Valérie Polonais

Functional Dissection of the Apicomplexan Glideosome Molecular Architecture

The glideosome of apicomplexan parasites is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45. We identify GAP40, a polytopic protein of the IMC, as an additional glideosome component and show that GAP45 is anchored to the PM and IMC via its N- and C-terminal extremities, respectively. While the C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. The orthologous Plasmodium falciparum GAP45 can fulfill this dual function, as shown by transgenera complementation, whereas the coccidian GAP45 homolog (designated here as) GAP70 specifically recruits the glideosome to the apical cap of the parasite.

BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei

While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens.

Plasticity between MyoC- and MyoA-Glideosomes: An Example of Functional Compensation in Toxoplasma gondii Invasion

The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.

Annotation of microsporidian genomes using transcriptional signals

High-quality annotation of microsporidian genomes is essential for understanding the biological processes that govern the development of these parasites. Here we present an improved structural annotation method using transcriptional DNA signals. We apply this method to re-annotate four previously annotated genomes, which allow us to detect annotation errors and identify a significant number of unpredicted genes. We then annotate the newly sequenced genome of Anncaliia algerae. A comparative genomic analysis of A. algerae permits the identification of not only microsporidian core genes, but also potentially highly expressed genes encoding membrane-associated proteins, which represent good candidates involved in the spore architecture, the invasion process and the microsporidian-host relationships. Furthermore, we find that the ten-fold variation in microsporidian genome sizes is not due to gene number, size or complexity, but instead stems from the presence of transposable elements. Such elements, along with kinase regulatory pathways and specific transporters, appear to be key factors in microsporidian adaptive processes.

Versatility in the acquisition of energy and carbon sources by the Apicomplexa

Members of the phylum Apicomplexa are motile and rapidly dividing intracellular parasites, able to occupy a large spectrum of niches by infecting diverse hosts and invading various cell types. As obligate intracellular parasites, most apicomplexans only survive for a short period extracellularly, and, during this time, have a high energy demand to power gliding motility and invasion into new host cells. Similarly, these fast‐replicating intracellular parasites are critically dependent on host‐cell nutrients as energy and carbon sources, noticeably for the extensive membrane biogenesis imposed during growth and division. To access host‐cell metabolites, the apicomplexans Toxoplasma gondii and Plasmodium falciparum have evolved strategies that exquisitely reflect adaptation to their respective niches. In the present review, we summarize and compare some recent findings regarding the energetic metabolism and carbon sources used by these two genetically tractable apicomplexans during host‐cell invasion and intracellular growth and replication.

Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii.

Toxoplasma gondii possesses 11 rather atypical myosin heavy chains. The only myosin light chain described to date is MLC1, associated with myosin A, and contributing to gliding motility. In this study, we examined the repertoire of calmodulin‐like proteins in Apicomplexans, identified six putative myosin light chains and determined their subcellular localization in T. gondii and Plasmodium falciparum. MLC2, only found in coccidians, is associated with myosin D via its calmodulin (CaM)‐like domain and anchored to the plasma membrane of T. gondii via its N‐terminal extension. Molecular modeling suggests that the MyoD–MLC2 complex is more compact than the reported structure of Plasmodium MyoA–myosin A tail‐interacting protein (MTIP) complex. Anchorage of this MLC2 to the plasma membrane is likely governed by palmitoylation.

Carbohydrate Moieties of Microsporidian Polar Tube Proteins Are Targeted by Immunoglobulin G in Immunocompetent Individuals

ABSTRACT Microsporidia of the Encephalitozoon species are frequently found as opportunistic pathogens of immunocompromised patients, but very little is known about the prevalence and significance of Encephalitozoon infection in immunocompetent individuals. It was reported previously that 8% of Dutch blood donors and 5% of pregnant French women had an immunoglobulin G (IgG) immune response against specific organelles of Encephalitozoon intestinalis. These organelles, the so-called polar tube and anchoring disk, are used to penetrate membranes of host cells during infection. The unexpectedly high percentage of immunocompetent individuals with IgG against these organelles suggested that infection of humans with microsporidia might be more common than previously recognized. In the present study, we analyzed this anti-Encephalitozoon IgG response by using indirect immunofluorescence, Western blotting, two-dimensional gel electrophoresis, and chemical deglycosylation. Our results show that the antibody response is directed against the posttranslational carbohydrate modification of the major polar tube protein (polar tube protein 1) and carbohydrate moieties of proteins in the anchoring region of the polar tube of Encephalitozoon. In addition, the antibodies were found to decrease the infectivity of E. intestinalis in vitro. The significance and possible origin of these prevalent antibodies are discussed.

The Microsporidian Polar Tube and Its Role in Invasion

The Microsporidia are a phylum of small unicellular eukaryotes comprising more than 150 genera and 1200 species. They are obligate intracellular parasites which are able to form environmentally resistant spores. Historically, Nosema bombycis, was the first described species in this phylum and is the etiological agent of “pebrine” disease that nearly destroyed the silk-worm industry in the nineteenth century. Although the majority of microsporidia that have been described are found in arthropods and fishes, being responsible of important economic losses in the apiculture and fish farms, there are several species of medical and veterinary significance which infect animals and humans.1,2 Cerebral microsporidian infections attributed to Encephalitozoon cuniculi were initially described in 1922 in rabbits with granulomatous encephalitis and several infections were then reported in most vertebrate groups. The first case of microsporidiosis in a human was identified in 1959 in a nine-year-old boy suffering from neurological disorders.3 While reports of humans infected with microsporidia were extremely rare before the AIDS epidemic, these organisms are now recognized as significant emerging pathogens in immunocompromised hosts (HIV-infected patients with AIDS and organ transplant recipients) and is a cause of intestinal, ocular, muscular and systemic diseases.2 Some clinical manifestations have been also reported in immunocompetent hosts. Serological studies with blood donors and pregnant women revealed a prevalence of about 8%,4 suggesting that infections by microsporidia may be common in humans. So far, species belonging to seven different genera Brachiola (recendy renamed Anncaliia), Encephalitozoon, Enterocytozoon, Nosema, Pleistophora, Trachipleistophora and Vittaforma have been found in human infections. Although the origin of infection and epidemiology still remain to be documented for human microsporidiosis, horizontal transmission of most microsporidia occurs by oral ingestion of spores, with the site of initial infection being the gastrointestinal tract.5

The Human Microsporidian Encephalitozoon hellem Synthesizes Two Spore Wall Polymorphic Proteins Useful for Epidemiological Studies

ABSTRACT Microsporidia are obligate intracellular fungus-related parasites considered as emerging opportunistic human pathogens. Their extracellular infective and resistance stage is a spore surrounded by a unique plasma membrane protected by a thick cell wall consisting of two layers: the electron-lucent inner endospore which contains chitin and protein components and the outer-electron-dense and mainly proteinaceous exospore. We identified the whole sequences of two spore wall proteins in the microsporidian species Encephalitozoon hellem, designated EhSWP1a and EhSWP1b. Isolation of the genes encoding these SWP1-like proteins was performed using degenerate oligonucleotides based on the amino acid sequence alignment of the previously reported Encephalitozoon cuniculi and Encephalitozoon intestinalis SWP1s. Sequences lacking the 5′ and 3′ ends were then identified by PCR and reverse transcription (RT)-PCR amplifications. The swp1a and swp1b genes encode proteins of 509 and 533 amino acids, respectively, which present an identical N-terminal domain of 382 residues and a variable C-terminal extension mainly characterized by a 26-amino-acid (aa) deletion/insertion containing glutamate- and lysine-rich repeats. Using polyclonal antibodies raised against recombinant polypeptides, we showed that EhSWP1a and EhSWP1b appear as dithiothreitol (DTT)-soluble bands of 55 and 60 kDa in size, respectively. Immunolocalization experiments by IFA and transmission electron microscopy (TEM) indicated that both proteins are present at the onset of sporogony and are specifically located to the spore wall exospore in mature spores. Analysis of four E. hellem human isolates revealed that the C-terminal regions of both EhSWP1a and EhSWP1b are polymorphic, which is of interest for epidemiological studies.

Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites

Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.