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.

mSphere of Influence: Where the Pathogen Proteins Are.

Aaron Reinke studies microsporidian evolution and how microsporidia interact with their hosts. In this mSphere of Influence article, he reflects on how the papers "A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells" (K. J. Roux, D. I. Kim, M. Raida, and B. Burke, J Cell Biol 196:801-810, 2012, https://doi.org/10.1083/jcb.201112098) and "Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging" (H.-W. Rhee, P. Zou, N. D. Udeshi, J. D. Martell, et al., Science 339:1328-1331, 2013, https://doi.org/10.1126/science.1230593) impacted his thinking on how to determine where proteins from intracellular pathogens are located within host cells.

Construction and heterologous overexpression of two chimeric proteins carrying outer hydrophilic loops of Vairimorpha ceranae and Nosema bombycis ATP/ADP carriers.

Microsporidia Nosema bombycis and Vairimorpha ceranae cause destructive epizootics of honey bees and silkworms. Insufficient efficiency of the antibiotic fumagillin against V. ceranae, its toxicity and the absence of effective methods of N. bombycis treatment demand the discovery of novel strategies to suppress infections of domesticated insects. RNA interference is one such novel treatment strategy. Another one implies that the intracellular development of microsporidia may be suppressed by single-chain antibodies (scFv fragments) against functionally important parasite proteins. Important components of microsporidian metabolism are non-mitochondrial, plastidic-bacterial ATP/ADP carriers. These membrane transporters import host-derived ATP and provide the capacity to pathogens for energy parasitism. Here, we analyzed membrane topology of four V. ceranae and three N. bombycis ATP/ADP transporters to construct two fusion proteins carrying their outer hydrophilic loops contacting with infected host cell cytoplasm. Interestingly, full-size genes of N. bombycis transporters may be derived from the Asian swallowtail Papilio xuthus genome sequencing project. Synthesis of the artificial genes was followed by overexpression of recombinant proteins in E. coli as insoluble inclusion bodies. The gene fragments encoding the loops of individual transporters were also effectively expressed in bacteria. The chimeric antigens may be used to construct immune libraries or select microsporidia-suppressing scFv fragments from synthetic, semisynthetic, naïve and immune antibody libraries. A further expression of such antibodies in insect cells may increase their resistance to microsporidial infections.

Single-cell Raman spectroscopy reveals microsporidia spore heterogeneity in various insect hosts.

Single-cell Raman spectroscopy was used to analyze the spore heterogeneity of 16 microsporidia strains from various insect hosts in order to better understand the basic biology of microsporidia. The Raman spectrum of a single spore revealed basic spore composition, and microsporidia spores in various hosts were found to be rich in trehalose. Principal component analysis and Raman intensity showed obvious heterogeneity in the trehalose, nucleic acid, and protein content of various spores; however, there was no correlation between various spore groups and host type. Trehalose content correlated with spore infectivity on Bombyx mori. Raman spectroscopy is an excellent tool for label-free investigation of intercellular molecular constituents, providing insight into the heterogeneity of microsporidia spores.

A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation.

Microsporidia are obligate intracellular parasites, existing in a wide variety of animal hosts. Here, we reported AlocSWP2, a novel protein identified from the spore wall of Antonospora locustae (formerly, Nosema locustae, and synonym, Paranosema locustae), containing four cysteines that are conserved among the homologues of several Microspodian pathogens in insects and mammals. AlocSWP2 was detected in the wall of mature spores via indirect immunofluorescence assay. In addition, immunocytochemistry localization experiments showed that the protein was observed in the wall of sporoblasts, sporonts, and meronts during sporulation within the host body, also in the wall of mature spores. AlocSWP2 was not detected in the fat body of infected locust until the 9th day after inoculating spores via RT-PCR experiments. Furthermore, the survival percentage of infected locusts injected with dsRNA of AlocSWP2 on the 15th, 16th, and 17th days after inoculation with microsporidian were significantly higher than those of infected locusts without dsRNA treatment. Conversely, the amount of spores in locusts infected with A. locustae after treated with RNAi AlocSWP2 was significantly lower than those of infected locusts without RNAi of this gene. This novel spore wall protein from A. locustae may be involved in sporulation, thus contributing to host mortality.

Brominated Variant of the Lantibiotic NAI-107 with Enhanced Antibacterial Potency.

We identified an Actinoallomurus strain producing NAI-107, a chlorinated lantibiotic effective against multidrug-resistant Gram-positive pathogens and previously reported from the distantly related genus Microbispora. Inclusion of KBr in the production medium of either the Actinoallomurus or the Microbispora producer readily afforded brominated variants of NAI-107, which were designated as NAI-108. The other post-translational modifications naturally occurring in this lantibiotic family (i.e., hydroxylation of Pro-14 and C-terminal decarboxylation) were unaffected by the presence of a brominated tryptophan. In addition to being the first example of a bromine-containing lantibiotic, NAI-108 displayed a small but consistent improvement in antibacterial activity against all tested strains. The brominated lantibiotic maintained the same rapid bactericidal activity as NAI-107 but at reduced concentrations, consistent with its increased potency and with the role played by the hydrophobicity of the first lanthionine ring. NAI-108 thus represents an interesting addition to a promising family of potent and effective lantibiotics.

[Biologically active compounds produced by microbial endophytes].

Microbial endophytes present a rich and not fully studied source of novel natural biologically active compounds of wide interest all over the world. The research of natural compounds with various biological effects produced by endophytes are described in the review.

[Use of the OTE-staining method for ultrathin sections on the example of microsporidia (Protozoa: Microsporidia)].

A novel method for staining ultrathin sections and examining organelles of taxonomic importance in microsporidian parasites was evaluated using oolong tea extract (OTE) and compared with traditional staining with uranyl acetate (UA). All basic intracellular structures of taxonomic significance were effectively stained with the OTE-staining method and additional layers of the polar filament with more clear boundaries between them were revealed. However, greater resolution and higher general contrast of several structures including membranes, layers of the envelope of mature spores, the structure of rough endoplasmic reticulum, Golgi complex, and nuclear chromatin were achieved with traditional UA-staining. The OTE-staining method has the advantage of being safe and preparations can be stored in light at room temperature with no loss in staining properties. However, greater staining time is required. We conclude that the OTE-staining method may be used as an alternative to traditional staining with UA with successful results.

Evidence of a reduced and modified mitochondrial protein import apparatus in microsporidian mitosomes.

Microsporidia are a group of highly adapted obligate intracellular parasites that are now recognized as close relatives of fungi. Their adaptation to parasitism has resulted in broad and severe reduction at (i) a genomic level by extensive gene loss, gene compaction, and gene shortening; (ii) a biochemical level with the loss of much basic metabolism; and (iii) a cellular level, resulting in lost or cryptic organelles. Consistent with this trend, the mitochondrion is severely reduced, lacking ATP synthesis and other typical functions and apparently containing only a fraction of the proteins of canonical mitochondria. We have investigated the mitochondrial protein import apparatus of this reduced organelle in the microsporidian Encephalitozoon cuniculi and find evidence of reduced and modified machinery. Notably, a putative outer membrane receptor, Tom70, is reduced in length but maintains a conserved structure chiefly consisting of tetratricopeptide repeats. When expressed in Saccharomyces cerevisiae, EcTom70 inserts with the correct topology into the outer membrane of mitochondria but is unable to complement the growth defects of Tom70-deficient yeast. We have scanned genomic data using hidden Markov models for other homologues of import machinery proteins and find evidence of severe reduction of this system.

Major O-glycans in the spores of two microsporidian parasites are represented by unbranched manno-oligosaccharides containing alpha-1,2 linkages.

Protein glycosylation in microsporidia, a fungi-related group comprising exclusively obligate intracellular parasitic species, is still poorly documented. Here, we have studied glycoconjugate localization and glycan structures in spores of Encephalitozoon cuniculi and Antonospora locustae, two distantly related microsporidians invading mammalian and insect hosts, respectively. The polar sac-anchoring disc complex or polar cap, an apical element of the sporal invasion apparatus, was strongly periodic acid-thiocarbohydrazide-Ag proteinate-positive. Mannose-binding lectins reacted with the polar cap and recognized several bands (from 20 to 160 kDa) on blots of E. cuniculi protein extracts. Physicochemical analyses provided the first determination of major glycostructures in microsporidia. O-linked glycans were demonstrated to be linear manno-oligosaccharides containing up to eight alpha1, 2-linked mannose residues, thus resembling those reported in some fungi such as Candida albicans. No N-linked glycans were detected. The data are in accordance with gene-based prediction of a minimal O-mannosylation pathway. Further identification of individual mannoproteins should help in the understanding of spore germination mechanism and host-microsporidia interactions.

New insights into myosin evolution and classification.

Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites.

Microsporidian polar tube proteins: highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups.

The spore polar tube is a unique organelle required for cell invasion by fungi-related microsporidian parasites. Two major polar tube proteins (PTP1 and PTP2) are encoded by two tandemly arranged genes in Encephalitozoon species. A look at Antonospora (Nosema) locustae contigs (http://jbpc.mbl.edu/Nosema/Contigs/) revealed significant conservation in the order and orientation of various genes, despite high sequence divergence features, when comparing with Encephalitozoon cuniculi complete genome. This syntenic relationship between distantly related Encephalitozoon and Antonospora genera has been successfully exploited to identify ptp1 and ptp2 genes in two insect-infecting species assigned to the Antonospora clade (A. locustae and Paranosema grylli). Targeting of respective proteins to the polar tube was demonstrated through immunolocalization experiments with antibodies raised against recombinant proteins. Both PTPs were extracted from spores with 100mM dithiothreitol. Evidence for PTP1 mannosylation was obtained in studied species, supporting a key role of PTP1 in interactions with host cell surface.

Microsporidian mitochondrial proteins: expression in Antonospora locustae spores and identification of genes coding for two further proteins.

Microsporidia are obligate intracellular parasites, phylogenetically allied to the fungi. Once considered amitochondriate, now a number of mitochondrion-derived genes have been described from various species, and the relict organelle was recently identified in Trachipleistophora hominis. We have investigated the expression of potential mitochondrial targeted proteins in the spore stage to determine whether the organelle is likely to have a role in the spore or early infection stage. To investigate whether the Antonospora locustae genome codes for a different complement of mitochondrial proteins than Encephalitozoon cuniculi an EST library was searched for putative mitochondrial genes that have not been identified in the E. cuniculi genome project. The spore is the infectious stage of microsporidia, but is generally considered to be metabolically dormant. Fourteen genes for putatively mitochondrion-targeted proteins were shown to be present in purified spore mRNA by 3'-rapid amplification of cDNA ends and EST sequencing. Pyruvate dehydrogenase E1alpha and mitochondrial glycerol-3-phosphate dehydrogenase proteins were also shown to be present in A. locustae and E. cuniculi spores, respectively, suggesting a role for these proteins in the early stages of infection, or within the spore itself. EST sequencing also revealed two mitochondrial protein-encoding genes in A. locustae that are not found in the genome of E. cuniculi. One encodes a possible pyruvate transporter, the other a subunit of the mitochondrial inner membrane peptidase. In yeast mitochondria, this protein is part of a trimeric complex that processes proteins targeted to the inner membrane and the intermembrane space, and its substrate in A. locustae is presently unknown.

A mitochondrial remnant in the microsporidian Trachipleistophora hominis.

Microsporidia are obligate intracellular parasites of several eukaryotes. They have a highly complex and unique infection apparatus but otherwise appear structurally simple. Microsporidia are thought to lack typical eukaryotic organelles, such as mitochondria and peroxisomes. This has been interpreted as support for the hypothesis that these peculiar eukaryotes diverged before the mitochondrial endosymbiosis, which would make them one of the earliest offshoots in eukaryotic evolution. But microsporidial nuclear genes that encode orthologues of typical mitochondrial heatshock Hsp70 proteins have been detected, which provides evidence for secondary loss of the organelle or endosymbiont. In addition, gene trees and more sophisticated phylogenetic analyses have recovered microsporidia as the relatives of fungi, rather than as basal eukaryotes. Here we show that a highly specific antibody raised against a Trachipleistophora hominis Hsp70 protein detects the presence, under light and electron microscopy, of numerous tiny ( approximately 50 x 90 nm) organelles with double membranes in this human microsporidial parasite. The finding of relictual mitochondria in microsporidia provides further evidence of the reluctance of eukaryotes to lose the mitochondrial organelle, even when its canonical function of aerobic respiration has been apparently lost.

rDNA phylogeny of Intrapredatorus barri (Microsporida: Amblyosporidae) parasitic to Culex fuscanus Wiedemann (Diptera: Culicidae).

Intrapredatorus barri, a microsporidian parasite of the predator mosquito Culex fuscanus Wiedemann has been shown to be heterosporous. In many features this species has been reported to be similar to Amblyospora trinus, another microsporidian parasite of a different predator mosquito which was thus proposed for reassignation to the genus Intrapredatorus. In this report small subunit rDNA has been sequenced from I. barri and phylogenetic analysis of 42 microsporidia sequences has been performed. The molecular data show that I. barri can not be a member of the genus Amblyospora. Furthermore, the phylogenetic analysis shows, with high confidence, that the Amblyospora spp. sequences in the present study are not monophyletic.

Lipids of three microsporidian species and multivariate analysis of the host-parasite relationship.

Sporal lipids of 3 microsporidia, Encephalitozoon cuniculi from mammals and Glugea atherinae and Spraguea lophii from fishes, were investigated. High phospholipid levels were found (54.8-64.5% of total lipids), which is in agreement with the presence of highly developed internal membranes in microsporidian spores. Sphingomyelin was not detected in G. atherinae. Triglycerides (less than 10% of total lipids), cholesterol, and free fatty acids were identified in all species. Analysis of fatty acids from the phospholipid fraction revealed the predominance of docosahexaenoic acid (30-40% of total phospholipid fatty acids) in G. atherinae and S. lophii and oleic acid (25.8% of total phospholipid fatty acids) in E. cuniculi. The 3 microsporidia possessed a significant amount of branched-chain fatty acids (iso and anteiso forms) not found in the hosts, supporting the existence of some parasite-specific metabolic steps for these fatty acids. On the basis of phospholipid fatty acid profiles, host-parasite relationships were investigated through correspondence factorial analysis. It shows 3 distinct clusters with the first corresponding to fishes, the second to fish parasites, and the third to E. cuniculi and its host cell. These data suggest that the mammal microsporidia developing within parasitophorous vacuoles are more dependent on host cells than the fish microsporidia that induce cystlike structures.

Phylogenetic relationships among microsporidia based on rDNA sequence data, with particular reference to fish-infecting Microsporidium balbiani 1884 species.

Recently, large discrepancies have been identified between microsporidian systematics based on molecular and traditional characteristics. In the current study the 530f-580r region of the rRNA gene of eight microsporidian species was cloned and sequenced. Included were two unclassified species of Microsporidium Balbiani, 1884 and an unidentified microsporidian that infects the musculature of different sea bream species. Sequence identities in excess of 98% indicated that these three species almost certainly are members of the same genus. Phylogenetic analyses of all microsporidian sequence data available for this region of the gene (20 species) and for partial small subunit sequences (51 species of 21 genera) revealed these species to be distinct from the family Pleistophoridae Doflein, 1901 and closely related them to the genus Sproguea Weissenberg, 1976. This clade was found to comprise a sister taxon to that containing the vast majority of fish-infecting species. Broad cladistic divisions were found between terrestrial insect-infecting and fish-infecting species, which together are distant from the aquatic insect-infecting microsporidia. The rRNA gene of certain fish-infecting genera was found to be more highly conserved than previously reported. This has implications for its utility in diagnostic assays and phylogenetic studies at, or close to, the species level.

[Pathogenic protozoans in man: differential characterization of Entamoeba histolytica/Entamoeba dispar complex, Acanthamoeba spp., Microsporidia]. / Protozoi patogeni per l'uomo: protocolli di caratterizzazione differenziale di genere e di specie nell'ambito del complesso Entamoeba histolytica/Entamoeba dispar, Acanthamoeba spp., Microsporidi.

The review summarizes the results in the main parasitological topics of our Lab: amoebic infections due to Entamoeba histolytica/Entamoeba dispar complex and to Acanthamoeba spp. respectively, and human infections caused by microsporidia. Different rapid and advanced techniques have been included in the standardized diagnostic protocols for each topic, and a critical comparison among them was made, in order to define the gold standard diagnostic method: a) E. histolytica/E. dispar: in vitro culture, zymodeme typization, biomolecular identification (PCR), immunoenzymatic assay (ELISA) for direct detection in stools of specific surface antigenic lectins; b) Acanthamoeba spp.: in vitro culture, light and ultrastructural characterization, species identification by immunofluorescence method with monoclonal antibodies, in vitro pharmacological studies; c) Microsporidia: ultrastructural (TEM), biomolecular (PCR), biochemical and immunological (SDS-PAGE, Immunoblotting) studies for species identification, use of advanced ultrastructural techniques ("freeze-etching", "deep-etching") in order to deepen the spore wall structure, to study the cytoskeletal function of actin and to define the mode of infection, in vitro pharmacological assays on some inhibitors of chitin-synthases.