The exception that proves the rule: Virulence gene expression at the onset of Plasmodium falciparum blood stage infections.

Controlled human malaria infections (CHMI) are a valuable tool to study parasite gene expression in vivo under defined conditions. In previous studies, virulence gene expression was analyzed in samples from volunteers infected with the Plasmodium falciparum (Pf) NF54 isolate, which is of African origin. Here, we provide an in-depth investigation of parasite virulence gene expression in malaria-naïve European volunteers undergoing CHMI with the genetically distinct Pf 7G8 clone, originating in Brazil. Differential expression of var genes, encoding major virulence factors of Pf, PfEMP1s, was assessed in ex vivo parasite samples as well as in parasites from the in vitro cell bank culture that was used to generate the sporozoites (SPZ) for CHMI (Sanaria PfSPZ Challenge (7G8)). We report broad activation of mainly B-type subtelomeric located var genes at the onset of a 7G8 blood stage infection in naïve volunteers, mirroring the NF54 expression study and suggesting that the expression of virulence-associated genes is generally reset during transmission from the mosquito to the human host. However, in 7G8 parasites, we additionally detected a continuously expressed single C-type variant, Pf7G8_040025600, that was most highly expressed in both pre-mosquito cell bank and volunteer samples, suggesting that 7G8, unlike NF54, maintains expression of some previously expressed var variants during transmission. This suggests that in a new host, the parasite may preferentially express the variants that previously allowed successful infection and transmission. Trial registration: ClinicalTrials.gov - NCT02704533; 2018-004523-36.

Functional inactivation of Plasmodium falciparum glycogen synthase kinase GSK3 modulates erythrocyte invasion and blocks gametocyte maturation.

Malaria is responsible for hundreds of thousands of deaths every year. The lack of an effective vaccine and the global spread of multidrug resistant parasites hampers the fight against the disease and underlines the need for new antimalarial drugs. Central to the pathogenesis of malaria is the proliferation of Plasmodium parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and the host cell. Posttranslational modifications such as protein phosphorylation are known to be key regulators in this process and are mediated by protein kinases. For several parasite kinases, including the Plasmodium falciparum glycogen synthase kinase 3 (PfGSK3), inhibitors have been shown to block erythrocyte invasion. Here, we provide an assessment of PfGSK3 function by reverse genetics. Using targeted gene disruption, we show the active gene copy, PfGSK3ß, is not essential for asexual blood stage proliferation, although it modulates efficient erythrocyte invasion. We found functional inactivation leads to a 69% decreased growth rate and confirmed this growth defect by rescue experiments with wildtype and catalytically inactive mutants. Functional knockout of PfGSK3ß does not lead to transcriptional upregulation of the second copy of PfGSK3. We further analyze expression, localization, and function of PfGSK3ß during gametocytogenesis using a parasite line allowing conditional induction of sexual commitment. We demonstrate PfGSK3ß-deficient gametocytes show a strikingly malformed morphology leading to the death of parasites in later stages of gametocyte development. Taken together, these findings are important for our understanding and the development of PfGSK3 as an antimalarial target.

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum.

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single-celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites.

Controlled human malaria infection with Plasmodium falciparum demonstrates impact of naturally acquired immunity on virulence gene expression.

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host's immune system, thus promoting overall parasite survival in the face of host immunity.

Mosquito Passage Dramatically Changes var Gene Expression in Controlled Human Plasmodium falciparum Infections.

Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase.

Tumstatin fragment selectively inhibits neutrophil infiltration in experimental asthma exacerbation.

BACKGROUND: Asthma is a chronic inflammatory disease with structural changes present. Burgess and colleagues recently found tumstatin markedly reduced in adult asthmatic lung tissue compared with nonasthmatics. ECM fragments such as tumstatin are named matrikines and act independently of the parent molecule. The role of Col IV matrikines in neutrophil inflammation (eg. exacerbation in asthma) has not been investigated to date. Severe adult asthma phenotypes are dominated by neutrophilic inflammation and show a high frequency of severe exacerbations. OBJECTIVE: This study sought to investigate the role of a novel active region within tumstatin (CP17) and its implication in neutrophil inflammatory responses related to asthma exacerbation. METHODS: For reactive oxygen production, isolated neutrophils were preincubated with peptides or vehicle for 1 hour and stimulated (PMA). Luminescence signal was recorded (integration over 10 seconds) for 1.5 hours. Neutrophil migration was performed according to the SiMA protocol. Mice were sensitized to OVA/Alumn by intraperitoneal (i.p.) injections. Mice were then treated with CP17, vehicle (PBS) or scrambled peptide (SP17) after OVA exposure (days 27 and 28, polyI:C stimulation). All animals were killed on day 29 with lung function measurement, histology and lavage. RESULTS: CP17 decreased total ROS production rate to 52.44% (0.5 µmol/L, P < 0.05 vs SP17), reduced the in vitro directionality (vs SP17, P = 1 × 10-6 ) and migration speed (5 µmol/L, P = 1 × 10-3 ). In vivo application of CP17 decreased neutrophil inflammation ~1.8-fold (P < 0.001 vs SP17) and reduced numbers of mucus-producing cells (-29%, P < 0.05). CONCLUSION: CP17 reduced the ROS production rate, migrational speed and selectively inhibited neutrophil accumulation in the lung interstitium and lumen. CLINICAL RELEVANCE: CP17 may serve as a potential precursor for drug development to combat overwhelming neutrophil inflammation.

Plasmodium falciparum STEVOR phosphorylation regulates host erythrocyte deformability enabling malaria parasite transmission.

Deformability of Plasmodium falciparum gametocyte-infected erythrocytes (GIEs) allows them to persist for several days in blood circulation and to ensure transmission to mosquitoes. Here, we investigate the mechanism by which the parasite proteins STEVOR (SubTElomeric Variable Open Reading frame) exert changes on GIE deformability. Using the microsphiltration method, immunoprecipitation, and mass spectrometry, we produce evidence that GIE stiffness is dependent on the cytoplasmic domain of STEVOR that interacts with ankyrin complex at the erythrocyte skeleton. Moreover, we show that GIE deformability is regulated by protein kinase A (PKA)-mediated phosphorylation of the STEVOR C-terminal domain at a specific serine residue (S324). Finally, we show that the increase of GIE stiffness induced by sildenafil (Viagra) is dependent on STEVOR phosphorylation status and on another independent mechanism. These data provide new insights into mechanisms by which phosphodiesterase inhibitors may block malaria parasite transmission.

Sporozoite Route of Infection Influences In Vitro var Gene Transcription of Plasmodium falciparum Parasites From Controlled Human Infections.

BACKGROUND: Antigenic variation in Plasmodium falciparum is mediated by the multicopy var gene family. Each parasite possesses about 60 var genes, and switching between active var loci results in antigenic variation. In the current study, the effect of mosquito and host passage on in vitro var gene transcription was investigated. METHODS: Thirty malaria-naive individuals were inoculated by intradermal or intravenous injection with cryopreserved, isogenic NF54 P. falciparum sporozoites (PfSPZ) generated from 1 premosquito culture. Microscopic parasitemia developed in 22 individuals, and 21 in vitro cultures were established. The var gene transcript levels were determined in early and late postpatient cultures and in the premosquito culture. RESULTS: At the early time point, all cultures preferentially transcribed 8 subtelomeric var genes. Intradermal infections had higher var gene transcript levels than intravenous infections and a significantly longer intrahost replication time (P = .03). At the late time point, 9 subtelomeric and 8 central var genes were transcribed at the same levels in almost all cultures. Premosquito and late postpatient cultures transcribed the same subtelomeric and central var genes, except for var2csa CONCLUSIONS: The duration of intrahost replication influences in vitro var gene transcript patterns. Differences between premosquito and postpatient cultures decrease with prolonged in vitro growth.

Evidence of promiscuous endothelial binding by Plasmodium falciparum-infected erythrocytes.

The adhesion of infected red blood cells (iRBCs) to human endothelium is considered a key event in the pathogenesis of cerebral malaria and other life-threatening complications caused by the most prevalent malaria parasite Plasmodium falciparum. In the past 30 years, 14 endothelial receptors for iRBCs have been identified. Exposing 10 additional surface proteins of endothelial cells to a mixture of P. falciparum isolates from three Ghanaian malaria patients, we identified seven new iRBC receptors, all expressed in brain vessels. This finding strongly suggests that endothelial binding of P. falciparum iRBCs is promiscuous and may use a combination of endothelial surface moieties.

A comparative study of the localization and membrane topology of members of the RIFIN, STEVOR and PfMC-2TM protein families in Plasmodium falciparum-infected erythrocytes.

BACKGROUND: Variant surface antigens (VSA) exposed on the membrane of Plasmodium falciparum infected erythrocytes mediate immune evasion and are important pathogenicity factors in malaria disease. In addition to the well-studied PfEMP1, the small VSA families RIFIN, STEVOR and PfMC-2TM are assumed to play a role in this process. METHODS: This study presents a detailed comparative characterization of the localization, membrane topology and extraction profile across the life cycle of various members of these protein families employing confocal microscopy, immunoelectron microscopy and immunoblots. RESULTS: The presented data reveal a clear association of variants of the RIFIN, STEVOR and PfMC-2TM proteins with the host cell membrane and topological studies indicate that the semi-conserved N-terminal region of RIFINs and some STEVOR proteins is exposed at the erythrocyte surface. At the Maurer's clefts, the semi-conserved N-terminal region as well as the variable stretch of RIFINs appears to point to the lumen away from the erythrocyte cytoplasm. These results challenge the previously proposed two transmembrane topology model for the RIFIN and STEVOR protein families and suggest that only one hydrophobic region spans the membrane. In contrast, PfMC-2TM proteins indeed seem to be anchored by two hydrophobic stretches in the host cell membrane exposing just a few, variable amino acids at the surface of the host cell. CONCLUSION: Together, the host cell surface exposure and topology of RIFIN and STEVOR proteins suggests members of these protein families may indeed be involved in immune evasion of the infected erythrocyte, whereas members of the PfMC-2TM family seem to bear different functions in parasite biology.

A novel computational pipeline for var gene expression augments the discovery of changes in the Plasmodium falciparum transcriptome during transition from in vivo to short-term in vitro culture.

The pathogenesis of severe Plasmodium falciparum malaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family of var genes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches for var gene profiling and classification of predicted binding phenotypes in clinical P. falciparum isolates (Wichers et al., 2021), which represented a major technical advance. Building on this, we report here a novel method for var gene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wichers et al., 2021, on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate the var transcriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role of var genes in malaria pathogenesis. We applied this new method to investigate changes in var gene expression through early transition of parasite isolates to in vitro culture, using paired sets of ex vivo samples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictable var gene switching and convergence towards var2csa were observed in culture, along with differential expression of 19% of the core transcriptome between paired ex vivo and generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences about in vivo phenotype and behaviour.

A Microtubule-Associated Protein Is Essential for Malaria Parasite Transmission.

Mature gametocytes of Plasmodium falciparum display a banana (falciform) shape conferred by a complex array of subpellicular microtubules (SPMT) associated with the inner membrane complex (IMC). Microtubule-associated proteins (MAPs) define MT populations and modulate interaction with pellicular components. Several MAPs have been identified in Toxoplasma gondii, and homologues can be found in the genomes of Plasmodium species, but the function of these proteins for asexual and sexual development of malaria parasites is still unknown. Here, we identified a novel subpellicular MAP, termed SPM3, that is conserved within the genus Plasmodium, especially within the subgenus Laverania, but absent in other Apicomplexa. Conditional knockdown and targeted gene disruption of Pfspm3 in Plasmodium falciparum cause severe morphological defects during gametocytogenesis, leading to round, nonfalciform gametocytes with an aberrant SPMT pattern. In contrast, Pbspm3 knockout in Plasmodium berghei, a species with round gametocytes, caused no defect in gametocytogenesis, but sporozoites displayed an aberrant motility and a dramatic defect in invasion of salivary glands, leading to a decreased efficiency in transmission. Electron microscopy revealed a dissociation of the SPMT from the IMC in Pbspm3 knockout parasites, suggesting a function of SPM3 in anchoring MTs to the IMC. Overall, our results highlight SPM3 as a pellicular component with essential functions for malaria parasite transmission. IMPORTANCE A key structural feature driving the transition between different life cycle stages of the malaria parasite is the unique three-membrane pellicle, consisting of the parasite plasma membrane (PPM) and a double membrane structure underlying the PPM termed the inner membrane complex (IMC). Additionally, there are numerous linearly arranged intramembranous particles (IMPs) linked to the IMC, which likely link the IMC to the subpellicular microtubule cytoskeleton. Here, we identified, localized, and characterized a novel subpellicular microtubule-associated protein unique to the genus Plasmodium. The knockout of this protein in the human-pathogenic species P. falciparum resulted in malformed gametocytes and aberrant microtubules. We confirmed the microtubule association in the P. berghei rodent malaria homologue and show that its knockout results in a perturbed microtubule architecture, aberrant sporozoite motility, and decreased transmission efficiency.

The essentials of protein import in the degenerate mitochondrion of Entamoeba histolytica.

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.

Highly co-ordinated var gene expression and switching in clinical Plasmodium falciparum isolates from non-immune malaria patients.

Antigenic variation to fool the immune system is one of the molecular tricks Plasmodium uses to maintain infection in its human host. The exclusive expression of the surface-exposed PfEMP1 molecules, encoded by var genes, is the best example for this. Central questions regarding the dynamics of antigenic variation, namely the rate of switching and the regulation of var gene expression in Plasmodium falciparum, are yet unanswered. To elucidate the in vivo situation, we studied var gene switching by analysing the var transcripts from parasites isolated from 20 non-immune malaria patients as well as during subsequent in vitro generations. Parasites were found to be highly co-ordinated as the whole population isolated from individual patients usually expressed only one dominant - preferentially group A -var gene. While some isolates have very low switching rates, others switched their var gene expression in every generation. However, during extended cultivation the co-ordinated expression and switching is lost resulting in random expression of all var gene groups. Switching as observed on the RNA level was also supported on the protein level using PfEMP1-specific antibodies. The results suggest that var genes switch in an ordered, hierarchical manner at much higher rates than previously described.

Analysis of var Gene Transcript Patterns by Quantitative Real-Time PCR.

Quantitative real-time PCR (qPCR) is a simple and sensitive method for determining the amount of a specific target DNA sequence present in a sample. Compared to RNA-seq, reverse transcription qPCR (RT-qPCR) is fast, requires only low input material and is easy to analyze. Therefore, qPCR is widely used to analyze gene expression in P. falciparum, including analyses of the multicopy gene families encoding variant surface antigens (VSAs), whose expression is clonally variant and prone to changes over time. In the recent years, several P. falciparum genomes of culture-adapted strains have been sequenced, providing the knowledge to design variable gene family-specific qPCR primers for each P. falciparum genetic background. Here, we describe the required materials, methods and key factors to perform RT-qPCR experiments to determine VSA transcript abundances in the P. falciparum clones 3D7/NF54, IT4, HB3, and 7G8.

The Putative Bromodomain Protein PfBDP7 of the Human Malaria Parasite Plasmodium Falciparum Cooperates With PfBDP1 in the Silencing of Variant Surface Antigen Expression.

Epigenetic regulation is a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite Plasmodium (P.) falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the P. falciparum bromodomain proteins (PfBDP) 1 and PfBDP2 (BDP1/BDP2 core complex) was previously shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7, because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, STEVOR, and PfMC-2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and established PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.

CD36-A Host Receptor Necessary for Malaria Parasites to Establish and Maintain Infection.

Plasmodium falciparum-infected erythrocytes (PfIEs) present P. falciparum erythrocyte membrane protein 1 proteins (PfEMP1s) on the cell surface, via which they cytoadhere to various endothelial cell receptors (ECRs) on the walls of human blood vessels. This prevents the parasite from passing through the spleen, which would lead to its elimination. Each P. falciparum isolate has about 60 different PfEMP1s acting as ligands, and at least 24 ECRs have been identified as interaction partners. Interestingly, in every parasite genome sequenced to date, at least 75% of the encoded PfEMP1s have a binding domain for the scavenger receptor CD36 widely distributed on host endothelial cells and many other cell types. Here, we discuss why the interaction between PfIEs and CD36 is optimal to maintain a finely regulated equilibrium that allows the parasite to multiply and spread while causing minimal harm to the host in most infections.

PMRT1, a Plasmodium-Specific Parasite Plasma Membrane Transporter, Is Essential for Asexual and Sexual Blood Stage Development.

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles, and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown, and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures-the food vacuole, the apicoplast, and the parasite plasma membrane-and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1; PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions. IMPORTANCE Plasmodium falciparum-infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes not only facilitate movement of nutrients, metabolites, and other molecules between these compartments, but also are common therapeutic targets and can confer antimalarial drug resistance. Orphan membrane transporters in P. falciparum without sequence homology to transporters in other evolutionary lineages and divergent from host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed their importance during asexual parasite growth by using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.

In vitro neutrophil migration is associated with inhaled corticosteroid treatment and serum cytokines in pediatric asthma.

Background: Different asthma phenotypes are driven by molecular endotypes. A Th1-high phenotype is linked to severe, therapy-refractory asthma, subclinical infections and neutrophil inflammation. Previously, we found neutrophil granulocytes (NGs) from asthmatics exhibit decreased chemotaxis towards leukotriene B4 (LTB4), a chemoattractant involved in inflammation response. We hypothesized that this pattern is driven by asthma in general and aggravated in a Th1-high phenotype. Methods: NGs from asthmatic nd healthy children were stimulated with 10 nM LTB4/100 nM N-formylmethionine-leucyl-phenylalanine and neutrophil migration was documented following our prior SiMA (simplified migration assay) workflow, capturing morphologic and dynamic parameters from single-cell tracking in the images. Demographic, clinical and serum cytokine data were determined in the ALLIANCE cohort. Results: A reduced chemotactic response towards LTB4 was confirmed in asthmatic donors regardless of inhaled corticosteroid (ICS) treatment. By contrast, only NGs from ICS-treated asthmatic children migrate similarly to controls with the exception of Th1-high donors, whose NGs presented a reduced and less directed migration towards the chemokines. ICS-treated and Th1-high asthmatic donors present an altered surface receptor profile, which partly correlates with migration. Conclusions: Neutrophil migration in vitro may be affected by ICS-therapy or a Th1-high phenotype. This may be explained by alteration of receptor expression and could be used as a tool to monitor asthma treatment.

The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost.

Intraerythrocytic malaria parasites proliferate bounded by a parasitophorous vacuolar membrane (PVM). The PVM contains nutrient permeable channels (NPCs) conductive to small molecules, but their relevance for parasite growth for individual metabolites is largely untested. Here we show that growth-relevant levels of major carbon and energy sources pass through the NPCs. Moreover, we find that NPCs are a gate for several antimalarial drugs, highlighting their permeability properties as a critical factor for drug design. Looking into NPC-dependent amino acid transport, we find that amino acid shortage is a reason for the fitness cost in artemisinin-resistant (ARTR) parasites and provide evidence that NPC upregulation to increase amino acids acquisition is a mechanism of ARTR parasites in vitro and in human infections to compensate this fitness cost. Hence, the NPCs are important for nutrient and drug access and reveal amino acid deprivation as a critical constraint in ARTR parasites.