Search

Different components of the symbiotic interface play an important role in providing positional information during rhizobial infection and nodule development: successive changes in cell morphology correspond to subsequent changes in the molecular architecture of the apoplast and the associated surface structures. The localisation and distribution of pectins, xyloglucans, and cell wall proteins in symbiotic nodules of Pisum sativum and Medicago truncatula were studied using immunofluorescence and immunogold analysis in wild-type and ineffective mutant nodules. As a result, the ontogenetic changes in the symbiotic interface in the nodules of both species were described. Some differences in the patterns of distribution of cell wall polysaccharides and proteins between wild-type and mutant nodules can be explained by the activation of defence reaction or premature senescence in mutants. The absence of fucosylated xyloglucan in the cell walls in the P. sativum nodules, as well as its predominant accumulation in the cell walls of uninfected cells in the M. truncatula nodules, and the presence of the rhamnogalacturonan I (unbranched) backbone in meristematic cells in P. sativum can be attributed to the most striking species-specific features of the symbiotic interface. [ABSTRACT FROM AUTHOR]

A morphophysiological study of tubular reabsorption and mechanisms of protein endocytosis in the kidney of frogs (Rana temporaria L.) during parasitic infection was carried out. Pseudoplasmodia and spores of myxosporidia, beforehand assigned to the genus Sphaerospora, were detected in Bowman's capsules and in the lumen of individual renal tubules by light and electron microscopy. Remarkable morphological alteration and any signs of pathology in kidney tissue related to this myxosporean infection have not been noted. At the same time, significant changes in protein reabsorption and distribution of molecular markers of endocytosis in the proximal tubule (PT) cells in infected animals were detected by immunofluorescence confocal microscopy. In lysozyme injection experiments, the endocytosed protein and megalin expression in the infected PTs were not revealed. Tubular expression of cubilin and clathrin decreased, but endosomal recycling marker Rab11 increased or remained unchanged. Thus, myxosporean infection resulted in the alterations in lysozyme uptake and expression of the main molecular determinants of endocytosis. The inhibition of receptor-mediated clathrin-dependent protein endocytosis in amphibian kidneys due to myxosporidiosis was shown for the first time. Established impairment of the endocytic process is a clear marker of tubular cell dysfunction that can be used to assess the functioning of amphibian kidneys during adaptation to adverse environmental factors. [ABSTRACT FROM AUTHOR]

Pisum sativum symbiotic mutant SGEFix−-2 carries the sym33-3 allele of the gene Sym33, encoding transcription factor PsCYCLOPS/PsIPD3. Previously, strong host cell defence reactions were identified in nodules of this mutant. In the present study, new manifestations of defence reactions were revealed in 28-day-old white nodules in which bacterial release had occurred. These nodules were investigated using histochemical staining of pectin and suberin and by immunogold localisation of three components of pectin: highly methyl-esterified homogalacturonan (HG) recognised by monoclonal antibody JIM7, low methyl-esterified HG recognised by JIM5 and linear (1–4)-β-D-galactan side-chain of rhamnogalacturonan I (RG I) recognised by LM5. In the mutant, but not in the wild-type, cell wall material was deposited around the vacuole in the uninfected cells, in cells containing infection threads and in the infected cells. The deposits around the vacuole were marked with JIM7 and LM5 antibodies but not with JIM5, suggesting that they contain newly formed cell wall material. Deposition was accompanied by suberin accumulation. This is the first report that deposition of cell wall material around the vacuole may be associated with the defence reaction in ineffective nodules. In addition, hypertrophic infection droplets labelled with JIM7 were identified. In the matrix of some infection threads, RG I recognised a pectic gel component. Callose deposits in the cell walls and in the walls of infection threads were occasionally observed. The observations suggest that an important function of transcriptional factor CYCLOPS/IPD3 is the suppression of defence reactions during establishment of the legume-rhizobial symbiosis. [ABSTRACT FROM AUTHOR]

Infection of host cells by nitrogen-fixing soil bacteria, known as rhizobia, involves the progressive remodelling of the plant–microbe interface. This process was examined by using monoclonal antibodies to study the subcellular localisation of pectins and arabinogalactan proteins (AGPs) in wild-type and ineffective nodules of Pisum sativum and Medicago truncatula. The highly methylesterified homogalacturonan (HG), detected by monoclonal antibody JIM7, showed a uniform localisation in the cell wall, regardless of the cell type in nodules of P. sativum and M. truncatula. Low methylesterified HG, recognised by JIM5, was detected mainly in the walls of infection threads in nodules of both species. The galactan side chain of rhamnogalacturonan I (RG-I), recognised by LM5, was present in the nodule meristem in both species and in the infection thread walls in P. sativum, but not in M. truncatula. The membrane-anchored AGP recognised by JIM1 was observed on the plasma membrane in nodules of P. sativum and M. truncatula. In P. sativum, the AGP epitope recognised by JIM1 was present on mature symbiosome membranes of wild-type nodules, but JIM1 labelling was absent from symbiosome membranes in the mutant Sprint-2Fix− (sym31) with undifferentiated bacteroids, suggesting a possible involvement of AGP in the maturation of symbiosomes. Thus, the common and species-specific traits of cell wall remodelling during nodule differentiation were demonstrated. [ABSTRACT FROM AUTHOR]

Microsporidia, a large group of fungi-related intracellular parasites, are characterized by drastically reduced metabolism. They possess genes encoding glycolysis components, and the glycerol–phosphate shuttle, but lack mitochondria, Krebs cycle, respiratory chain and pyruvate-converting enzymes, except α and β subunits of E1 enzyme of pyruvate dehydrogenase (PDH) complex. Here, we have expressed PDH subunits from the microsporidum Paranosema ( Antonospora) locustae in Escherichia coli. Western blot analysis with antibodies raised against recombinant proteins has revealed their specific accumulation in mature spores of P. locustae but not in the intracellular development stages. Two subunits were coprecipitated as a single heterooligomeric complex by anti-α or anti-β PDH antibodies. Ultracentrifugation of spore homogenate has shown the presence of PDH in the soluble fraction. Relocalization of the mitochondrial protein in microsporidial spore cytoplasm was confirmed by immunoelectron microscopy of ultrathin cryosections with affinity-purified anti-α PDH antibodies. On cryosections, parasite enzyme was found partly associated with the cytoplasmic side of ER and other intraspore membranes, suggesting that electrons might be transferred to any membrane acceptor and finally to oxygen in the parasite cell. [ABSTRACT FROM AUTHOR]

[Display omitted] Participation of molecular determinants of endocytosis in the processes of glomerular filtration and tubular reabsorption of albumin and lysozyme in the mesonephros of grass frogs (Rana temporaria L.), lake frogs (Rana ridibunda P.), and newts (Triturus vulgaris L.) is investigated. In all studied species, the constitutive expression of endocytic receptors in proximal tubule (PT) cells is established using immunofluorescence microscopy and immunoblotting. The certain stages of lysozyme and albumin endocytosis involving megalin/LRP2, cubilin, clathrin and protein Rab11 are detailed, and the central role of ligand-induced megalin/LRP2 activity in this process is shown. Increased ligand-induced expression for clathrin and Rab11was also found. In grass frogs, the different patterns of endocytic receptors and both absorbed proteins in the initial parts of proximal tubules suggest the proximo-distal specialization of absorptive processes along these tubule segments, similar to this in more complex mammalian nephrons. This data, as well as the revealed peculiarities of ligand-receptor interactions during intracellular trafficking of proteins prove that megalin is mainly involved in the absorption of lysozyme. At the same time, albumin absorption is mediated by both receptors, or cubilin contributes the most. The detection of endocytic receptor in glomerular structural elements in frogs and newts suggests the participation of filtration barrier components in endocytosis of filterable proteins. The results represent a new contribution to the study of the fundamental mechanisms of renal protein uptake in the amphibian mesonephros as a more primitive kidney compared to mammalian metanephros. [ABSTRACT FROM AUTHOR]

Despite global warming, the influence of heat on symbiotic nodules is scarcely studied. In this study, the effects of heat stress on the functioning of nodules formed by Rhizobium leguminosarum bv. viciae strain 3841 on pea (Pisum sativum) line SGE were analyzed. The influence of elevated temperature was analyzed at histological, ultrastructural, and transcriptional levels. As a result, an unusual apical pattern of nodule senescence was revealed. After five days of exposure, a senescence zone with degraded symbiotic structures was formed in place of the distal nitrogen fixation zone. There was downregulation of various genes, including those associated with the assimilation of fixed nitrogen and leghemoglobin. After nine days, the complete destruction of the nodules was demonstrated. It was shown that nodule recovery was possible after exposure to elevated temperature for 3 days but not after 5 days (which coincides with heat wave duration). At the same time, the exposure of plants to optimal temperature during the night leveled the negative effects. Thus, the study of the effects of elevated temperature on symbiotic nodules using a well-studied pea genotype and Rhizobium strain led to the discovery of a novel positional response of the nodule to heat stress. [ABSTRACT FROM AUTHOR]

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs. [ABSTRACT FROM AUTHOR]

Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs. [ABSTRACT FROM AUTHOR]

Larvae of Chironomus plumosus, collected in North-Western Russia in September 2008, were infected with a microsporidium possessing broadly oval uninucleate spores in sporophorous vesicles. Sporogony and spore ultrastructure of this microsporidium differed from that of known microsporidian species, suggesting establishment of a new species, Anisofilariata chironomi, being a type species of a new genus. Sporogony di-, tetra-, octo-, and 16-sporoblastic. Fixed and stained spores are 4.7–6.8 × 3.4–5.4 µm in size, the spore measurements varying depending upon the number of spores in the sporophorous vesicle. The polaroplast is bipartite, with anterior and posterior parts composed of very thin and thick lamellae, respectively, and occupies the major volume of the spore. The polar filament is anisofilar, with two broad proximal and 10–13 narrow distal coils arranged in 2–4 layers. The sporophorous vesicle is bounded by a thin membrane and contains multiple tubular structures. Small subunit ribosomal DNA phylogeny showed basal position of the new microsporidium to a cluster uniting microsporidia infecting ciliates ( Euplotespora binucleata), microcrustaceans ( Glugoides intestinalis, Mrazekia macrocyclopis), lepidopteran insects ( Cystosporogenes spp., Endoreticulatus spp.) and human ( Vittaforma corneae), nested within Clade IV sensu Vossbrinck and Debrunner-Vossbrinck (2005 Folia Parasitol 52:131–142). No close phylogenetic relationships were found between A. chironomi and microsporidia from other dipteran hosts. [ABSTRACT FROM AUTHOR]

In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view. In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view. [ABSTRACT FROM AUTHOR]

[Display omitted] • scFv library against microsporidia Vairimorpha ceranae hexokinase was constructed. • representativeness of constructed mouse immune library is 5 million transformants. • scFv-antibody inhibiting V. ceranae growth was selected by the library panning. • V. ceranae growth in Sf9 cell culture was confirmed by reverse transcription PCR. Secretion of hexokinase (HK) by microsporidia into infected cells suggests an important role for this enzyme for the intracellular development of parasites. To verify whether the expression of HK-specific antibodies in the host cell cytoplasm can suppress the growth of microsporidia, we constructed an immune library of recombinant scFv fragments against the enzyme of the honey bee pathogen Vairimorpha (Nosema) ceranae (Vc HK) with a representativeness of about 5 million bacterial transformants. Two variants of Vc HK-specific recombinant antibodies were selected by library panning and expressed in lepidopteran Sf9 cell line. Infecting of cells expressing two selected and control scFv fragments with V. ceranae spores was followed by their cultivation for 4 days. Analysis of parasite β-tubulin as well as spore wall protein SWP32 transcripts in infected cultures by reverse transcription PCR and real-time qPCR showed (1) V. ceranae growth in cells heterologous to bee pathogens, (2) its inhibition by one of the selected Vc HK-specific recombinant antibodies. The latter result once again emphasizes an important role of microsporidia hexokinases in their relationships with infected host cells and suggests further focusing on the mechanisms of such suppression, as well as on the search for new V. ceranae - inhibiting scFv fragments. [ABSTRACT FROM AUTHOR]

Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, γCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II. [ABSTRACT FROM AUTHOR]