During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. ...Understanding each element in this process increases the potential to block the parasite's life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1) an early heparin-blockable interaction which weakly deforms the erythrocyte, 2) EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite's actin-myosin motor, 3) a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4) an AMA1-RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine.
The malaria parasite is the most important member of the Apicomplexa, a large and highly successful phylum of intracellular parasites. Invasion of host cells allows apicomplexan parasites access to a ...rich source of nutrients in a niche that is largely protected from host defenses. All Apicomplexa adopt a common mode of host-cell entry, but individual species incorporate unique features and utilize a specific set of ligand-receptor interactions. These adhesins ultimately connect to a parasite actin-based motor, which provides the power for invasion. While some Apicomplexa can invade many different host cells, the disease-associated blood-stage form of the malaria parasite is restricted to erythrocytes.
Malaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effector proteins exported from the parasite into the ...host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment.
Whilst evidence of use of face masks in reducing COVID-19 cases is increasing, the impact of mandatory use across a large population has been difficult to assess. Introduction of mandatory mask use ...on July 22, 2020 during a resurgence of COVID-19 in Melbourne, Australia created a situation that facilitated an assessment of the impact of the policy on the epidemic growth rate as its introduction occurred in the absence of other changes to restrictions.
Exponential epidemic growth or decay rates in daily COVID-19 diagnoses were estimated using a non-weighted linear regression of the natural logarithm of the daily cases against time, using a linear spline model with one knot (lspline package in R v 3.6.3). The model's two linear segments pivot around the hinge day, on which the mask policy began to take effect, 8 days following the introduction of the policy. We used two forms of data to assess change in mask usage: images of people wearing masks in public places obtained from a major media outlet and population-based survey data. Potential confounding factors (including daily COVID-19 tests, number of COVID-19 cases among population subsets affected differentially by the mask policy-e.g., healthcare workers) were examined for their impact on the results. Daily cases fitted an exponential growth in the first log-linear segment (k = +0.042, s.e. = 0.007), and fitted an exponential decay in the second (k = -0.023, s.e. = 0.017) log-linear segment. Over a range of reported serial intervals for SARS-CoV-2 infection, these growth rates correspond to a 22-33% reduction in an effective reproduction ratio before and after mandatory mask use. Analysis of images of people in public spaces showed mask usage rose from approximately 43% to 97%. Analysis of survey data found that on the third day before policy introduction, 44% of participants reported "often" or "always" wearing a mask; on the fourth day after, 100% reported "always" doing so. No potentially confounding factors were associated with the observed change in growth rates.
The mandatory mask use policy substantially increased public use of masks and was associated with a significant decline in new COVID-19 cases after introduction of the policy. This study strongly supports the use of masks for controlling epidemics in the broader community.
In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better ...understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.
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•CD8+ tissue-resident memory T cells (Trm cells) can be found in the murine liver•These liver Trm cells survey the liver from within the sinusoids•A prime-and-trap vaccination strategy efficiently induces liver Trm cells•Liver Trm cells are essential for protection against liver-stage malaria after vaccination
While various intervention strategies have reduced morbidity and mortality from malaria, further improvement is likely to depend on an effective vaccine. Fernandez-Ruiz et al. identify liver-resident memory CD8+ T cells as vital for liver-stage immunity and describe a protective vaccination strategy that drives their formation.
The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing
Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we ...use video microscopy to address the kinetics of RBC invasion in the human malaria parasite
Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1
min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6
s after primary contact. This period can be divided into two distinct phases. The first is an ∼11
s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17
s period. After invasion, a third ‘echinocytosis’ phase commences when about 36
s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4
s, after which the infected RBC recovered over a 5–11
min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite
Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the
Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.
Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host ...cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1). Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA). Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion.
Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cell's physiology. The Plasmodium export element (PEXEL) motif contained within the N-terminus of most ...exported proteins directs the trafficking of those proteins into the erythrocyte. To reach the host cell, the PEXEL motif of exported proteins is processed by the endoplasmic reticulum (ER) resident aspartyl protease plasmepsin V. Then, following secretion into the parasite-encasing parasitophorous vacuole, the mature exported protein must be unfolded and translocated across the parasitophorous vacuole membrane by the Plasmodium translocon of exported proteins (PTEX). PTEX is a protein-conducting channel consisting of the pore-forming protein EXP2, the protein unfoldase HSP101, and structural component PTEX150. The mechanism of how exported proteins are specifically trafficked from the parasite's ER following PEXEL cleavage to PTEX complexes on the parasitophorous vacuole membrane is currently not understood. Here, we present evidence that EXP2 and PTEX150 form a stable subcomplex that facilitates HSP101 docking. We also demonstrate that HSP101 localises both within the parasitophorous vacuole and within the parasite's ER throughout the ring and trophozoite stage of the parasite, coinciding with the timeframe of protein export. Interestingly, we found that HSP101 can form specific interactions with model PEXEL proteins in the parasite's ER, irrespective of their PEXEL processing status. Collectively, our data suggest that HSP101 recognises and chaperones PEXEL proteins from the ER to the parasitophorous vacuole and given HSP101's specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte.
A highly-effective, long-lasting vaccine, targeting multiple stages of the Plasmodium falciparum life cycle, is likely to be important for the elimination of this pathogen. Key antigens of this ...vaccine would produce host antibodies that block the ligands required for merozoite invasion of erythrocytes, thereby curtailing the expansion of parasitemia and symptomatic disease. Recent live cell imaging of invading Plasmodium falciparum merozoites with various receptor–ligand interactions inhibited has provided new information about the function, sequence, and timing of these events, providing a rationale for a vaccine containing multiple antigens that inhibit the sequential steps of invasion.
Abstract
In order to facilitate a number of processes including nutrient acquisition and immune evasion, malaria parasites extensively remodel their host erythrocyte. This remodelling is to a large ...extent accomplished through protein export, a crucial process mediated by the Plasmodium translocon for exported proteins (PTEX) translocon which is comprised of three core components, HSP101, PTEX150 and EXP2. EXP2 has been structurally and electrophysiologically shown to form the pore that spans the vacuole membrane enveloping the parasite. Here, we biochemically investigate the structure and function of EXP2. By differential alkylation we provide direct evidence that cysteines C113 and C140 form an intramolecular disulphide bond, while C201 is predominantly in a reduced state. We demonstrate that EXP2 possesses a protease resistant, membrane-associated, N-terminal region of ∼20 kDa that does not project into the infected erythrocyte cytosol; however, its C-terminus does project into the vacuole space. We show that a putative transmembrane peptide derived from the N-terminal region of EXP2 is haemolytic and in a polymer-based osmotic protection assay, we demonstrate that this peptide forms a discrete haemolytic pore. This work provides further biochemical insight into the role, function and cellular arrangement of EXP2 as the pore-forming component for protein translocation.
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