Biomedical subjects

Kai Matuschewski

Publications and source records attributed to Kai Matuschewski.

At least 19 recordsLinked to original sources

PubMed2026

Plasmodium thiamine pyrophosphokinase is essential for sporozoite formation and activation of an antiplasmodial thiamine analogue.

Oxythiamine, a thiamine analogue, inhibits Plasmodium falciparum proliferation by acting as an antimetabolite of vitamin B1. To elucidate in more detail its underlying mechanism of action, in vitro drug pressure was employed to generate oxythiamine-resistant P. falciparum lines. Whole-genome sequencing revealed that resistance was conferred by a single-point mutation in the thiamine pyrophosphokinase (TPK) gene. The mutated TPK has reduced activity, thereby likely limiting the conversion of oxythiamine into its active toxic form. To investigate the functional role of TPK across the parasite life cycle, a TPK-knockout line was generated in Plasmodium berghei. TPK-knockout parasites displayed a minor fitness cost during intraerythrocytic proliferation that could be overcome by infecting reticulocytes, but their sensitivity to oxythiamine was reduced fivefold in vivo, consistent with the hypothesis that activation of oxythiamine via TPK is essential for its antiplasmodial activity. In the Anopheles vector, TPK-knockout parasites produced a similar number of oocysts as wild-type parasites, but oocyst maturation was impaired and sporozoite formation was completely inhibited. These findings underscore an essential role for TPK in mediating the antiplasmodial activity of oxythiamine and reveal its critical function in sporogony within the mosquito, supporting its potential as a transmission-blocking target for antimalarial intervention.

Animals↗

PubMed2006

Regulation of apicomplexan microfilament dynamics by a minimal set of actin-binding proteins.

Efficient and rapid host cell invasion is a prerequisite for an intracellular parasitic life style. Pathogens typically induce receptor-mediated endocytosis and hijack the force-transducing system of a host cell to gain access to a replication-competent niche. In striking contrast, apicomplexan parasites such as Plasmodium, the causative agent of malaria, and the human and animal pathogens Toxoplasma and Cryptosporidium employ their own actomyosin motor machinery to propel themselves into prospective host cells. Understanding the regulation and dynamics of actin-based motility of these parasites is therefore central to understanding their pathogenesis. The parasite genomes harbour surprisingly few potential actin-regulatory proteins indicating that a basic repertoire meets the requirements to regulate actin dynamics. In this article, we summarize our current knowledge of Plasmodium microfilament dynamics and describe its potential players.

Actin Cytoskeleton↗

PubMed2006

Vaccine development against malaria.

Malaria vaccine development aims to significantly reduce mortality and morbidity in the two high-risk groups: young children and pregnant women in sub-Saharan Africa. A pre-erythrocytic subunit vaccine, RTS,S, was recently evaluated in a first Phase IIb clinical trial in young children that live in a mesoendemic area of malaria transmission. The identification of a major parasite-encoded ligand that was found to be involved in pregnancy-associated malaria allows for the rational development of a tailor-made subunit vaccine for young women. The generation of transgenic yellow fever viruses and defined attenuated vaccine lines by reverse genetics are complementary approaches that might further boost the development of safe vaccines that elicit protective immune responses in this population.

Animals↗

PubMed2006

Plasmodium motility: actin not actin' like actin.

Apicomplexan parasites such as Plasmodium and Toxoplasma display actomyosin-dependent motility in the absence of readily detectable actin polymers. Three recent studies indicate that parasite actin polymers, either harvested from parasites or formed from purified recombinant proteins, are exceptionally short ( approximately 100 nm). We propose that parasite motility could be directed by the transient formation of short actin filament scaffolds. Parasite actin polymers that support transmembrane receptors are pulled, by myosin interaction, backwards along the parasite periphery, resulting in forward movement.

Actin Cytoskeleton↗

PubMed2006

Getting infectious: formation and maturation of Plasmodium sporozoites in the Anopheles vector.

Research on Plasmodium sporozoite biology aims at understanding the developmental program steering the formation of mature infectious sporozoites - the transmission stage of the malaria parasite. The recent identification of genes that are vital for sporozoite egress from oocysts and subsequent targeting and transmigration of the mosquito salivary glands allows the identification of mosquito factors required for life cycle completion. Mature sporozoites appear to be equipped with the entire molecular repertoire for successful transmission and subsequent initiation of liver stage development. Innovative malaria intervention strategies that target the early, non-pathogenic phases of the life cycle will crucially depend on our insights into sporozoite biology and the underlying molecular mechanisms that lead the parasite from the mosquito midgut to the liver.

Animals↗

PubMed2005

Transgenic Plasmodium berghei sporozoites expressing beta-galactosidase for quantification of sporozoite transmission.

Malaria transmission occurs during a blood-meal of an infected Anopheles mosquito. Visualization and quantification of sporozoites along the journey from the mosquito midgut, where they develop, to the vertebrate liver, their final target organ, is important for understanding many aspects of sporozoite biology. Here we describe the generation of Plasmodium berghei parasites that express the reporter gene lacZ as a stable transgene, under the control of the sporozoite-specific CSP promoter. Transgenic sporozoites expressing beta-galactosidase can be simply visualized and quantified in an enzymatic assay. In addition, these sporozoites can be used to quantify sporozoites deposited in subcutaneous tissue during natural infection.

Animals↗

PubMed2005

High efficiency transfection of Plasmodium berghei facilitates novel selection procedures.

The use of transfection in the study of the biology of malaria parasites has been limited due to poor transfection efficiencies (frequency of 10(-6) to 10(-9)) and a paucity of selection markers. Here, a new method of transfection, using non-viral Nucleofector technology, is described for the rodent parasite Plasmodium berghei. The transfection efficiency obtained (episomal and targeted integration into the genome) is in the range of 10(-2) to 10(-3). Such high transfection efficiency strongly reduces the time, number of laboratory animals and amount of materials required to generate transfected parasites. Moreover, it allows different experimental strategies for reverse genetics to be developed and we demonstrate direct selection of stably and non-reversibly transformed, fluorescent protein (FP)-expressing parasites using FACS. Since there is no need to use a drug-selectable marker, this method increases the (low) number of selectable markers available for transformation of P. berghei and can in principle be extended to utilise additional FP. Furthermore the FACS-selected, FP-expressing parasites may serve as easily visualized reference lines that may still be genetically manipulated with the existing drug-selectable markers. The combination of enhanced transfection efficiency and a versatile rodent model provides a basis for the further development of novel tools for high throughput genome manipulation.

Animals↗

PubMed2005

A malarial cysteine protease is necessary for Plasmodium sporozoite egress from oocysts.

The Plasmodium life cycle is a sequence of alternating invasive and replicative stages within the vertebrate and invertebrate hosts. How malarial parasites exit their host cells after completion of reproduction remains largely unsolved. Inhibitor studies indicated a role of Plasmodium cysteine proteases in merozoite release from host erythrocytes. To validate a vital function of malarial cysteine proteases in active parasite egress, we searched for target genes that can be analyzed functionally by reverse genetics. Herein, we describe a complete arrest of Plasmodium sporozoite egress from Anopheles midgut oocysts by targeted disruption of a stage-specific cysteine protease. Our findings show that sporozoites exit oocysts by parasite-dependent proteolysis rather than by passive oocyst rupture resulting from parasite growth. We provide genetic proof that malarial cysteine proteases are necessary for egress of invasive stages from their intracellular compartment and propose that similar cysteine protease-dependent mechanisms occur during egress from liver-stage and blood-stage schizonts.

Animals↗

PubMed2005

A Plasmodium actin-depolymerizing factor that binds exclusively to actin monomers.

ADF/cofilins (AC) are essential F- and G-actin binding proteins that modulate microfilament turnover. The genome of Plasmodium falciparum, the parasite causing malaria, contains two members of the AC family. Interestingly, P. falciparum ADF1 lacks the F-actin binding residues of the AC consensus. Reverse genetics in the rodent malaria model system suggest that ADF1 performs vital functions during the pathogenic red blood cell stages, whereas ADF2 is not present in these stages. We show that recombinant PfADF1 interacts with monomeric actin but does not bind to actin polymers. Although other AC proteins inhibit nucleotide exchange on monomeric actin, the Plasmodium ortholog stimulates nucleotide exchange. Thus, PfADF1 differs in its biochemical properties from previously known AC proteins and seems to promote turnover exclusively by interaction with actin monomers. These findings provide important insights into the low cytosolic abundance and unique turnover characteristics of actin polymers in parasites of the phylum Apicomplexa.

Actins↗

PubMed2005

Intravital observation of Plasmodium berghei sporozoite infection of the liver.

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.

Aedes↗

PubMed2005

Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface.

Plasmodium parasites of mammals, including the species that cause malaria in humans, infect the liver first and develop there into clinically silent liver stages. Liver stages grow and ultimately produce thousands of first-generation merozoites, which initiate the erythrocytic cycles causing malaria pathology. Here, we present a Plasmodium protein with a critical function for complete liver stage development. UIS4 (up-regulated in infective sporozoites gene 4) is expressed exclusively in infective sporozoites and developing liver stages, where it localizes to the parasitophorous vacuole membrane. Targeted gene disruption of UIS4 in the rodent model malaria parasite Plasmodium berghei generated knockout parasites that progress through the malaria life cycle until after hepatocyte invasion but are severely impaired in further liver stage development. Immunization with UIS4 knockout sporozoites completely protects mice against subsequent infectious WT sporozoite challenge. Genetically attenuated liver stages may thus induce immune responses, which inhibit subsequent infection of the liver with WT parasites.

Animals↗

PubMed2005

Unusual properties of Plasmodium falciparum actin: new insights into microfilament dynamics of apicomplexan parasites.

Plasmodium falciparum, the etiologic agent of malaria, is a facultative intracellular parasite of the phylum Apicomplexa. A limited turnover of microfilaments takes place beneath the parasite plasma membrane, but the cytoplasm of apicomplexans is virtually devoid of F-actin. We produced Plasmodium actin in yeast. Purified recombinant Plasmodium actin polymerized inefficiently unless both gelsolin and phalloidin were added. The resulting actin polymers appeared fragmented in the fluorescence microscope. Plasmodium actin bound DNaseI about 200 times weaker than bovine non-muscle actin. Our findings suggest that the unique properties of Plasmodium actin can explain some of the unusual features of apicomplexan parasite microfilaments.

Actins↗

PubMed2004

Genetically modified Plasmodium parasites as a protective experimental malaria vaccine.

Malaria is a mosquito-borne disease that is transmitted by inoculation of the Plasmodium parasite sporozoite stage. Sporozoites invade hepatocytes, transform into liver stages, and subsequent liver-stage development ultimately results in release of pathogenic merozoites. Liver stages of the parasite are a prime target for malaria vaccines because they can be completely eliminated by sterilizing immune responses, thereby preventing malarial infection. Using expression profiling, we previously identified genes that are only expressed in the pre-erythrocytic stages of the parasite. Here, we show by reverse genetics that one identified gene, UIS3 (upregulated in infective sporozoites gene 3), is essential for early liver-stage development. uis3-deficient sporozoites infect hepatocytes but are unable to establish blood-stage infections in vivo, and thus do not lead to disease. Immunization with uis3-deficient sporozoites confers complete protection against infectious sporozoite challenge in a rodent malaria model. This protection is sustained and stage specific. Our findings demonstrate that a safe and effective, genetically attenuated whole-organism malaria vaccine is possible.

Animals↗

PubMed2004

Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins.

Invasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage-specific pre-erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite-specific genes encoding the circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low-molecular-mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite-red blood cell interactions.

Amino Acid Sequence↗

PubMed2003

Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites.

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.

Actin Cytoskeleton↗

PubMed2003

The Plasmodium sporozoite journey: a rite of passage.

Sporozoites are the most versatile of the invasive stages of the Plasmodium life cycle. During their passage within the mosquito vector and the vertebrate host, sporozoites display diverse behaviors, including gliding locomotion and invasion of, migration through and egress from target cells. At the end of the journey, sporozoites invade hepatocytes and transform into exoerythrocytic stages, marking the transition from the pre-erythrocytic to the erythrocytic part of the life cycle. This article discusses recent work, mostly done with rodent malaria parasites, that has contributed to a better understanding of the sporozoites' complex biology and which has opened up new avenues for future sporozoite research.

Animals↗

PubMed2002

Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage.

Injection of Plasmodium salivary gland sporozoites into the vertebrate host by Anopheles mosquitoes initiates malaria infection. Sporozoites develop within oocysts in the mosquito midgut and then enter and mature in the salivary glands. Although morphologically similar, oocyst sporozoites and salivary gland sporozoites differ strikingly in their infectivity to the mammalian host, ability to elicit protective immune responses, and cell motility. Here, we show that differential gene expression coincides with these dramatic phenotypic differences. Using suppression subtractive cDNA hybridization we identified highly up-regulated mRNAs transcribed from 30 distinct genes in salivary gland sporozoites. Of those genes, 29 are not significantly expressed in the parasite's blood stages. The most frequently recovered transcript encodes a protein kinase. Developmental up-regulation of specific mRNAs in the infectious transmission stage of Plasmodium indicates that their translation products may have unique roles in hepatocyte infection and/or development of liver stages.

Animals↗

PubMed2002

Plasmodium sporozoite invasion into insect and mammalian cells is directed by the same dual binding system.

Plasmodium sporozoites, the transmission form of the malaria parasite, successively invade salivary glands in the mosquito vector and the liver in the mammalian host. Sporozoite capacity to invade host cells is mechanistically related to their ability to glide on solid substrates, both activities depending on the transmembrane protein TRAP. Here, we show that loss-of- function mutations in two adhesive modules of the TRAP ectodomain, an integrin-like A-domain and a thrombospondin type I repeat, specifically decrease sporozoite invasion of host cells but do not affect sporozoite gliding and adhesion to cells. Irrespective of the target cell, i.e. in mosquitoes, rodents and cultured human or hamster cells, sporozoites bearing mutations in one module are less invasive, while those bearing mutations in both modules are non-invasive. In Chinese hamster ovary cells, the TRAP modules interact with distinct cell receptors during sporozoite invasion, and thus act as independently active pass keys. As these modules are also present in other members of the TRAP family of proteins in Apicomplexa, they may account for the capacity of these parasites to enter many cell types of phylogenetically distant origins.

Amino Acid Sequence↗