The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na+ regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na+ homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na+ homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.
Apicomplexan host cell invasion and gliding motility depend on the parasite's actomyosin system located beneath the plasma membrane of invasive stages. Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein. A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex. Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA. Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP. In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA. The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane. The head domain of the immobilized MyoA moves actin filaments that,directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family. The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.
The causative agents of malaria have developed a sophisticated machinery for entering multiple cell types in the human and insect hosts. In this machinery, a critical interaction occurs between the unusual myosin motor MyoA and the MyoA-tail Interacting Protein (MTIP). Here we present one crystal structure that shows three different conformations of Plasmodium MTIP, one of these in complex with the MyoA-tail, which reveal major conformational changes in the C-terminal domain of MTIP upon binding the MyoA-tail helix, thereby creating several hydrophobic pockets in MTIP that are the recipients of key hydrophobic side chains of MyoA. Because we also show that the MyoA helix is able to block parasite growth, this provides avenues for designing antimalarials.cell invasion machinery ͉ myosin-tail-interacting protein ͉ Plasmodium ͉ gliding motility
Although AAV vectors show promise for hepatic gene therapy, the optimal transcriptional regulatory elements have not yet been identified. In this study, we show that an AAV vector with the CMV enhancer/chicken b -actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1a promoter, and 137-fold higher expression than an AAV vector with the CMV promoter/enhancer. Although induction of the acute-phase response with the administration of lipopolysaccharide (LPS) activated the CMV promoter/enhancer from the context of an adenoviral vector in a previous study, LPS resulted in only a modest induction of this promoter from an AAV vector in vivo. An AAV vector with the CMV-b -actin promoter upstream of the coagulation protein human factor X (hFX) was injected intravenously into neonatal mice. This resulted in expression of hFX at 548 ng/ml (6.8% of normal) for up to 1.2 years, and 0.6 copies of AAV vector per diploid genome in the liver at the time of sacrifice. Neonatal intramuscular injection resulted in expression of hFX at 248 ng/ml (3.1% of normal), which derived from both liver and muscle. We conclude that neonatal gene therapy with an AAV vector with the CMV-b -actin promoter might correct hemophilia due to hFX deficiency. OVERVIEW SUMMARYOptimization of gene expression from an AAV vector might allow higher levels of expression to be achieved, which will be necessary for effective gene therapy for some genetic deficiencies. It might also allow a lower dose of vector to be administered, which would reduce the risk of insertional mutagenesis or germ line transmission. Neonatal gene transfer might reduce the chance of inducing an immune response, and would lead to a more immediate correction of a genetic disease than would transfer into adults. We demonstrate here that the CMV-b -actin promoter is expressed well from an AAV vector in the liver. Neonatal intravenous administration of an AAV vector that expresses the coagulation protein factor X from this promoter results in therapeutic levels of factor X for more than 1 year in mice. Neonatal gene therapy with an AAV vector may allow effective gene therapy to be achieved for hemophilia.
For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 ؋ 10 6 infectious units of recombinant adeno-associated virus encoding the human -glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adenoassociated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.
In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP1 42 ) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/⌬1 and MSP9/⌬2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/⌬1 and MSP9/⌬2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP1 42 , the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.
Since the developmental stages of malarial parasites which replicate within erythrocytes are responsible for the morbidity and mortality associated with this disease, antigens produced by these stages have been proposed as candidates for a vaccine. One surface protein of merozoites (MSP-1) has been shown to immunize both rodents and primates against virulent challenge infection in experimental systems. However, little is known of relevant epitopes on the molecule, and attempts to obtain recombinant MSP-1 polypeptides in a native configuration have proven difficult. We have found that the cysteine-rich, carboxyl-terminal region of the MSP-1 protein from the rodent malarial parasite Plasmodiumyoeliiyoelii can be expressed in a native configuration as a fusion protein in Escherichia coli. This recombinant polypeptide containing 15 kDa of the predicted 197-kDa protein elicits antibodies in mice which recognize the native parasite MSP-1. Most significantly, both inbred and outbred mice immunized with the fusion protein in Ribi adjuvant are partially and in some cases completely protected against challenge infection with an otherwise lethal parasite strain. This is the first observation of such significant protection obtained with a small portion of the MSP-1 produced in recombinant systems.
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