key for identification of potent inhibitors or antibodies demonstrating transmission-blocking activity.

The Ins and Outs of Plasmodium Rhoptries, Focusing on the Cytosolic Side.

Parasites of the genus Plasmodium cause human and animal malaria, leading to significant health and economic impacts. A key aspect of the complex life cycle of Plasmodium parasites is the invasion of the parasite into its host cell, which is mediated by secretory organelles. The largest of these organelles, the rhoptry, undergoes rapid and profound physiological changes when it secretes its contents during merozoite and sporozoite invasion of the host erythrocyte and hepatocyte, respectively. Here we discuss recent advancements in our understanding of the dynamic rhoptry biology during the parasite's invasive stages, with a focus on the roles of cytosolically exposed rhoptry-interacting proteins (C-RIPs). We explore potential similarities between the molecular mechanisms driving merozoite and sporozoite rhoptry function.

Crystalloids: Fascinating Parasite Organelles Essential for Malaria Transmission.

Crystalloids are malaria parasite organelles exclusive to the ookinete and young oocyst life stages that infect the mosquito. The organelles have key roles in sporozoite development and infectivity but the way this is facilitated on a molecular level remains poorly understood. Recent discoveries have shed new light on these processes.

Vesicle dynamics during the egress of malaria gametocytes from the red blood cell.

Malaria parasites are obligate intracellular pathogens that live in human red blood cells harbored by a parasitophorous vacuole. The parasites need to exit from the red blood cell to continue life-cycle progression and ensure human-to-mosquito transmission. Two types of blood stages are able to lyse the enveloping red blood cell to mediate egress, the merozoites and the gametocytes. The intraerythrocytic parasites exit the red blood cell via an inside-out mode during which the membrane of the parasitophorous vacuole ruptures prior to the red blood cell membrane. Membrane rupture is initiated by the exocytosis of specialized secretory vesicles following the perception of egress triggers. The molecular mechanisms of red blood cell egress have particularly been studied in malaria gametocytes. Upon activation by external factors, gametocytes successively discharge at least two types of vesicles, the osmiophilic bodies needed to rupture the parasitophorous vacuole membrane and recently identified egress vesicles that are important for the perforation of the erythrocyte membrane. In recent years, important components of the signaling cascades leading to red blood cell egress have been investigated and several proteins of the osmiophilic bodies have been identified. We here report on the newest findings on the egress of gametocytes from the red blood cell. We further focus on the content and function of the egress-related vesicles and discuss the molecular machinery that might drive vesicle discharge.

mSphere of Influence: an Army Marching on Its Stomach-Malaria Parasites Sense and Subvert Host Nutrition.

Clare Harding works on the metal biology of the parasite Toxoplasma gondii In this mSphere of Influence article, she reflects on how two papers from the laboratory of Maria Mota, "Host-mediated regulation of superinfection in malaria" by Portugal et al. (S. Portugal, C. Carret, M. Recker, A. E. Armitage, et al., Nat Med 17:732-737, 2011, https://doi.org/10.1038/nm.2368) and "Nutrient sensing modulates malaria parasite virulence" by Mancio-Silva et al. (L. Mancio-Silva, K. Slavic, M. T. Grilo Ruivo, A. R. Grosso, et al., Nature 547:213-216, 2017, https://doi.org/10.1038/nature23009), made an impact on her understanding of host-pathogen interactions by examining the complex interplay between parasites and their hosts' nutritional status.

Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.

Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species.

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

Retrospective Analysis Using Pharmacokinetic/Pharmacodynamic Modeling and Simulation Offers Improvements in Efficiency of the Design of Volunteer Infection Studies for Antimalarial Drug Development.

Volunteer infection studies using the induced blood stage malaria (IBSM) model have been shown to facilitate antimalarial drug development. Such studies have traditionally been undertaken in single-dose cohorts, as many as necessary to obtain the dose-response relationship. To enhance ethical and logistic aspects of such studies, and to reduce the number of cohorts needed to establish the dose-response relationship, we undertook a retrospective in silico analysis of previously accrued data to improve study design. A pharmacokinetic (PK)/pharmacodynamic (PD) model was developed from initial fictive-cohort data for OZ439 (mixing the data of the three single-dose cohorts as: n = 2 on 100 mg, 2 on 200 mg, and 4 on 500 mg). A three-compartment model described OZ439 PKs. Net growth of parasites was modeled using a Gompertz function and drug-induced parasite death using a Hill function. Parameter estimates for the PK and PD models were comparable for the multidose single-cohort vs. the pooled analysis of all cohorts. Simulations based on the multidose single-cohort design described the complete data from the original IBSM study. The novel design allows for the ascertainment of the PK/PD relationship early in the study, providing a basis for rational dose selection for subsequent cohorts and studies.

γδ T cells in malaria: a double-edged sword.

Malaria remains a devastating global health problem, resulting in many annual deaths due to the complications of severe malaria. However, in endemic regions, individuals can acquire 'clinical immunity' to malaria, characterized by a decrease in severe malaria episodes and an increase of asymptomatic Plasmodium falciparum infections. Recently, it has been reported that tolerance to 'clinical malaria' and reduced disease severity correlates with a decrease in the numbers of circulating Vγ9Vδ2 T cells, the major subset of γδ T cells in the human peripheral blood. This is particularly interesting as this population typically undergoes dramatic expansions during acute Plasmodium infections and was previously shown to play antiparasitic functions. Thus, regulated γδ T-cell responses may be critical to balance immune protection with severe pathology, particularly as both seem to rely on the same pro-inflammatory cytokines, most notably TNF and IFN-γ. This has been clearly demonstrated in mouse models of experimental cerebral malaria (ECM) based on Plasmodium berghei ANKA infection. Furthermore, our recent studies suggest that the natural course of Plasmodium infection, mimicked in mice through mosquito bite or sporozoite inoculation, includes a major pathogenic component in ECM that depends on γδ T cells and IFN-γ production in the asymptomatic liver stage, where parasite virulence is seemingly set and determines pathology in the subsequent blood stage. Here, we discuss these and other recent advances in our understanding of the complex-protective versus pathogenic-functions of γδ T cells in malaria.

Biogenesis and discharge of the rhoptries: Key organelles for entry and hijack of host cells by the Apicomplexa.

Rhoptries are specialized secretory organelles found in the Apicomplexa phylum, playing a central role in the establishment of parasitism. The rhoptry content includes membranous as well as proteinaceous materials that are discharged into the host cell in a regulated fashion during parasite entry. A set of rhoptry neck proteins form a RON complex that critically participates in the moving junction formation during invasion. Some of the rhoptry bulb proteins are associated with the membranous materials and contribute to the formation of the parasitophorous vacuole membrane while others are targeted into the host cell including the nucleus to subvert cellular functions. Here, we review the recent studies on Toxoplasma and Plasmodium parasites that shed light on the key steps leading to rhoptry biogenesis, trafficking, and discharge.

Molecular approaches to Malaria 2020.

Twenty years ago the Molecular Approaches to Malaria conference was conceived as a forum to present the very latest advances in malaria research and to consolidate and forge new collaborative links between international researchers. The 6th MAM conference, held in February 2020 in Australia, provided 5 days of stimulating scientific exchange and highlighted the incredible malaria research conducted globally that is providing the critical knowledge and cutting-edge technological tools needed to control and ultimately eliminate malaria.

Battleground midgut: The cost to the mosquito for hosting the malaria parasite.

In eco-evolutionary studies of parasite-host interactions, virulence is defined as a reduction in host fitness as a result of infection relative to an uninfected host. Pathogen virulence may either promote parasite transmission, when correlated with higher parasite replication rate, or decrease the transmission rate if the pathogen quickly kills the host. This evolutionary mechanism, referred to as 'trade-off' theory, proposes that pathogen virulence evolves towards a level that most benefits the transmission. It has been generally predicted that pathogens evolve towards low virulence in their insect vectors, mainly due to the high dependence of parasite transmission on their vector survival. Therefore, the degree of virulence which malaria parasites impose on mosquito vectors may depend on several external and internal factors. Here, we review briefly (i) the role of mosquito in parasite development, with a particular focus on mosquito midgut as the battleground between Plasmodium and the mosquito host. We aim to point out (ii) the histology of the mosquito midgut epithelium and its role in host defence against parasite's countermeasures in the three main battle sites, namely (a) the lumen (microbiota and biochemical environment), (b) the peritrophic membrane (physical barrier) and (c) the tubular epithelium including the basal membrane (physical and biochemical barrier). Lastly, (iii) we describe the impact which malaria parasite and its virulence factors have on mosquito fitness.

High fidelity defines the temporal consistency of host-parasite interactions in a tropical coastal ecosystem.

Host-parasite interactions represent a selective force that may reduce hosts' lifespan, their reproductive success and survival. Environmental conditions can affect host-parasite communities, leading to distinct patterns of interactions with divergent ecological and evolutionary consequences for their persistence. Here, we tested whether climatic oscillation shapes the temporal dynamics of bird-haemosporidian associations, assessing the main mechanisms involved in the temporal dissimilarity of their interactions' networks. For two years, we monthly sampled birds in a tropical coastal ecosystem to avian malaria molecular diagnosis. The studied networks exhibited high specialization, medium modularity, with low niche overlap among parasites lineages. Moreover, alpha and ß-diversity of hosts, parasites and their interactions, as well as the structure of their networks were temporally consistent, i.e., stable under fluctuations in temperature or precipitation over seasons. The structure and temporal consistency of the studied antagonistic networks suggest a high fidelity between partners, which is likely relevant for their evolutionary persistence.

Advances in the research on the targets of anti-malaria actions of artemisinin.

Malaria has been a global epidemic health threat since ancient times. It still claims roughly half a million lives every year in this century. Artemisinin and its derivatives, are frontline antimalarial drugs known for their efficacy and low toxicity. After decades of wide use, artemisinins remain our bulwark against malaria. Here, we review decades of efforts that aim to understand the mechanism of action (MOA) of artemisinins, which help explain the specificity and potency of this anti-malarial drug. We summarize the methods and approaches employed to unravel the MOA of artemisinin over the last three decades, showing how the development of advanced techniques can help provide mechanistic insights and resolve some long-standing questions in the field of artemisinin research. We also provide examples to illustrate how to better repurpose artemisinins for anti-cancer therapies by leveraging on MOA. These examples point out a practical direction to engineer artemisinin for broader applications beyond malaria.