Bacterial catalase in the microsporidian Nosema locustae: implications for microsporidian metabolism and genome evolution

Microsporidia constitute a group of extremely specialized intracellular parasites that infect virtually all animals. They are highly derived, reduced fungi that lack several features typical of other eukaryotes, including canonical mitochondria, flagella, and peroxisomes. Consistent with the absence of peroxisomes in microsporidia, the recently completed genome of the microsporidian Encephalitozoon cuniculi lacks a gene for catalase, the major enzymatic marker for the organelle. We show, however, that the genome of the microsporidian Nosema locustae, in contrast to that of E. cuniculi, encodes a group II large-subunit catalase. Surprisingly, phylogenetic analyses indicate that the N. locustae catalase is not specifically related to fungal homologs, as one would expect, but is instead closely related to proteobacterial sequences. This finding indicates that the N. locustae catalase is derived by lateral gene transfer from a bacterium. The catalase gene is adjacent to a large region of the genome that appears to be far less compact than is typical of microsporidian genomes, a characteristic which may make this region more amenable to the insertion of foreign genes. The N. locustae catalase gene is expressed in spores, and the protein is detectable by Western blotting. This type of catalase is a particularly robust enzyme that has been shown to function in dormant cells, indicating that the N. locustae catalase may play some functional role in the spore. There is no evidence that the N. locustae catalase functions in a cryptic peroxisome.     

An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae

The mitochondrion is one of the defining characteristics of eukaryotic cells, and to date, no eukaryotic lineage has been shown to have lost mitochondria entirely. In certain anaerobic or microaerophilic lineages, however, the mitochondrion has become severely reduced that it lacks a genome and no longer synthesizes ATP. One example of such a reduced organelle, called the mitosome, is found in microsporidian parasites. Only a handful of potential mitosomal proteins were found to be encoded in the complete genome of the microsporidian Encephalitozoon cuniculi, and significantly no proteins of the mitochondrial carrier family were identified. These carriers facilitate the transport of solutes across the inner mitochondrial membrane, are a means of communication between the mitochondrion and cytosol, and are abundant in organisms with aerobic mitochondria. Here, we report the characterization of a mitochondrial carrier protein in the microsporidian Antonospora locustae and demonstrate that the protein is heterologously targeted to mitochondria in Saccharomyces cerevisiae. The protein is phylogenetically allied to the NAD⁺ transporter of S. cerevisiae, but we show that it has high specificity for ATP and ADP when expressed in Escherichia coli. An ADP/ATP carrier may provide ATP for essential ATP-dependent mitosomal processes such as Hsp70-dependent protein import and export of iron-sulfur clusters to the cytosol.     

Comparative analysis of sequences encoding ABC systems in the genome of the microsporidian Encephalitozoon cuniculi

Microsporidia are amitochondriate eukaryotic microbes with fungal affinities and a common status of obligate intracellular parasites. A set of 13 potential genes encoding ATP-binding cassette (ABC) systems was identified in the fully sequenced genome of Encephalitozoon cuniculi. Our analyses of multiple alignments, phylogenetic trees and conserved motifs support a distribution of E. cuniculi ABC systems within only four subfamilies. Six half transporters are homologous to the yeast ATM1 mitochondrial protein, a finding which is in agreement with the hypothesis of a cryptic mitochondrion-derived compartment playing a role in the synthesis and transport of Fe-S clusters. Five half transporters are similar to the human ABCG1 and ABCG2 proteins, involved in regulation of lipid trafficking and anthracyclin resistance respectively. Two proteins with duplicated ABC domains are clearly candidate to non-transport ABC systems: the first is homologous to mammalian RNase L inhibitor and the second to the yeast translation initiation regulator GCN20. An unusual feature of ABC systems in E. cuniculi is the lack of homologs of P-glycoprotein and other ABC transporters which are involved in multiple drug resistance in a large number of eukaryotic microorganisms.     

Confirmation of the Human-Pathogenic Microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in Water

Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of Encephalitozoon intestinalis was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of Enterocytozoon bieneusi was confirmed in surface water; and the presence of Vittaforma corneae was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.     

Characterization of the mRNA capping apparatus of the microsporidian parasite Encephalitozoon cuniculi.

A scheme of eukaryotic phylogeny has been suggested based on the structure and physical linkage of the enzymes that catalyze mRNA cap formation. Here we show that the intracellular parasite Encephalitozoon cuniculi encodes a complete mRNA capping apparatus consisting of separate triphosphatase (EcCet1), guanylyltransferase (EcCeg1), and methyltransferase (Ecm1) enzymes, which we characterize biochemically and genetically. The triphosphatase EcCet1 belongs to a metal-dependent phosphohydrolase family that includes the triphosphatase components of the capping apparatus of fungi, DNA viruses, and the malaria parasite Plasmodium falciparum. These enzymes are structurally and mechanistically unrelated to the metal-independent cysteine phosphatase-type RNA triphosphatases found in metazoans and plants. Our findings support the proposed evolutionary connection between microsporidia and fungi, and they place fungi and protozoa in a common lineage distinct from that of metazoans and plants. RNA triphosphatase presents an attractive target for antiprotozoal/antifungal drug development.     

Encephalitozoon cuniculi: quantitation of parasites and evaluation of viability

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.     

Distinct localization patterns of two putative mitochondrial proteins in the microsporidian Encephalitozoon cuniculi

Microsporidia were once considered amitochondriate, but have now been found to retain relict mitochondria called mitosomes. These organelles have been identified by immunolocalization in Trachipleistophora hominis, whereas most data on function have been inferred from the presence of mitochondrial protein-encoding sequences in the genome of Encephalitozoon cuniculi. Here we describe the localization of two such enzymes in E. cuniculi cells. Immunofluorescent localization of ferredoxin involved in mitochondrial iron-sulfur cluster assembly reveals a punctate distribution as expected for mitochondria. In contrast, localization of mitochondrial glycerol-3-phosphate dehydrogenase suggests a cytoplasmic distribution in E. cuniculi and possible relocalization of this typically mitochondrial protein.     

Characteristics of the microsporidian Enterocytozoon bieneusi: a consequence of its development within short-living enterocytes.

Ultrastructural studies were done on developmental stages of Enterocytozoon bieneusi obtained from HIV seropositive patients suffering from diarrhea. The presence of elaborate multilamellar structures suggest that they give rise to various membrane systems needed for rapid production of disseminating stages.     

Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.     

Natural and experimental infection of immunocompromised rhesus macaques (Macaca mulatta) with the microsporidian Enterocytozoon bieneusi genotype D

Microsporidia are obligate intracellular parasites that cause opportunistic infections in AIDS and other immunocompromised patients. Eight simian immunodeficiency virus (SIV)-infected rhesus macaque monkeys (Macaca mulatta) were inoculated orally with Enterocytozoon bieneusi spores isolated from intestinal lavage fluid of an AIDS patient (genotype D) to study the natural history of this infection. Four monkeys were already naturally infected with E. bieneusi (also genotype D), and were included to determine if a second inoculum affected the course of illness. Spore shedding was detected in feces of all eight monkeys within the first week of experimental infection. Five monkeys died within 3.5 months of experimental E. bieneusi inoculation. Three of these five monkeys began the study with CD4+CD29+ T cell levels well below 20% of total T lymphocytes. Deaths were due to a variety of AIDS-related manifestations. Microsporidia did not appear to directly contribute to mortality but may have contributed to morbidity. At necropsy, microsporidia were found in bile and tissue sections of the gallbladder but not in the gut, kidneys, or liver. The percent CD4+CD29+ levels of the last three monkeys remained near the level observed at the time of inoculation. These monkeys lived more than 2 years after the end of the study and continued to shed spores. This study corroborates previous reports that E. bieneusi can be reliably transmitted to SIV-infected rhesus monkeys but indicates that the use of SIV-infected monkeys for the study of microsporidiosis is complicated by the confounding effect of other opportunistic or AIDS-related infections.     

A novel nested multiplex PCR for the simultaneous detection and differentiation of Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon intestinalis

Cryptosporidium spp. and microsporidia are opportunistic parasites affecting a wide range of hosts in which they can be potentially life threatening in immunocompromised individuals. Diagnosis usually relies on the identification of the stained Cryptosporidium oocyst or microsporidial spores, but these methods lack sensitivity and require highly trained technicians to perform and interpret the results. Molecular diagnosis offers an alternative with both superior sensitivity and specificity as compared to microscopy. Although replacing microscopy with nucleic acid based methods is hampered by the higher costs, in particular in developing countries, multiplexing the detection of more than one parasite in a single test has been found to be very effective and would decrease the cost of the test without the need for new equipment, as it would be the case for quantitative PCR. The method shown in this report for the simultaneous detection of Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon intestinalis by multiplex nested PCR, has proved to have several advantages versus microscopy such as higher sensitivity and specificity, low subjectivity and a minimal need for specialist's training to interpret the results.The present multiplex assay can fill an important gap to identify other possible causative agents of several diarrheal diseases which until present remain undiagnosed and can improve the epidemiology of the disease with a more reliable detection method.     

On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi , a major PTP was isolated from two-dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline-rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26-amino-acid repeats. An N-terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli , purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.     

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.     

Genomic parasites and genome evolution

A report of the Second International Conference/Workshop on the Genomic Impact of Eukaryotic Transposable Elements, Pacific Grove, USA, 6-10 February 2009.