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The extent to which dengue virus has been circulating in Africa is largely unknown. Testing available blood samples from previous cross-sectional serological surveys offers a convenient strategy to investigate past dengue infections, as such serosurveys provide the ideal data to reconstruct the age-dependent immunity profile of the population and to estimate the average per-capita annual risk of infection; the force of infection (FOI), which is a fundamental measure of transmission intensity.In this study, we present a novel methodological approach to inform the size and age distribution of blood samples to test when samples are acquired from previous surveys. The method was used to inform a dengue seroprevalence survey which is currently being conducted in Ghana by the Drug for Neglected disease initiative, utilizing samples previously collected for a SARS-CoV-2 serosurvey.The method described in this paper can be employed to determine sample sizes and testing strategies for different diseases and transmission settings.Author summaryThe historical circulation of dengue virus in Africa is still poorly understood, and age-stratified seroprevalence surveys can provide the data to quantify population exposure to dengue and its transmission intensity.In this work, we developed a simulation-based method that can be used to identify the sample sizes and age-distribution of the samples needed to obtain informative estimates of dengue force of infection from existing blood samples. We apply this method to data obtained from a SARS-CoV-2 serological survey, previously conducted in three cities in Ghana.The proposed method can be used to design serological surveys for other pathogens when using existing blood samples with accompanying age and location are available.

Objectives Specific serological tests are mandatory for reliable SARS‐CoV‐2 diagnostics and seroprevalence studies. Here, we assess the specificities of four commercially available SARS‐CoV‐2 IgG ELISAs in serum/plasma panels originating from Africa, South America, and Europe. Methods 882 serum/plasma samples collected from symptom‐free donors before the COVID‐19 pandemic in three African countries (Ghana, Madagascar, Nigeria), Colombia, and Germany were analysed with three nucleocapsid‐based ELISAs (Euroimmun Anti‐SARS‐CoV‐2‐NCP IgG, EDITM Novel Coronavirus COVID‐19 IgG, Mikrogen recomWell SARS‐CoV‐2 IgG), one spike/S1‐based ELISA (Euroimmun Anti‐SARS‐CoV‐2 IgG), and in‐house common cold CoV ELISAs. Results High specificity was confirmed for all SARS‐CoV‐2 IgG ELISAs for Madagascan (93.4%‐99.4%), Colombian (97.8%‐100.0%), and German (95.9%‐100.0%) samples. In contrast, specificity was much lower for the Ghanaian and Nigerian serum panels (Ghana: NCP‐based assays 77.7%‐89.7%, spike/S1‐based assay 94.3%; Nigeria: NCP‐based assays 39.3%‐82.7%, spike/S1‐based assay 90.7%). 15 of 600 African sera were concordantly classified as positive in both the NCP‐based and the spike/S1‐based Euroimmun ELISA, but did not inhibit spike/ACE2 binding in a surrogate virus neutralization test. IgG antibodies elicited by previous infections with common cold CoVs were found in all sample panels, including those from Madagascar, Colombia, and Germany and thus do not inevitably hamper assay specificity. Nevertheless, high levels of IgG antibodies interacting with OC43 NCP were found in all 15 SARS‐CoV‐2 NCP/spike/S1 ELISA positive sera. Conclusions Depending on the chosen antigen and assay protocol, SARS‐CoV‐2 IgG ELISA specificity may be significantly reduced in certain populations probably due to interference of immune responses to endemic pathogens like other viruses or parasites.

This study aims to explore the critical prerequisites for accelerating the distribution of the COVID‐19 vaccine in developing countries by using Ghana as a case study. A qualitative study method and content analysis approach was used. In‐depth interviews were conducted with health experts from the Ghana Health Service, World Health Organization (WHO), AstraZeneca, Novartis, and Medtronic Inc. in Ghana. Our analysis of data revealed that new structures, committees, advisory bodies and lines of communication in government evolved during this pandemic and are underlying the current strategy development and decision‐making on COVID‐19 vaccines. The interviews gave insights into six major factors that will aid COVID‐19 vaccine acceleration in Ghana. These factors are: (1) Access to vaccines through delivery, (2) national manufacturing of vaccines, (3) choosing the best vaccine candidates, (4) financial resources, (5) transparency, and (6) vaccine roll‐out and administration. These results could guide policymakers and other relevant stakeholders in prioritizing activities that will aid COVID‐19 vaccine acceleration in Ghana and other lower‐middle‐income countries, tailored to their specific context. As a recommendation, the Ghanaian government should embrace a multisectoral synergy approach to fight the disease. The study also provides insights into how vaccine adoption can be accelerated in the case of future pandemics.

Background Malaria presents with unspecific clinical symptoms that frequently overlap with other infectious diseases and is also a risk factor for coinfections, such as non-Typhi Salmonella. Malaria rapid diagnostic tests are sensitive but unable to distinguish between an acute infection requiring treatment and asymptomatic malaria with a concomitant infection. We set out to test whether cytokine profiles could predict disease status and allow the differentiation between malaria and a bacterial bloodstream infection. Methods We created a classification model based on cytokine concentration levels of pediatric inpatients with either Plasmodium falciparum malaria or a bacterial bloodstream infection using the Luminex platform. Candidate markers were preselected using classification and regression trees, and the predictive strength was calculated through random forest modeling. Results Analyses revealed that a combination of 7–15 cytokines exhibited a median disease prediction accuracy of 88% (95th percentile interval, 73%–100%). Haptoglobin, soluble Fas-Ligand, and complement component C2 were the strongest single markers with median prediction accuracies of 82% (with 95th percentile intervals of 71%–94%, 62%–94%, and 62%–94%, respectively). Conclusions Cytokine profiles possess good median disease prediction accuracy and offer new possibilities for the development of innovative point-of-care tests to guide treatment decisions in malaria-endemic regions.

The cause of acute encephalitis/meningoencephalitis in pediatric patients remains often unexplained despite extensive investigations for large panel of pathogens. To explore a possible viral implication, we investigated the virome of cerebrospinal fluid specimens of 70 febrile pediatric inpatients with clinical compatible encephalitis/meningoencephalitis. Using viral metagenomics, we detected and genetically characterized three novel human Torque teno mini virus (TTMV) species (TTMV-G1-3). Phylogenetically, TTMV-G1-3 clustered in three novel monophyletic lineages within genus Betatorquevirus of the Anelloviridae family. TTMV-G1-3 were highly prevalent in diseased children, but absent in the healthy cohort which may indicate an association of TTMV species with febrile illness. With 2/3 detected malaria co-infection, it remains unclear if these novel anellovirus species are causative agents or increase disease severity by interaction with malaria parasites. The presence of the viruses 28 days after initiating antimalarial and/or antibiotic treatment suggests a still active viral infection likely as effect of parasitic and/or bacterial co-infection that may have initiated a modulated immune system environment for viral replication or a defective virus clearance. This study increases the current knowledge on the genetic diversity of TTMV and strengthens that human anelloviruses can be considered as biomarkers for strong perturbations of the immune system in certain pathological conditions.

Summary We identify a selective nanomolar inhibitor of blood-stage malarial proliferation from a screen of microbial natural product extracts. The responsible compound, PDE-I2, is a precursor of the anticancer duocarmycin family that preserves the class's sequence-specific DNA binding but lacks its signature DNA alkylating cyclopropyl warhead. While less active than duocarmycin, PDE-I2 retains comparable antimalarial potency to chloroquine. Importantly, PDE-I2 is >1,000-fold less toxic to human cell lines than duocarmycin, with mitigated impacts on eukaryotic chromosome stability. PDE-I2 treatment induces severe defects in parasite nuclear segregation leading to impaired daughter cell formation during schizogony. Time-of-addition studies implicate parasite DNA metabolism as the target of PDE-I2, with defects observed in DNA replication and chromosome integrity. We find the effect of duocarmycin and PDE-I2 on parasites is phenotypically indistinguishable, indicating that the DNA binding specificity of duocarmycins is sufficient and the genotoxic cyclopropyl warhead is dispensable for the parasite-specific selectivity of this compound class.

A better understanding of disease-specific biomarker profiles during acute infections could guide the development of innovative diagnostic methods to differentiate between malaria and alternative causes of fever. We investigated autoantibody (AAb) profiles in febrile children (≤ 5 years) admitted to a hospital in rural Ghana. Serum samples from 30 children with a bacterial bloodstream infection and 35 children with Plasmodium falciparum malaria were analyzed using protein microarrays (Protoplex Immune Response Assay, ThermoFisher). A variable selection algorithm was applied to identify the smallest set of AAbs showing the best performance to classify malaria and bacteremia patients. The selection procedure identified 8 AAbs of which IFNGR2 and FBXW5 were selected in repeated model run. The classification error was 22%, which was mainly due to non-Typhi Salmonella (NTS) diagnoses being misclassified as malaria. Likewise, a cluster analysis grouped patients with NTS and malaria together, but separated malaria from non-NTS infections. Both current and recent malaria are a risk factor for NTS, therefore, a better understanding about the function of AAb in disease-specific immune responses is required in order to support their application for diagnostic purposes.

Background The increasing incidence of multi-antibiotic-resistant bacterial infections, coupled with the risk of co-infections in malaria-endemic regions, complicates accurate diagnosis and prolongs hospitalization, thereby increasing the total cost of illness. Further, there are challenges in making the correct choice of antibiotic treatment and duration, precipitated by a lack of access to microbial culture facilities in many hospitals in Ghana. The aim of this case report is to highlight the need for blood cultures or alternative rapid tests to be performed routinely in malaria patients, to diagnose co-infections with bacteria, especially when symptoms persist after antimalarial treatment. Case presentation A 6-month old black female child presented to the Agogo Presbyterian Hospital with fever, diarrhea, and a 3-day history of cough. A rapid diagnostic test for malaria and Malaria microscopy was positive for P. falciparum with a parasitemia of 224 parasites/μl. The patient was treated with Intravenous Artesunate, parental antibiotics (cefuroxime and gentamicin) and oral dispersible zinc tablets in addition to intravenous fluids. Blood culture yielded Acinetobacter baumanii, which was resistant to all of the third-generation antibiotics included in the susceptibility test conducted, but sensitive to ciprofloxacin and gentamicin. After augmenting treatment with intravenous ciprofloxacin, all symptoms resolved. Conclusion Even though this study cannot confirm whether the bacterial infection was nosocomial or otherwise, the case highlights the necessity to test malaria patients for possible co-infections, especially when fever persists after parasites have been cleared from the bloodstream. Bacterial blood cultures and antimicrobial susceptibility testing should be routinely performed to guide treatment options for febril illnesses in Ghana in order to reduce inappropriate use of broad-spectrum antibiotics and limit the development of antimicrobial resistance.

A short motif termed Plasmodium export element (PEXEL) or vacuolar targeting signal (VTS) characterizes Plasmodium proteins exported into the host cell. These proteins mediate host cell modifications essential for parasite survival and virulence. However, several PEXEL-negative exported proteins indicate that the currently predicted malaria exportome is not complete and it is unknown whether and how these proteins relate to PEXEL-positive export. Here we show that the N-terminal 10 amino acids of the PEXEL-negative exported protein REX2 (ring-exported protein 2) are necessary for its targeting and that a single-point mutation in this region abolishes export. Furthermore we show that the REX2 transmembrane domain is also essential for export and that together with the N-terminal region it is sufficient to promote export of another protein. An N-terminal region and the transmembrane domain of the unrelated PEXEL-negative exported protein SBP1 (skeleton-binding protein 1) can functionally replace the corresponding regions in REX2, suggesting that these sequence features are also present in other PEXEL-negative exported proteins. Similar to PEXEL proteins we find that REX2 is processed, but in contrast, detect no evidence for N-terminal acetylation.

Plasmodium falciparum, the causative agent of malaria, relies on a complex protein-secretion system for protein targeting into numerous subcellular destinations. Recently, a homologue of the Golgi re-assembly stacking protein (GRASP) was identified and used to characterise the Golgi organisation in this parasite. Here, we report on the presence of a splice variant that leads to the expression of a GRASP isoform. Although the first GRASP protein (GRASP1) relies on a well-conserved myristoylation motif, the variant (GRASP2) displays a different N-terminus, similar to GRASPs found in fungi. Phylogenetic analyses between GRASP proteins of numerous taxa point to an independent evolution of the unusual N-terminus that could reflect unique requirements for Golgi-dependent protein sorting and organelle biogenesis in P. falciparum. Golgi association of GRASP2 depends on the hydrophobic N-terminus that resembles a signal anchor, leading to a unique mode of Golgi targeting and membrane attachment.

The Golgi apparatus forms the heart of the secretory pathway in eukaryotic cells where proteins are modified, processed and sorted. The transport of proteins from the endoplasmic reticulum (ER) to the cis-side of the Golgi complex takes place at specialized ER sub-domains known as transitional ER (tER). We used the Plasmodium falciparum orthologue of Sec13p to analyse tER organization. We show that the distribution of PfSec13p is restricted to defined areas of the ER membrane. These foci are juxtaposed to the Golgi apparatus and might represent tER sites. To further analyse cis- to trans-Golgi architecture, we generated a double transfectant parasite line that expresses the Golgi marker Golgi reassembly stacking protein (GRASP) as a green fluorescent protein fusion and the trans-Golgi marker Rab6 as a DsRed fusion protein. Our data demonstrate that Golgi multiplication is closely linked to tER multiplication, and that parasite maturation is accompanied by the spatial separation of the cis- and trans- face of this organelle.

The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBA-GFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.

Summary The malaria parasite Plasmodium falciparum har- bours a relict plastid (termed the apicoplast) that has evolved by secondary endosymbiosis. The apicoplast is surrounded by four membranes, the outermost of which is believed to be part of the endomembrane system. Nuclear-encoded apicoplast proteins have a two-part N-terminal extension that is necessary and sufficient for translocation across these four membranes. The first domain of this N-terminal exten- sion resembles a classical signal peptide and medi- ates translocation into the secretory pathway, whereas the second domain is homologous to plant chloroplast transit peptides and is required for the remaining steps of apicoplast targeting. We explored the initial, secretory pathway component of this targeting process using green fluorescent reporter protein constructs with modified leaders. We exchanged the apicoplast signal peptide with signal peptides from other secretory proteins and observed correct targeting, demonstrating that apicoplast tar- geting is initiated at the general secretory pathway of P. falciparum. Furthermore, we demonstrate by immu- nofluorescent labelling that the apicoplast resides on a small extension of the endoplasmic reticulum (ER) that is separate from the cis-Golgi. To define the posi- tion of the apicoplast in the endomembrane pathway in relation to the Golgi we tracked apicoplast protein targeting in the presence of the secretory inhibitor Brefeldin A (BFA), which blocks traffic between the ER and Golgi. We observe apicoplast targeting in the presence of BFA despite clear perturbation of ER to Golgi traffic by the inhibitor, which suggests that the apicoplast resides upstream of the cis-Golgi in the parasite's endomembrane system. The addition of an ER retrieval signal (SDEL) - a sequence recognized by the cis-Golgi protein ERD2 - to the C-terminus of an apicoplast-targeted protein did not markedly affect apicoplast targeting, further demonstrating that the apicoplast is upstream of the Golgi. Apicoplast transit peptides are thus dominant over an ER retention signal. However, when the transit peptide is rendered non-functional (by two point mutations or by com- plete deletion) SDEL-specific ER retrieval takes over, and the fusion protein is localized to the ER. We speculate either that the apicoplast in P. falciparum resides within the ER directly in the path of the general secretory pathway, or that vesicular traffick- ing to the apicoplast directly exits the ER.

Transit peptides mediate protein targeting into plastids and are only poorly understood. We extracted amino acid features from transit peptides that target proteins to the relict plastid (apicoplast) of malaria parasites. Based on these amino acid characteristics, we identified 466 putative apicoplast proteins in the Plasmodium falciparum genome. Altering the specific charge characteristics in a model transit peptide by site-directed mutagenesis severely disrupted organellar targeting in vivo. Similarly, putative Hsp70 (DnaK) binding sites present in the transit peptide proved to be important for correct targeting.

During liver-stage malaria infection, Plasmodium sporozoites must invade a hepatocyte in order to produce thousands of merozoites, the stage that infects erythrocytes and causes malarial pathology. One of the first steps of hepatocyte infection is the invagination of the host cell membrane, which shelters the parasite within a parasitophorous vacuole (PV) and allows parasite development inside the host cell. Sporozoites also have another peculiar type of cell invasion, in which the parasite breaches the host cell plasma membrane and transmigrates, free in the cytoplasm, through the host cell. This cell wounding and crossing process is termed cell traversal. Here, using live-cell imaging techniques, fluorescent markers of the host cell plasma membrane and a mutant parasite lacking cell traversal activity, we describe a new type of sporozoite transcellular migration. We have observed wild-type Plasmodium berhgei sporozoites moving through hepatocytes while surrounded by host cell membrane. Additionally, a small percentage of sporozoites drag host cell membrane with them when they leave hepatocytes, forming aggregates of membrane we have termed membrane "bobbins." Membrane-surrounded migrating sporozoites and membrane bobbins are also observed during transmigration of primary liver sinusoidal endothelial cells. As cell traversal deficient sporozoites are still able to move across the cell surrounded by host cell plasma membrane, we define this new transcellular migration as being distinct from cell traversal. We hypothesize that this form of transmigration starts with the PV membrane formation similar to invasion with the exception that these sporozoites continue migration, ultimately leaving the cell by membrane fusion or disruption of the vacuole membrane and the plasma membrane.

International audience; Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red-fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. Therefore, we have generated two additional transgenic parasite lines expressing the novel red-fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.

The transfer of genes from an endosymbiont to its host typically requires acquisition of targeting signals by the gene product to ensure its return to the endosymbiont for function. Many hundreds of plastid-derived genes must have acquired transit peptides for successful relocation to the nucleus. Here, we explore potential evolutionary origins of plastid transit peptides in the malaria parasite Plasmodium falciparum . We show that exons of the P. falciparum genome could serve as transit peptides after exon shuffling. We further demonstrate that numerous randomized peptides and even whimsical sequences based on English words can also function as transit peptides in vivo . Thus, facile acquisition of transit peptides from existing sequence likely expedited endosymbiont integration through intracellular gene transfer.

One of the key processes in the pathobiology of the malaria parasite is the invasion and subsequent modification of the human erythrocyte. In this complex process, an unknown number of parasite proteins are involved, some of which are leading vaccine candidates. The majority of the proteins that play pivotal roles in invasion are either stored in the apical secretory organelles or located on the surface of the merozoite, the invasive stage of the parasite. Using transcriptional and structural features of these known proteins, we performed a genomewide search that identified 49 hypothetical proteins with a high probability of being located on the surface of the merozoite or in the secretory organelles. Of these candidates, we characterized a novel leucine zipper-like protein in Plasmodium falciparum that is conserved in Plasmodium spp. This protein is expressed in late blood stages and localizes to the rhoptries of the parasite. We demonstrate that this Plasmodium sp.-specific protein has a high degree of conservation within field isolates and that it is refractory to gene knockout attempts and thus might play an important role in invasion.

Plasmodium falciparum, the causative agent of malaria, relies on a sophisticated protein secretion system for host cell invasion and transformation. Although the parasite displays a secretory pathway similar to those of all eukaryotic organisms, a classical Golgi apparatus has never been described. We identified and characterised the putative Golgi matrix protein PfGRASP, a homologue of the Golgi re-assembly stacking protein (GRASP) family. We show that PfGRASP is expressed as a 70 kDa protein throughout the asexual life cycle of the parasite. We generated PfGRASP-GFP-expressing transgenic parasites and showed that this protein is localised to a single, juxtanuclear compartment in ring-stage parasites. The PfGRASP compartment is distinct from the ER, restricted within the boundaries of the parasite and colocalises with the cis-Golgi marker ERD2. Correct subcellular localisation of this Golgi matrix protein depends on a cross-species conserved functional myristoylation motif and is insensitive to Brefeldin A. Taken together our results define the Golgi apparatus in Plasmodium and depict the morphological organisation of the organelle throughout the asexual life cycle of the parasite.

The apicoplast and mitochondrion of the malaria parasite Plasmodium falciparumare important intracellular organelles and targets of several anti-malarial drugs. In recent years, our group and others have begun to piece together the metabolic pathways of these organelles, with a view to understanding their functions and identifying further anti-malarial targets. This has involved localization of putative organellar proteins using fluorescent reporter proteins such as green fluorescent protein (GFP). A major limitation to such an approach is the difficulties associated with using existing plasmids to genetically modify P. falciparum. In this paper, we present a novel series of P. falciparumtransfection vectors based around the Gateway TM recombinatorial cloning system. Our system makes it considerably easier to construct fluorescent reporter fusion proteins, as well as allowing the use of two selectable markers. Using this approach, we localize proteins involved in isoprenoid biosynthesis and the posttranslational processing of apicoplast-encoded proteins to the apicoplast, and a protein putatively involved in the citric acid cycle to the mitochondrion. To confirm the localization of these proteins, we have developed a new immunofluorescence assay (IFA) protocol using antibodies specific to the apicoplast and mitochondrion. In comparison with published IFA methods, we find that ours maintains considerably better structural preservation, while still allowing sufficient antibody binding as well as preserving reporter protein fluorescence. In summary, we present two important new tools that have enabled us to characterize some of the functions of the apicoplast and mitochondrion, and which will be of use to the wider malaria research community in elucidating the localization of other P. falciparumproteins. © 2004 Elsevier B.V. All rights reserved.

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies., Author Summary Malaria is one of the most lethal parasitic diseases worldwide, causing more than 1 million fatalities per annum. Drug resistance is widespread, and a vaccine is not available. One of the leading blood stage vaccine candidates is the apical membrane antigen 1 (AMA-1), which is well-conserved among apicomplexan parasites. Although this protein plays an essential role in the invasion of human red blood cells, little is known about the molecular mechanisms underlying its function. In this study we use live video microscopy in the presence and absence of an invasion-inhibiting molecule to investigate the function of AMA-1 in real time. We establish a complementation assay based on strain-specific AMA-1 inhibition that allows functional characterization of this protein on a molecular level. Using this approach, we provide evidence that the intracellular cytoplasmic domain of AMA-1 is directly involved in the invasion process and that this domain is phosphorylated. We conclude that the phosphorylation of AMA-1 might represent an attractive target for novel therapeutic drug strategies independent of the polymorphic extracellular domain.

Apicomplexan pathogens are obligate intracellular parasites. To enter cells, they must bind with high affinity to host cell receptors and then uncouple these interactions to complete invasion. Merozoites of Plasmodium falciparum, the parasite responsible for the most dangerous form of malaria, invade erythrocytes using a family of adhesins called Duffy binding ligand-erythrocyte binding proteins (DBL-EBPs). The best-characterized P. falciparum DBL-EBP is erythrocyte binding antigen 175 (EBA-175), which binds erythrocyte surface glycophorin A. We report that EBA-175 is shed from the merozoite at around the point of invasion. Shedding occurs by proteolytic cleavage within the transmembrane domain (TMD) at a site that is conserved across the DBL-EBP family. We show that EBA-175 is cleaved by PfROM4, a rhomboid protease that localizes to the merozoite plasma membrane, but not by other rhomboids tested. Mutations within the EBA-175 TMD that abolish cleavage by PfROM4 prevent parasite growth. Our results identify a crucial role for intramembrane proteolysis in the life cycle of this pathogen.