The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism.

The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG (Cdc45, Mcm2-7, GINS). CMG is "built" on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 "double hexamer." In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol ϵ and lagging Pol α-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins.

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC.

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host-derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole-host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative 'meront' stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (< 10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC-1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno-EM revealed that the ATP-delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria-vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP-delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite.

Branching network of proteinaceous filaments within the parasitophorous vacuole of Encephalitozoon cuniculi and Encephalitozoon hellem.

The microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculi developing spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

Patterns of 5' untranslated region length distribution in Encephalitozoon cuniculi: implications for gene regulation and potential links between transcription and splicing.

Encephalitozoon cuniculi, a eukaryotic intracellular parasite belonging to the group Microsporidia, has a highly reduced and compacted genome. Its mRNA transcripts have been found to differ between the two life stages, the spore and meront, of the parasite. Spore transcripts generally have more transcription start sites, longer 5' untranslated regions (UTRs), and overlap more frequently with upstream genes than those of meronts. A previous analysis of 31 meront gene transcripts showed that most have short 5'UTRs, and intron-containing genes, mostly ribosomal protein genes, exclusively have very short 5'UTRs. Here we analyzed a larger set of transcripts from meronts, and we find a pattern of 5'UTR length distribution similar to other reduced genomes. There is an abundance of very short 5'UTRs that are <20 bp in length, and very few 5'UTRs that are much longer. We also find a relationship between gene categories and 5'UTR length: intron-containing genes and ribosomal protein genes have exclusively short 5'UTRs. We suggest that the abundance of short 5'UTRs may be related to a class of highly expressed genes that benefit the parasite's growth cycle. Also, the longer 5'UTRs may be playing a role in down-regulating expression of genes that require temporal or environment-induced expression.

The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26.

Binding of elongation factor Spt6 to Iws1 provides an effective means for coupling eukaryotic mRNA synthesis, chromatin remodelling and mRNA export. We show that an N-terminal region of Spt6 (Spt6N) is responsible for interaction with Iws1. The crystallographic structures of Encephalitozoon cuniculi Iws1 and the Iws1/Spt6N complex reveal two conserved binding subdomains in Iws1. The first subdomain (one HEAT repeat; HEAT subdomain) is a putative phosphoprotein-binding site most likely involved in an Spt6-independent function of Iws1. The second subdomain (two ARM repeats; ARM subdomain) specifically recognizes a bipartite N-terminal region of Spt6. Mutations that alter this region of Spt6 cause severe phenotypes in vivo. Importantly, the ARM subdomain of Iws1 is conserved in several transcription factors, including TFIIS, Elongin A and Med26. We show that the homologous region in yeast TFIIS enables this factor to interact with SAGA and the Mediator subunits Spt8 and Med13, suggesting the molecular basis for TFIIS recruitment at promoters. Taken together, our results provide new structural information about the Iws1/Spt6 complex and reveal a novel interaction domain used for the formation of transcription networks.

tRNA binding properties of eukaryotic translation initiation factor 2 from Encephalitozoon cuniculi.

A critical consequence of the initiation of translation is the setting of the reading frame for mRNA decoding. In eukaryotic and archaeal cells, heterotrimeric initiation factor e/aIF2, in its GTP form, specifically binds Met-tRNA(i)(Met) throughout the translation initiation process. After start codon recognition, the factor, in its GDP-bound form, loses affinity for Met-tRNA(i)(Met) and eventually dissociates from the initiation complex. The role of each aIF2 subunit in tRNA binding has been extensively studied in archaeal systems. The isolated archaeal γ subunit is able to bind tRNA, but the α subunit is required for strong binding. Until now, difficulties during purification have hampered the study of the role of each of the three subunits of eukaryotic eIF2 in specific binding of the initiator tRNA. Here, we have produced the three subunits of eIF2 from Encephalitozoon cuniculi, isolated or assembled into heterodimers or into the full heterotrimer. Using assays following protection of Met-tRNA(i)(Met) against deacylation, we show that the eukaryotic γ subunit is able to bind by itself the initiator tRNA. However, the two peripheral α and ß subunits are required for strong binding and contribute equally to tRNA binding affinity. The core domains of α and ß probably act indirectly by stabilizing the tRNA binding site on the γ subunit. These results, together with those previously obtained with archaeal aIF2 and yeast eIF2, show species-specific distributions of the roles of the peripheral subunits of e/aIF2 in tRNA binding.

Glycosylation of the major polar tube protein of Encephalitozoon cuniculi.

To infect their host cells the Microsporidia use a unique invasion organelle, the polar tube complex. During infection, the organism is injected into the host cell through the hollow polar tube formed during spore germination. Currently, three proteins, PTP1, PTP2, and PTP3 have been identified by immunological and molecular techniques as being components of this structure. Genomic data suggests that Microsporidia are capable of O-linked, but not N-linked glycosylation as a post-translational protein modification. Cells were infected with Encephalitozoon cunicuili, labeled with radioactive mannose or glucosamine, and the polar tube proteins were examined for glycosylation. PTP1 was clearly demonstrated to be mannosylated consistent with 0-glycosylation. In addition, it was evident that several other proteins were mannosylated, but no labeling was seen with glucosamine. The observed post-translational mannosylation of PTP1 may be involved in the functional properties of the polar tube, including its adherence to host cells during penetration.

Metabolism of an alkyl polyamine analog by a polyamine oxidase from the microsporidian Encephalitozoon cuniculi.

Encephalitozoon cuniculi is a microsporidium responsible for systemic illness in mammals. In the course of developing leads to new therapy for microsporidiosis, we found that a bis(phenylbenzyl)3-7-3 analog of spermine, 1,15-bis{N-[o-(phenyl)benzylamino}-4,12-diazapentadecane (BW-1), was a substrate for an E. cuniculi amine oxidase activity. The primary natural substrate for this oxidase activity was N'-acetylspermine, but BW-1 had activity comparable to that of the substrate. As the sole substrate, BW-1 gave linear reaction rates over 15 min and K(m) of 2 microM. In the presence of N'-acetylspermine, BW-1 acted as a competitive inhibitor of oxidase activity and may be a subversive substrate, resulting in increased peroxide production. By use of (13)C-labeled BW-1 as a substrate and nuclear magnetic resonance analysis, two products were determined to be oxidative metabolites, a hydrated aldehyde or dicarboxylate and 2(phenyl)benzylamine. These products were detected after exposure of (13)C-labeled BW-1 to E. cuniculi preemergent spore preparations and to uninfected host cells. In previous studies, BW-1 was curative in a rodent model of infection with E. cuniculi. The results in this study demonstrate competitive inhibition of oxidase activity by BW-1 and support further studies of this oxidase activity by the parasite and host.

A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi.

Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont. Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins. This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities. The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia, to import ATP from the cytosol of their eukaryotic host cells. The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi, by reductive evolution and analogous gene replacement. Instead of the mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle.

The nascent parasitophorous vacuole membrane of Encephalitozoon cuniculi is formed by host cell lipids and contains pores which allow nutrient uptake.

Microsporidia are obligate intracellular pathogens which enter host cells by the discharge of a hollow tube through which the sporoplasma is extruded into the host cell. Since this invasion mechanism is very different from common entry strategies, the formation of the parasitophorous vacuole (PV) in Encephalitozoon species is likely to be distinct from known principles. We investigated the origin of the nascent Encephalitozoon cuniculi PV membrane with the aid of fluorescent lipid probes. When Bodipy 500/510-C(12)-HPC-labeled spores were used for infection, the emerging PV membrane was unlabeled, suggesting that sporoplasma-derived lipids do not significantly contribute to the formation of the PV membrane. In contrast, when raft and nonraft microdomains of the host cell plasma membrane were selectively labeled with DiIC(16) and Speedy DiO, both tracers were detectable in the nascent PV membrane shortly after infection, indicating that the bulk lipids of the PV membrane are host cell derived. Time-lapse fluorescence microscopy revealed that the formation of the PV membrane is a fast event (<1.3 s), which occurred simultaneously with the extrusion of the sporoplasma. The portion of the discharged tube which is in contact with the host cell was found to be coated with labeled host cell lipids, which might be an indication for a plasma membrane invagination at the contact site. To investigate the presence of pores in the E. cuniculi PV membrane, we microinjected fluorescent dyes of different sizes into infected host cells. A 0.5-kDa dextran as well as 0.8- to 1.1-kDa peptides could rapidly enter the PV, while a 10-kDa dextran was stably excluded from the PV lumen, indicating that the PV membrane possesses pores with an exclusion size of <10 kDa, which should allow metabolite exchange.

Identification of a new spore wall protein from Encephalitozoon cuniculi.

Microsporidia form environmentally resistant spores that are critical for their host-to-host transmission and persistence in the environment. The spore walls of these organisms are composed of two layers, the exospore and the endospore. Two spore wall proteins (SWP1 and SWP2) have been previously identified in members of the Encephalitozoonidae family. These proteins localize to the exospore. The endospore is known to contain chitin, and a putative glycosylphosphatidylinositol (GPI)-anchored chitin deacetylase has been localized to the plasmalemma-endospore interface. Using proteomic techniques, we have identified a new spore wall protein (SWP3) that is located in the endospore. The gene for this protein is located on chromosome 1 and corresponds to the open reading frame ECU01_1270. SWP3 is predicted to have a signal peptide and to be GPI anchored. Consistent with these modifications, two-dimensional electrophoresis demonstrated that SWP3 has an acidic pI and a molecular mass of <20 kDa. By immunoelectron microscopy, this protein was found on the cell surface during sporogony and in the endospore in mature spores. SWP3 has several potential O-glycosylation sites, and it is possible that it is a mannosylated protein like the major polar tube protein (PTP1).

RNA-binding domain proteins in Kinetoplastids: a comparative analysis.

RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein.

The parasitophorous vacuole membrane of Encephalitozoon cuniculi lacks host cell membrane proteins immediately after invasion.

Microsporidia of the genus Encephalitozoon develop inside a parasitophorous vacuole (PV) of unknown origin. Using colocalization studies, the PV was found to be absent from the endocytic pathway markers early endosomal autoantigen 1, transferrin receptor, and lysosome-associated membrane protein 1 and for the endoplasmic reticulum marker calnexin. The nonfusiogenic characteristic of the PV appears to be acquired as early as 1 min postinfection and is not reversed by drug treatment with albendazole or fumagillin.

GoArrays: highly dynamic and efficient microarray probe design.

MOTIVATION: The use of oligonucleotide microarray technology requires a very detailed attention to the design of specific probes spotted on the solid phase. These problems are far from being commonplace since they refer to complex physicochemical constraints. Whereas there are more and more publicly available programs for microarray oligonucleotide design, most of them use the same algorithm or criteria to design oligos, with only little variation. RESULTS: We show that classical approaches used in oligo design software may be inefficient under certain experimental conditions, especially when dealing with complex target mixtures. Indeed, our biological model is a human obligate parasite, the microsporidia Encephalitozoon cuniculi. Targets that are extracted from biological samples are composed of a mixture of pathogen transcripts and host cell transcripts. We propose a new approach to design oligonucleotides which combines good specificity with a potentially high sensitivity. This approach is original in the biological point of view as well as in the algorithmic point of view. We also present an experimental validation of this new strategy by comparing results obtained with standard oligos and with our composite oligos. A specific E.cuniculi microarray will overcome the difficulty to discriminate the parasite mRNAs from the host cell mRNAs demonstrating the power of the microarray approach to elucidate the lifestyle of an intracellular pathogen using mix mRNAs.

Guanosine distribution and oxidation resistance in eight eukaryotic genomes.

Reactive oxygen species that attack DNA are continuously generated in living cells. Both the guanosine (G) mole fraction and its distribution should affect the stability of genomes and their parts to oxidation. At a lesser G content, genomes should be more oxidation resistant or "ennobled". Oxidant scavenging by G's in nonessential parts of introns and intergenic domains should decrease G oxidation in the essential exons. To determine whether genomes are indeed ennobled and whether oxidant-scavenging domains exist in genomes, the relative rates of guanosine oxidation in average exons, introns, and intergenic domains were estimated. Comparison among genomes indicated that average exons are ennobled in the genomes of Caenorhabditis (worm), Arabidopsis (plant), Saccharomyces (yeast), Schizosaccharomyces (yeast), and Plasmodium (malaria parasite), and that average introns and intergenic domains are ennobled in these genomes and in the genome of Drosophila (fly). The exon oxidation rates estimated for these genomes were less than the rate for the hypothetical "standard" genome, with a 0.25 mole fraction of uniformly distributed G. For Plasmodium the rate was half of that estimated for the standard genome. Average exons were not ennobled in the human or fly genomes; their G distributions were comparable to that in the standard genome. Instead, their exons were situated between introns and intergenic domains that could protect them by oxidant scavenging, the G's of their introns and intergenic domains outnumbering those of their exons 50-fold in humans and 4-fold in flies. The G distribution in the Encephalitozoon (parasite) genome was not protective relative to that of the standard genome.

Functional and evolutionary analysis of a eukaryotic parasitic genome.

The DNA sequences of the 11 linear chromosomes of the approximately 2.9 Mbp genome of Encephalitozoon cuniculi, an obligate intracellular parasite of mammals, include approximately 2000 putative protein-coding genes. The compactness of this genome is associated with the length reduction of various genes. Essential functions are dependent on a minimal set of genes. Phylogenetic analysis supports the hypotheses that microsporidia are related to fungi and have retained a mitochondrion-derived organelle, the mitosome.

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites infesting many animal groups. Lacking mitochondria and peroxysomes, these unicellular eukaryotes were first considered a deeply branching protist lineage that diverged before the endosymbiotic event that led to mitochondria. The discovery of a gene for a mitochondrial-type chaperone combined with molecular phylogenetic data later implied that microsporidia are atypical fungi that lost mitochondria during evolution. Here we report the DNA sequences of the 11 chromosomes of the approximately 2.9-megabase (Mb) genome of Encephalitozoon cuniculi (1,997 potential protein-coding genes). Genome compaction is reflected by reduced intergenic spacers and by the shortness of most putative proteins relative to their eukaryote orthologues. The strong host dependence is illustrated by the lack of genes for some biosynthetic pathways and for the tricarboxylic acid cycle. Phylogenetic analysis lends substantial credit to the fungal affiliation of microsporidia. Because the E. cuniculi genome contains genes related to some mitochondrial functions (for example, Fe-S cluster assembly), we hypothesize that microsporidia have retained a mitochondrion-derived organelle.

Encephalitozoon cuniculi (Microspora): characterization of a phospholipid metabolic pathway potentially linked to therapeutics.

Phospholipid metabolism of the microsporidian Encephalitozoon cuniculi, an obligate intracellular parasite, has been investigated. Labeled precursor incorporation experiments have shown that phosphatidylserine decarboxylase and phosphatidylethanolamine N-methyltransferase are more active in cells infected by E. cuniculi than in uninfected cells. In contrast, no difference was observed in the activity of Kennedy pathway's enzymes, the mammalian pathway. This suggests the occurrence in microsporidia of a bacteria- and fungi-typical pathway for phospholipid synthesis, which is supported by the identification of two genes implicated in this pathway, the cds gene encoding the key enzyme CDP-diacylglycerol synthase (E.C. 2.7.7.41) and the pss gene for CDP-alcohol phosphatidyltransferase. The pss gene could encode phosphatidylserine synthase (E.C. 2.7.8.8.), which catalyses the de novo synthesis of phosphatidylserine in bacteria and fungi. The complete CDP-diacylglycerol synthase messenger has been isolated and shows very short 5' and 3' untranslated regions. This is strong evidence for the functionality of a metabolic pathway which could be a potential target against microsporidia which infect humans.

Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.