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.

An improved procedure for Percoll gradient separation of sporogonial stages in Encephalitozoon cuniculi (Microsporidia).

Intracellular development of microsporidian parasites comprises a proliferative phase (merogony) followed by a differentiation phase (sporogony) leading to the release of resistant spores. Sporogony implies, successively, meront-to-sporont transformation, sporont division into sporoblasts, and sporogenesis. We report a procedure improving the separation of sporogonial stages of Encephalitozoon cuniculi, a species that develops inside parasitophorous vacuoles of mammalian cells. Supernatants of E. cuniculi-infected Madin-Darby canine kidney cell cultures provided a large number of parasites mixed with host-cell debris. This material was gently homogenized in phosphate-buffered saline containing 0.05% saponin and 0.05% Triton X-100 then filtered through glass wool columns. Centrifugation of the filtrate on 70% Percoll-0.23 M sucrose gradient gave a reproducible pattern of bands at different densities. Transmission electron microscopy showed that three of the four collected fractions were free of visible contaminants. Corresponding prominent cell stages were early sporoblasts (fraction B), late sporoblasts plus immature spores (fraction C), and mature spores (fraction D). Further centrifugation of the lightest fraction (A) on 30% Percoll-0.23 M sucrose gradient generated a sporont-rich fraction (A2). First analysis of proteins from fractions A2 and D by two-dimensional gel electrophoresis suggested a potential use of the described method for proteomic profiling.

What does the microsporidian E. cuniculi tell us about the origin of the eukaryotic cell?

The relationship among the three cellular domains Archaea, Bacteria, and Eukarya has become a central problem in unraveling the tree of life. This relationship can now be studied as the completely sequenced genomes of representatives of these cellular domains become available. We performed a bioinformatic investigation of the Encephalitozoon cuniculi proteome. E. cuniculi has the smallest sequenced eukaryotic genome, 2.9 megabases coding for 1997 proteins. The proteins of E. cuniculi were compared with a previously characterized set of eukaryotic signature proteins (ESPs). ESPs are found in a eukaryotic cell, whether from an animal, a plant, a fungus, or a protozoan, but are not found in the Archaea and the Bacteria. We demonstrated that 85% of the ESPs have significant sequence similarity to proteins in E. cuniculi. Hence, E. cuniculi, a minimal eukaryotic cell that has removed all inessential proteins, still preserves most of the ESPs that make it a member of the Eukarya. The locations and functions of these ESPs point to the earliest history of eukaryotes.

Gene loss, protein sequence divergence, gene dispensability, expression level, and interactivity are correlated in eukaryotic evolution.

Lineage-specific gene loss, to a large extent, accounts for the differences in gene repertoires between genomes, particularly among eukaryotes. We derived a parsimonious scenario of gene losses for eukaryotic orthologous groups (KOGs) from seven complete eukaryotic genomes. The scenario involves substantial gene loss in fungi, nematodes, and insects. Based on this evolutionary scenario and estimates of the divergence times between major eukaryotic phyla, we introduce a numerical measure, the propensity for gene loss (PGL). We explore the connection among the propensity of a gene to be lost in evolution (PGL value), protein sequence divergence, the effect of gene knockout on fitness, the number of protein-protein interactions, and expression level for the genes in KOGs. Significant correlations between PGL and each of these variables were detected. Genes that have a lower propensity to be lost in eukaryotic evolution accumulate fewer substitutions in their protein sequences and tend to be essential for the organism viability, tend to be highly expressed, and have many interaction partners. The dependence between PGL and gene dispensability and interactivity is much stronger than that for sequence evolution rate. Thus, propensity of a gene to be lost during evolution seems to be a direct reflection of its biological importance.

Discrimination between viable and dead Encephalitozoon cuniculi (Microsporidian) spores by dual staining with sytox green and calcofluor white M2R.

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled) Encephalitozoon cuniculi spores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P = 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P = 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.