New insights into Microsporidia polar tube function and invasion mechanism.

Microsporidia comprise a large phylum of single-cell and obligate intracellular parasites that can infect a wide range of invertebrate and vertebrate hosts including humans. These fungal-related parasites are characterized by a highly reduced genome, a strong energy dependence on their host, but also by their unique invasion organelle known as the polar tube which is coiled within the resistant spore. Upon appropriate environmental stimulation, the long hollow polar tube (ranging from 50 to 500 µm in length) is extruded at ultra-fast speeds (300 µm/s) from the spore acting as a harpoon-like organelle to transport and deliver the infectious material or sporoplasm into the host cell. To date, seven polar tube proteins (PTPs) with distinct localizations along the extruded polar tube have been described. For example, the specific location of PTP4 and PTP7 at the tip of the polar tube supports their role in interacting with cellular receptor(s). This chapter provides a brief overview on the current understanding of polar tube structure and dynamics of extrusion, primarily through recent advancements in cryo-tomography and 3D reconstruction. It also explores the various mechanisms used for host cell invasion. Finally, recent studies on the structure and maturation of sporoplasm and its moving through the tube are discussed.

MicroAnnot: A Dedicated Workflow for Accurate Microsporidian Genome Annotation.

With nearly 1700 species, Microsporidia represent a group of obligate intracellular eukaryotes with veterinary, economic and medical impacts. To help understand the biological functions of these microorganisms, complete genome sequencing is routinely used. Nevertheless, the proper prediction of their gene catalogue is challenging due to their taxon-specific evolutionary features. As innovative genome annotation strategies are needed to obtain a representative snapshot of the overall lifestyle of these parasites, the MicroAnnot tool, a dedicated workflow for microsporidian sequence annotation using data from curated databases of accurately annotated microsporidian genes, has been developed. Furthermore, specific modules have been implemented to perform small gene (<300 bp) and transposable element identification. Finally, functional annotation was performed using the signature-based InterProScan software. MicroAnnot's accuracy has been verified by the re-annotation of four microsporidian genomes for which structural annotation had previously been validated. With its comparative approach and transcriptional signal identification method, MicroAnnot provides an accurate prediction of translation initiation sites, an efficient identification of transposable elements, as well as high specificity and sensitivity for microsporidian genes, including those under 300 bp.

The role of microsporidian polar tube protein 4 (PTP4) in host cell infection.

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these 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.

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.

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.

Identification and localization of polar tube proteins in the extruded polar tube of the microsporidian Anncaliia algerae.

Microsporidia are obligate intracellular parasites able to infect a wide range of hosts from invertebrates to vertebrates. The success of their invasion process is based on an original organelle, the polar tube, which is suddenly extruded from the spore to inoculate the sporoplasm into the host cytoplasm. The polar tube is mainly composed of proteins named polar tube proteins (PTPs). A comparative analysis allowed us to identify genes coding for 5 PTPs (PTP1 to PTP5) in the genome of the microsporidian Anncaliia algerae. While PTP1 and PTP2 are found on the whole polar tube, PTP3 is present in a large part of the extruded polar tube except at its end-terminal part. On the contrary, PTP4 is specifically detected at the end-terminal part of the polar tube. To complete PTPs repertoire, sequential sporal protein extractions were done with high concentration of reducing agents. In addition, a method to purify polar tubes was developed. Mass spectrometry analysis conducted on both samples led to the identification of a PTP3-like protein (PTP3b), and a new PTP (PTP7) only found at the extremity of the polar tube. The specific localization of PTPs asks the question of their roles in cell invasion processes used by A. algerae.

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.

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.

The human microsporidian Encephalitozoon hellem synthesizes two spore wall polymorphic proteins useful for epidemiological studies.

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.

Draft Genome Sequence of Tubulinosema ratisbonensis, a Microsporidian Species Infecting the Model Organism Drosophila melanogaster.

We present the draft genome sequence of Tubulinosema ratisbonensis, a microsporidium species infecting Drosophila melanogaster A total of 3,013 protein-encoding genes and an array of transposable elements were identified. This work represents a necessary step to develop a novel model of host-parasite relationships using the highly tractable genetic model D. melanogaster.

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project.

Brachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity.

Comparative genomics of microsporidian genomes reveals a minimal non-coding RNA set and new insights for transcription in minimal eukaryotic genomes.

Microsporidia are ubiquitous intracellular pathogens whose opportunistic nature led to their increased recognition with the rise of the AIDS pandemic. As the RNA world was largely unexplored in this parasitic lineage, we developed a dedicated in silico methodology to carry out exhaustive identification of ncRNAs across the Encephalitozoon and Nosema genera. Thus, the previously missing U1 small nuclear RNA (snRNA) and small nucleolar RNAs (snoRNAs) targeting only the LSU rRNA were highlighted and were further validated using 5' and 3'RACE-PCR experiments. Overall, the 15 ncRNAs that were found shared between Encephalitozoon and Nosema spp. may represent the minimal core set required for parasitic life. Interestingly, the systematic presence of a CCC- or GGG-like motif in 5' of all ncRNA and mRNA gene transcripts regardless of the RNA polymerase involved suggests that the RNA polymerase machineries in microsporidia species could use common factors. Our data provide additional insights in accordance with the simplification processes observed in these reduce genomes and underline the usefulness of sequencing closely related species to help identify highly divergent ncRNAs in these parasites.

EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall.

Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.

The Prediction and Validation of Small CDSs Expand the Gene Repertoire of the Smallest Known Eukaryotic Genomes.

The proper prediction of the gene catalogue of an organism is essential to obtain a representative snapshot of its overall lifestyle, especially when it is not amenable to culturing. Microsporidia are obligate intracellular, sometimes hard to culture, eukaryotic parasites known to infect members of every animal phylum. To date, sequencing and annotation of microsporidian genomes have revealed a poor gene complement with highly reduced gene sizes. In the present paper, we investigated whether such gene sizes may have induced biases for the methodologies used for genome annotation, with an emphasis on small coding sequence (CDS) gene prediction. Using better delineated intergenic regions from four Encephalitozoon genomes, we predicted de novo new small CDSs with sizes ranging from 78 to 255 bp (median 168) and corroborated these predictions by RACE-PCR experiments in Encephalitozoon cuniculi. Most of the newly found genes are present in other distantly related microsporidian species, suggesting their biological relevance. The present study provides a better framework for annotating microsporidian genomes and to train and evaluate new computational methods dedicated at detecting ultra-small genes in various organisms.

Draft genome sequence of the intestinal parasite Blastocystis subtype 4-isolate WR1.

The intestinal protistan parasite Blastocystis is characterized by an extensive genetic variability with 17 subtypes (ST1-ST17) described to date. Only the whole genome of a human ST7 isolate was previously sequenced. Here we report the draft genome sequence of Blastocystis ST4-WR1 isolated from a laboratory rodent at Singapore.

Microsporidian genomes harbor a diverse array of transposable elements that demonstrate an ancestry of horizontal exchange with metazoans.

Microsporidian genomes are the leading models to understand the streamlining in response to a pathogenic lifestyle; they are gene-poor and often possess small genomes. In this study, we show a feature of microsporidian genomes that contrasts this pattern of genome reduction. Specifically, genome investigations targeted at Anncaliia algerae, a human pathogen with a genome size of 23 Mb, revealed the presence of a hitherto undetected diversity in transposable elements (TEs). A total of 240 TE families per genome were identified, exceeding that found in many free-living fungi, and searches of microsporidian species revealed that these mobile elements represent a significant portion of their coding repertoire. Their phylogenetic analysis revealed that many cases of ancestry involve recent and bidirectional horizontal transfers with metazoans. The abundance and horizontal transfer origin of microsporidian TEs highlight a novel dimension of genome evolution in these intracellular pathogens, demonstrating that factors beyond reduction are at play in their diversification.

Identification of two new polar tube proteins related to polar tube protein 2 in the microsporidian Antonospora locustae.

Microsporidia are obligate intracellular eukaryotic parasites with a broad host spectrum characterized by a unique and highly sophisticated invasion apparatus, the polar tube (PT). In a previous study, two PT proteins, named AlPTP1 (50 kDa) and AlPTP2 (35 kDa), were identified in Antonospora locustae, an orthoptera parasite that is used as a biological control agent against locusts. Antibodies raised against AlPTP2 cross-reacted with a band migrating at ~70 kDa, suggesting that this 70-kDa antigen is closely related to AlPTP2. A blastp search against the A. locustae genome database allowed the identification of two further PTP2-like proteins named AlPTP2b (568 aa) and AlPTP2c (599 aa). Both proteins are characterized by a specific serine- and glycine-rich N-terminal extension with elastomeric structural features and share a common C-terminal end conserved with AlPTP2 (~88% identity for the last 250 aa). MS analysis of the 70-kDa band revealed the presence of AlPTP2b. Specific anti-AlPTP2b antibodies labelled the extruded PTs of the A. locustae spores, confirming that this antigen is a PT component. Finally, we showed that several PTP2-like proteins are also present in other phylogenetically related insect microsporidia, including Anncaliia algerae and Paranosema grylli.

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.