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Immune response of Drosophila melanogaster to infection with the flagellate parasite Crithidia spp.

Insects are able to recognize invading microorganisms and to mount an immune response to bacterial and fungal infections. Recently, the fruitfly Drosophila melanogaster has emerged as a promising invertebrate model to investigate innate immunity because of its well-characterized genetics. Insects are also vectors of numerous parasites which can trigger an immune response. We have investigated the interaction of Drosophila melanogaster with the flagellate protozoan Crithidia spp. We show that a per os parasitic infection triggers the synthesis of several antimicrobial peptides. By reverse phase HPLC and mass spectrometry, peptides were shown to be present in the hemolymph and not in the gut tissue, suggesting the presence of immune messengers between the site of the infection, namely the gut, and the fat body, the main site of synthesis for antimicrobial peptides. Interestingly, we have identified one molecule which is specifically induced in the hemolymph after infection with Crithidia, but not with bacteria, suggesting that Drosophila can discriminate between pathogens. When flagellates were injected into the hemolymph, a low synthesis of antimicrobial peptides was observed together with phagocytosis of parasites by circulating hemocytes. The data presented here suggest that Drosophila-Crithidia spp. represents an interesting model to study host defense against protozoan parasites.

Animals↗

In vitro effect of essential oils from Cinnamomum aromaticum, Citrus limon and Allium sativum on two intestinal flagellates of poultry, Tetratrichomonas gallinarum and Histomonas meleagridis.

Essential oils may be effective preventive or curative treatments against several flagelated poultry parasites and may become primordial either to organic farms, or as more drugs are bannished. The anti-flagellate activity of essential oils obtained from fresh leaves of Cinnamomum aromaticum, Citrus limon pericarps and Allium sativum bulbs was investigated in vitro on Tetratrichomonas gallinarum and Histomonas meleagridis. On T. gallinarum, the minimal lethal concentration (MLC) at 24 hours was 0.25 microliter/ml for C. aromaticum oil, and 0.125 microliter/ml for C. limon and A. sativum oils. On H. meleagridis, MLC was 0.5 microliter/ml for C. aromaticum oil and 1 microliter/ml for C. limon and A. sativum oils at 24 and 48 hours. Moreover, no synergistic effects were evidenced in vitro. The essential oil constituents, based on their GC retention times have been also identified. The major component is trans-cinnamaldehyde (79%) for C. aromaticum; limonene for C. limon (71%) and diallyl tri- and disulfide (79%) for A. sativum. Even if concentration and protocol adaptations are required for successful in vivo treatments, it appears that these oils may be useful as chemotherapeutic agents against several poultry parasites.

Animals↗

Foraging mechanisms in excavate flagellates shed light on the functional ecology of early eukaryotes.

The phagotrophic flagellates described as "typical excavates" have been hypothesized to be morphologically similar to the Last Eukaryotic Common Ancestor and understanding the functional ecology of excavates may therefore help shed light on the ecology of these early eukaryotes. Typical excavates are characterized by a posterior flagellum equipped with a vane that beats in a ventral groove. Here, we combined flow visualization and observations of prey capture in representatives of the three clades of excavates with computational fluid dynamic modeling, to understand the functional significance of this cell architecture. We record substantial differences amongst species in the orientation of the vane and the beat plane of the posterior flagellum. Clearance rate magnitudes estimated from flow visualization and modeling are both like that of other similarly sized flagellates. The interaction between a vaned flagellum beating in a confinement is modeled to produce a very efficient feeding current at low energy costs, irrespective of the beat plane and vane orientation and of all other morphological variations. Given this predicted uniformity of function, we suggest that the foraging systems of typical excavates studied here may be good proxies to understand those potentially used by our distant ancestors more than 1 billion years ago.

Flagella↗

Analysis of expressed sequence tags (ESTs) from the scaly green flagellate Scherffelia dubia Pascher emend. Melkonian et Preisig.

Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery, generation of molecular markers and characterization of transcription patterns. We have constructed an EST-database of the scaly green flagellate Scherffelia dubia (Chlorophyta) containing 361 sequences. cDNAs were obtained from interphase cells and from cells regenerating flagella. Analysis of the ESTs identified 138 EST-groups with significant similarity to known sequences. 134 EST-groups showed no significant similarity to any sequences in the databases. Most of the ESTs with similarity to known proteins are associated with typical interphase cell functions of a photosynthetic plant cell: assimilation of nutrients and biosynthesis of proteins. Others are related to the activation of the secretory pathway or the biogenesis of scales (e.g. kdo-synthase). Comparison of S. dubia ESTs with the genome of Arabidopsis thaliana and the EST database of Chlamydomonas reinhardtii revealed that S. dubia ESTs with similarity to known proteins were more similar to sequences in C. reinhardtii than to those of A. thaliana. Additionally, ESTs for guanylyl cyclase and cGMP phosphodiesterase are present in the two flagellates, but so far these gene products have not been found in embryophytes.

3' Untranslated Regions↗

Two calmodulins in Naegleria flagellates: characterization, intracellular segregation, and programmed regulation of mRNA abundance during differentiation.

Flagellates of Naegleria gruberi contain two calmodulins that differ in apparent molecular weight and intracellular location. Calmodulin-1, localized in flagella, has an apparent molecular weight of approximately 16,000, approximately the size of other protozoan calmodulins, whereas calmodulin-2, localized in cell bodies, is 15,300. Both proteins, purified, are calmodulins by several criteria, including Ca2+-dependent stimulation of calmodulin-dependent cyclic nucleotide phosphodiesterase and affinity for antibodies to vertebrate calmodulin. The finding of two calmodulins is unusual. Since the only known difference is apparent molecular weight, one calmodulin could be derived from the other, except that both calmodulins are synthesized in a wheat germ, cell-free system directed by RNA from differentiating Naegleria. Translatable mRNAs encoding calmodulins 1 and 2, not detected in amebas, appear and subsequently disappear concurrently during the 100-min differentiation of Naegleria from amebas to flagellates. Furthermore, these mRNAs increase and then decrease in abundance concurrently with those for flagellar tubulins, which suggests the possibility that the expression of the unrelated genes for calmodulin and tubulin may be under coordinate control during differentiation.

Animals↗

Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates.

We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.

Actin Cytoskeleton↗

A novel 95-kD protein is located in a linker between cytoplasmic microtubules and basal bodies in a green flagellate and forms striated filaments in vitro.

The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH2- and COOH-terminal domains flanking a central domain of approximately 650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.

Amino Acid Sequence↗

Flagellated ectosymbiotic bacteria propel a eucaryotic cell.

A devescovinid flagellate from termites exhibits rapid gliding movements only when in close contact with other cells or with a substrate. Locomotion is powered not by the cell's own flagella nor by its remarkable rotary axostyle, but by the flagella of thousands of rod bacteria which live on its surface. That the ectosymbiotic bacteria actually propel the protozoan was shown by the following: (a) the bacteria, which lie in specialized pockets of the host membrane, bear typical procaryotic flagella on their exposed surface; (b) gliding continues when the devescovinid's own flagella and rotary axostyle are inactivated; (c) agents which inhibit bacterial flagellar motility, but not the protozoan's motile systems, stop gliding movements; (d) isolated vesicles derived from the surface of the devescovinid rotate at speeds dependent on the number of rod bacteria still attached; (e) individual rod bacteria can move independently over the surface of compressed cells; and (f) wave propagation by the flagellar bundles of the ectosymbiotic bacteria is visualized directly by video-enhanced polarization microscopy. Proximity to solid boundaries may be required to align the flagellar bundles of adjacent bacteria in the same direction, and/or to increase their propulsive efficiency (wall effect). This motility-linked symbiosis resembles the association of locomotory spirochetes with the Australian termite flagellate Mixotricha (Cleveland, L. R., and A. V. Grimstone, 1964, Proc. R. Soc. Lond. B Biol. Sci., 159:668-686), except that in our case propulsion is provided by bacterial flagella themselves. Since bacterial flagella rotate, an additional novelty of this system is that the surface bearing the procaryotic rotary motors is turned by the eucaryotic rotary motor within.

Animals↗

The rumen flagellate Piromonas communis: its life-history and invasion of plant material in the rumen.

The rumen flagellat Piromonas communis is the zoospore of a phycomycete fungus inhabiting the rumen. Zoosporogenesis was stimulated by a dietary component (the inducer), and inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The zoospores showed taxis towards the tissues surrounding the inflorescence of Lolium perenne L. in the rumen, invading principally the stomata and damaged tissues. The zoospores germinated on this substratum and the rhizoids of the developing vegetative stage penetrated the tissue, taking up C14 from labelled plant material, which was incorporated into the fungal cells. The conditions for maximum flagellate production (39 degrees C, pH 6-0 to 7-0, high concentration of CO2, absence of O2) resembled those found in the rumen. The organism was cultured in an undefined medium in vitro in the absence of other flagellates.

Animals↗

Bleomycin-induced flagellate dermatitis: a clinical and histopathological review.

We report a case of bleomycin-induced flagellate dermatitis. Our patient developed pruritic linear lesions 4 days after her first dose of bleomycin. A closed patch test was performed, and was negative. However, on rechallenge, the linear eruption recurred in the previously involved sites, and in new sites, within 24 h. Flagellate dermatitis is a characteristic reaction to bleomycin use, but varying histological features and clinical presentations may be seen.

Adult↗

Bleomycin-induced flagellate erythema.

A 56-year-old woman who developed widespread pruritus and flagellate erythema after attempted pleuredesis with bleomycin is described. The raised linear lesions of flagellate erythema could not be reproduced by scratching, and histopathological examination revealed a lymphocytic vasculitis. The rash faded spontaneously over several weeks to leave streaks of post-inflammatory melanoderma which remained for 6 months. The role of scratching and dermographism in the pathogenesis of the bleomycin-specific eruption is discussed.

Bleomycin↗

The pathogenic amoeboflagellate Naegleria fowleri: environmental isolations, competitors, ecologic interactions, and the flagellate-empty habitat hypothesis.

From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C. Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling. The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45 degrees C. Heat tolerant competitors were much more common than N. fowleri. Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient. Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45-49 degrees C) amoebae, and one thermophilic (52 degrees C) Acanthamoeba. Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N. fowleri from almost all other amoebae on agar plates. The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing.

Amoeba↗

Fine structure and taxonomic position of the giant amoeboid flagellate Pelomyxa palustris.

Specimens of Pelomyxa palustris from five collecting sites had numerous nonmotile flagella. The structures are called flagella because of morphological similarities to flagella and because P. palustris has affinities with amoeboid flagellates. Flagella were photographed on living cells and studied by transmission and scanning electron microscopy. From 64 to 742 flagella per cell were estimated from scanning electron microscopy of ten cells 204 to 1269 micron in length. The nonmotile flagella arise from basal granules which were, in one strain, surrounded by radiating electron-dense microtubules. This strain also had excess axonemal microtubules. Abundant cytoplasmic microtubules were arranged in several different patterns. In about half of the P. palustris cells in which nuclei were studied, microtubules were either apposed to the nuclear membrane in a parallel alignment (with some also radiating) or radiating from the nuclear membrane (with none parallel). Bacteria associated with nuclei were of three characteristic types: Gram-negative rods, Gram-positive rods, and large rods. All nuclei within a given trophozoite had similar perinuclear features. Recent proposals for separation of Pelomyxa to its own phylum (based on its proposed primitive, unique nature) can not be justified. Pelomyxa is a complex, highly specialized organism adapted to live in a specific fresh-water environment. Mastigamoebid amoeboid flagellates of the genera Mastigamoeba, Mastigella, Mastigina, and possibly Dinamoeba are placed with Pelomyxa within the order Pelobiontida Page, 1976, emend., containing two families. Pelomyxidae Schulze, 1877, and Mastigamoebidae Goldschmidt, 1907.

Amoeba↗

Molecular phylogeny of methanogens associated with flagellated protists in the gut and with the gut epithelium of termites.

The molecular phylogeny of methanogenic archaea associated with the flagellated protist species Dinenympha and Microjoenia in the gut of termites, Reticulitermes speratus and Hodotermopsis sjoestedti, and those attached to the gut epithelium was examined based on PCR-amplified small-subunit ribosomal RNA genes. The sequences identified were classified into six groups within the genus Methanobrevibacter, including groups of yet uncharacterized novel species. Closely related methanogens were shared between Microjoenia and some Dinenympha cells in each termite. The methanogens harbored by the flagellates were phylogenetically different from the methanogens associated with the gut epithelium, suggesting that distinct methanogen species showed distinct spatial distributions in the termite gut.

Journal Article↗

Induction of flagellation and a novel agar-penetrating flagellar structure in Salmonella enterica grown on solid media: possible consequences for serological identification.

Salmonella enterica grown on solid medium containing iron, thiosulfate and 100 mM hexoses and amino acids underwent cell surface differentiation involving increased flagellation (electrophoretic isotypes 60, 54 and 50 kDa), conversion from rough to smooth lipopolysaccharide, and assembly of a matrix that penetrated 1.4% agar. Flagellation was also induced in the avian pathogen S. enterica var Pullorum, which is diagnostically defined as aflagellate. Induction correlated closely with a simple colonial color change when Hektoen Enteric agar was used as the basal growth medium. Group D1 egg-contaminating Salmonella grown under inducing conditions deviated from their expected H-antigen immunoreactivity, suggesting possible consequences for the interpretation of the Kauffman-White identification scheme.

Agar↗

Effects of pH of the medium on flagellation of Vibrio parahaemolyticus.

Formation of the lateral flagella by Vibrio parahaemolyticus was inhibited under an alkaline condition of the medium. However, flagellation of the polar monotrichous flagellum was not affected in the same condition. Flagellation of the lateral flagella depended on the pH of the medium.

Culture Media↗

Cellulose Metabolism by the Termite Flagellate Trichomitopsis termopsidis.

The end products of cellulose metabolism by the trichomonad flagellate Trichomitopsis termopsidis from the termite Zootermopsis sp. were investigated by growing axenic flagellates on [C]cellulose. The growth of T. termopsidis resulted in the release of label into the supernatant fraction of the culture fluid, and > 75% was volatile under acid conditions. The label was analyzed for CO(2) and for [C]volatile compounds by vacuum distillation under acid and alkaline conditions in disposable micro-distillation vessels. The distillate and undistilled culture supernatant fluid were chromatographed on cellulose thin layers to identify the labeled end product. T. termopsidis produced CO(2) and [C]acetate which accounted for 25 to 30% and 55 to 60% of the labeled end products, respectively. The ratio of label in CO(2) to acetate suggests that they are produced in equimolar amounts. No neutral volatile compounds were produced. The remaining unidentified end product (10 to 20%) was not volatile nor extractable into ether. Hydrogen was produced by T. termopsidis, and the cells were killed by the drug metronidazole. Enzymatic activities were found which account for these end products: pyruvate:ferredoxin oxidoreductase and hydrogenase. The results indicate that acetate is the end product of T. termopsidis cellulose metabolism and is available to the termite for energy metabolism and biosynthesis.

Journal Article↗

Bacterial filament formation, a defense mechanism against flagellate grazing, is growth rate controlled in bacteria of different phyla.

A facultatively filamentous bacterium was isolated from eutrophic lake water and was identified as Flectobacillus sp. strain MWH38 (a member of the Cytophaga-Flavobacterium-Bacteroides phylum) by comparative 16S rRNA gene sequence analysis. Filament formation by Flectobacillus sp. strain MWH38 and filament formation by Flectobacillus major, the closest known relative of strain MWH38, were studied in chemostat cultures under grazing pressure by the bacterivorous flagellate Ochromonas sp. strain DS and without predation at several growth rates. The results clearly demonstrated that filament formation by the two flectobacilli is growth rate controlled and thus independent of the presence of a predator. However, flagellate grazing positively influenced bacterial growth rates by decreasing bacterial biomass and thus indirectly stimulated filament formation. The results of investigations of cell elongation and filament formation by Comamonas acidovorans PX54 (a member of the beta subclass of the class Proteobacteria) supported the recent proposal that in this species the mechanism of filament formation is growth rate controlled. The finding that the grazing defense mechanism consisting of filament formation is growth rate controlled in the flectobacilli investigated and C. acidovorans PX54 (i.e., in bacteria belonging to divergent evolutionary phyla) may indicate that this mechanism is a phylogenetically widely distributed defense strategy against grazing.

Animals↗