Host cell invasion by apicomplexan parasites: the junction conundrum

No abstract available

Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes

Poverty in the 1990s Evidence from the 1994 Living in Ireland survey

SIGLEAvailable from British Library Document Supply Centre-DSC:4111.1682(170) / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Country report Yugoslavia (Serbia-Montenegro) 3rd quarter 1998

Available from British Library Document Supply Centre-DSC:3481.901455(1998/3) / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Mutants of interest in studies on host cell invasion by apicomplexans.

<p>Mutants of interest in studies on host cell invasion by apicomplexans.</p

Molecular models of apicomplexan gliding and invasion.

<p><b>A.</b> The parasite motor (glideosome) is located in the space between the parasite plasma membrane (PPM) and the inner membrane complex (IMC) apposed to the microtubules. Gliding motility is mediated by the binding of the ectodomain of transmembrane TRAP-family proteins to a solid substrate, while the cytoplasmic tail of the protein is linked to the parasite motor. The integrity of the glideosome is maintained by the gliding-associated protein 45 (GAP45), which is anchored to the PPM at one end and to the IMC, via GAPs 40 and 50, at the other end. The link between the GAPs, and ultimately the IMC, to actin is provided by Myosin A (MyoA) and the MyoA Light Chain 1 (MLC1). The movement of the cell is the consequence of the capping, by myosin-actin activity, of the TRAP-family protein. <b>B.</b> The model of invasion seen as the junction structured by the AMA1-RON complex. The figure on the left shows a <i>Toxoplasma</i> tachyzoite invading a host cell. The arrow indicates the direction of movement. Immunostaining of surface MIC2 (sMIC2) stains the part of the zoite cell still extracellular (blue), while the rest of the cell, already internalized, is not stained. Immunostaining of total RON4 (tRON4, red) marks the junction as a ring at the point of constriction, indicated by the circle, and the rhoptries at the apical pole of the zoite cell. After a first step of adhesion to the host cell plasma membrane (HCPM) mediated by parasite surface adhesins and host cell surface receptors, the binding of the transmembrane protein AMA1 to RON2, inserted at the host cell membrane and complexed with RONs 4 and 5, forms the junction. The link to the parasite motor is as in (A), while host actin recruited at the junction provides the link to the host cell cytoskeleton. The movement of the zoite towards the interior of the newly formed parasitophorous vacuole membrane (PVM) is thus a consequence of the capping of AMA1, which would be anchored at the junction by binding to RON2. <b>C.</b> Models of zoite invasion in which the functions of AMA1 and RONs are dissociated. Color codes and acronyms are as in (A) and (B). After a first step of adhesion mediated by parasite surface adhesins and host cell surface receptors, AMA1 binding to a host cell receptor provides a strong attachment between the zoite and host cell membranes, possibly leading to reorientation of the zoite to allow junction formation. Three different hypotheses could then explain junction formation: 1. A still-unknown transmembrane parasite protein binds to the motor and to RON2, taking the place previously assigned to AMA1. 2. Unknown proteins structure the junction and connect the parasite motor to the host cell cortical actin, in which case the role of the RONs at the junction is not structural. 3. Unknown proteins structure the junction without a role of the parasite motor during invasion.</p

UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii

Nucleo-cytoplasmic RNA export is an essential post-transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans

Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion

Abstract Background Apicomplexan parasites employ a unique form of movement, termed gliding motility, in order to invade the host cell. This movement depends on the parasite\u2019s actomyosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system, since mutants for core components of the gliding machinery, such as parasite actin or subunits of the MyoA-motor complex (the glideosome), remain motile and invasive, albeit at significantly reduced efficiencies. While compensatory mechanisms and unusual polymerisation kinetics of parasite actin have been evoked to explain these findings, the actomyosin system could also play a role distinct from force production during parasite movement. Results In this study, we compared the phenotypes of different mutants for core components of the actomyosin system in Toxoplasma gondii to decipher their exact role during gliding motility and invasion. We found that, while some phenotypes (apicoplast segregation, host cell egress, dense granule motility) appeared early after induction of the act1 knockout and went to completion, a small percentage of the parasites remained capable of motility and invasion well past the point at which actin levels were undetectable. Those act1 conditional knockout (cKO) and mlc1 cKO that continue to move in 3D do so at speeds similar to wildtype parasites. However, these mutants are virtually unable to attach to a collagen-coated substrate under flow conditions, indicating an important role for the actomyosin system of T. gondii in the formation of attachment sites. Conclusion We demonstrate that parasite actin is essential during the lytic cycle and cannot be compensated by other molecules. Our data suggest a conventional polymerisation mechanism in vivo that depends on a critical concentration of G-actin. Importantly, we demonstrate that the actomyosin system of the parasite functions in attachment to the surface substrate, and not necessarily as force generator

Supplementary information files for A pharmacokinetic–pharmacodynamic model for chemoprotective agents against malaria

Supplementary files for article A pharmacokinetic–pharmacodynamic model for chemoprotective agents against malaria Chemoprophylactics are a vital tool in the fight against malaria. They can be used to protect populations at risk, such as children younger than the age of 5 in areas of seasonal malaria transmission or pregnant women. Currently approved chemoprophylactics all present challenges. There are either concerns about unacceptable adverse effects such as neuropsychiatric sequalae (mefloquine), risks of hemolysis in patients with G6PD deficiency (8-aminoquinolines such as tafenoquine), or cost and daily dosing (atovaquone–proguanil). Therefore, there is a need to develop new chemoprophylactic agents to provide more affordable therapies with better compliance through improving properties such as pharmacokinetics to allow weekly, preferably monthly, dosing. Here we present a pharmacokinetic–pharmacodynamic (PKPD) model constructed using DSM265 (a dihydroorotate dehydrogenase inhibitor with activity against the liver schizonts of malaria, therefore, a prophylaxis candidate). The PKPD model mimics the parasite lifecycle by describing parasite dynamics and drug activity during the liver and blood stages. A major challenge is the estimation of model parameters, as only blood-stage parasites can be observed once they have reached a threshold. By combining qualitative and quantitative knowledge about the parasite from various sources, it has been shown that it is possible to infer information about liver-stage growth and its initial infection level. Furthermore, by integrating clinical data, the killing effect of the drug on liver- and blood-stage parasites can be included in the PKPD model, and a clinical outcome can be predicted. Despite multiple challenges, the presented model has the potential to help translation from preclinical to late development for new chemoprophylactic candidates. </p