A comparison of Chryseobacterium indologenes pathogenicity to the soft tick Ornithodoros moubata and hard tick Ixodes ricinus

A yellow-pigmented Gram-negative bacterium, Chryseobacterium indologenes, was found in the gut contents of about 65% of soft ticks Ornithodoros moubata from a perishing laboratory colony. The isolated putative pathogen, C. indologenes, was susceptible to cotrimoxazol and addition of this antibiotic (Biseptol 480) to the blood meal significantly decreased the tick mortality rate. The artificial infection of healthy O. moubata by membrane feeding on blood contaminated with C. indologenes was lethal to all ticks at concentrations 10(6) bacteria/ml. On the contrary, a similar infection dose applied to the hard tick Ixodes ricinus by capillary feeding did not cause significant mortality. Examination of guts dissected from infected O. moubata and I. ricinus revealed that C. indologenes was exponentially multiplied in the soft tick but were completely cleared from the gut of the hard ticks within 1 day. In both tick species, C. indologenes were found to penetrate from the gut into the hemocoel. The phagocytic activity of hemocytes from both tick species was tested by intrahaemocoelic microinjection of C. indologenes and evaluated by indirect fluorescent microscopy using antibodies raised against whole bacteria. Hemocytes from both tick species displayed significant phagocytic activity against C. indologenes. All O. moubata injected with C. indologenes died within 3 days, whereas the increase of the mortality rate of I. ricinus was insignificant. Our results indicate that hard ticks possess much more efficient defense system against infection with C. indologenes than the soft ticks. Thus, C. indologenes infection has the potential to be a relevant comparative model for the study of tick immune reactions to transmitted pathogens.     

The major tick salivary gland proteins and toxins from the soft tick,Ornithodoros savignyi,are part of the tick Lipocalin family: implications for the origins of tick toxicoses

The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.     

Molecular cloning and comparative analysis of fibrinogen-related proteins from the soft tick Ornithodoros moubata and the hard tick Ixodes ricinus

Among disease-vectors, the evolution of the tick innate immune system is still lagging when compared to insects. Such an investigation, which was initiated, by first cloning and sequencing lectins associated in the innate immunity of invertebrates and having fibrinogen related domains, helped in the sequencing of cDNA encoding for OMFREP from the soft tick, Ornithodoros moubata. Also obtained were Ixoderin A and Ixoderin B cDNA sequences from the hard tick Ixodes ricinus. Tissue-specific expression of OMFREP showed that it was present primarily in the hemocytes and salivary glands. Ixoderin A besides sharing a similar expression profile was also expressed in the midgut. Both showed significantly high homology to the lectin Dorin M, from O. moubata. Further, phylogenetic comparisons between these molecules of the soft and hard ticks showed their relatedness to Tachylectins 5A and 5B, involved in the innate immunity of Tachypleus tridentatus and ficolins from both vertebrates and invertebrates. Ixoderin B showing tissue-specific expression only in the salivary glands and the sequence displaying certain motif differences in homology point towards a possible function different from the other two molecules. This is the first report of lectin-like sequences, with a fibrinogen-domain, from the hard tick I. ricinus and a preliminary phylogenetic study of these tick sequences with related fibrinogen-domain containing sequences highlights a possible role for them in the innate immunity of the ticks.     

Heart tissue contains small and large aggregates of ferritin subunits

Ferritin purified from horse heart and applied to nondenaturing polyacrylamide gel electrophoresis migrated as a single band that stained for both iron and protein. This ferritin contained almost equal amounts of fast- and slow-sedimenting components of 58 S and 3-7 S, which could be separated on sucrose density gradients. Iron removal reduced the sedimentation coefficient of the fast-sedimenting ferritin to 18 S, and sedimentation equilibrium gave a molecular weight 650,000, with some preparations containing ferritin of 500,000 molecular weight as well. Sedimentation rates of the 3 S and 7 S ferritins were not affected by iron removal, and sedimentation equilibrium data were consistent with Mr's 40,000 and 180,000, respectively. Preparations of ferritin extracted from horse spleen contained only 67 S (holo) or 16 S (apo) ferritin and no slow-sedimenting species. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all of the ferritins contained the usual H and L subunits (23 and 20 kDa, respectively), but the slow-sedimenting (3 S and 7 S) heart apoferritins also contained appreciable quantities (ca 25%) of three larger subunits of 42, 55, and 65 kDa. All the subunits reacted positively in Western blots to polyclonal antibodies made against specially purified large heart or spleen ferritins containing only 20- and 23-kDa subunits. Similar results were obtained for ferritins from rat heart. The results indicate that mammalian heart tissue is peculiar not just in having an abnormally large iron-rich ferritin but also in having iron-poor ferritins of much lower molecular weight, partly composed of larger subunits.     

Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari

Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5′-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5′-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.     

Crassostrea gigas ferritin: cDNA sequence analysis for two heavy chain type subunits and protein purification

Ferritin has been shown as being the principal iron storage in the majority of living organisms. In marine species, ferritin is also involved in high-level accumulation of (210)Po. As part of our work on the investigation of these radionuclides' concentration in natural environment, ferritin was searched at the gene and protein level. Ferritin was purified from the visceral mass of the oyster Crassostrea gigas by ion-exchange chromatography and HPLC. SDS-PAGE revealed one band of 20 kDa. An Expressed Sequence Tag (EST) library was screened and led to the identification of two complementary DNA (cDNA) involved in ferritin subunit expression. The complete coding sequences and the untranslated regions (UTRs) of the two genes were obtained and a 5' Rapid Amplification of cDNA Ends (RACE) was used to obtain the two iron-responsive elements (IREs) with the predicted stem-loop structures usually present in the 5'-UTR of ferritin mRNA. Sequence alignment in amino acid of the two new cDNA showed an identity with Pinctada fucata (85.4-88.3%), Lymnaea stagnalis (79.3-82.2%) and Helix pomatia (79.1-79.1%). The residues responsible for the ferroxidase center, conserved in all vertebrate H-ferritins, are present in the two oyster ferritin subunits. Oyster ferritins do not present the special characteristics of other invertebrate ferritins like insect ferritins but have some functional similarities with the vertebrate H chains ferritin.     

Structural homology between mouse liver and horse spleen ferritins

Mouse liver ferritin is composed almost exclusively of polypeptide chains similar in molecular mass (22 kDa) to that characteristic of the major chain (H) found in heart ferritin isolated from human, horse or rat. In these species the predominant polypeptide of liver (L) is smaller (about 20 kDa). Here we show that mouse liver and horse spleen ferritins and apoferritins exhibit extensive structural homology as judged by the similarity in the diffraction patterns of their crystals grown from cadmium sulphate solutions. Implications of this finding are discussed.     

Cellular location of rat muscle ferritins and their preferential loss during cell isolation

Heart and other muscles of the rat contain two forms of ferritin separable in polyacrylamide gel electrophoresis. The cellular location of the fast- and slow-migrating ferritins was investigated using primary cultures of hindlimb skeletal muscle, and isolated myocardial cell populations. Muscle and non-muscle cells were isolated in good yield from hearts of adult rats pretreated with large doses of iron to increase their ferritin content. In virtually all cases, the isolated muscle cells contained traces only of the fast-migrating species and the non-muscle cells contained small amounts of the slow-migrating ferritin. During cell isolation, 90-100% of both ferritins was lost and could be recovered in the perfusates and solutions employed, while one third of the total tissue protein, and a larger percentage of creatine phosphokinase, was recovered in the isolated cells. Primary cultures of thigh muscle from adult rats which had differentiated into multi-nucleated myotubes, were incubated for 1-3 days with chelated iron. These cells contained substantial amounts of the electrophoretically fast migrating ferritin, with its characteristic larger Stokes' radius (determined by quantitative polyacrylamide gel electrophoresis). None of the slow-migrating ferritin species was detected, although hindlimb muscle from iron-treated rats contained both forms. It is concluded that the fast-migrating ferritin of muscle, which is much larger and more asymmetric than other ferritins, is confined to the muscle cell population, while the other form is predominantly or exclusively in the non-muscle cells. Both ferritins are lost preferentially over other proteins during procedures which injure muscle tissue.     

Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals.

The presence, internal distribution, and phylogenetic position of endosymbiotic bacteria from four species of specific-pathogen-free ticks were studied. These included the hard ticks Ixodes scapularis (the black-legged tick), Rhipicephalus sanguineus (the brown dog tick), and Haemaphysalis longicornis and the African soft tick Ornithodoros moubata. PCR assays for bacteria, using two sets of general primers for eubacterial 16S and 23S rRNA genes (rDNAs) and seven sets of specific primers for wolbachial, rickettsial, or Francisella genes, indicated that I. scapularis possessed symbiotic rickettsiae in the ovaries and that the other species harbored eubacteria in both the ovaries and Malpighian tubules. Phylogenetic analysis based on the sequence of 16S rDNA indicated that the symbiont of I. scapularis belonged to the alpha subgroup of proteobacteria and was closely related to the members of the genus Rickettsia. The other species had similar microorganisms in the ovaries and Malpighian tubules, which belonged to the gamma subgroup of proteobacteria, and formed a monophyletic group with the Q-fever pathogen, Coxiella burnetii. O. moubata harbored another symbiont, which formed a monophyletic group with Francisella tularensis and Wolbachia persica, the latter a symbiont previously isolated from Malpighian tubules of the soft tick Argas (Persicargas) arboreus. Thus, the symbionts of these four tick species were not related to the Wolbachia species found in insects. The two symbionts that live in the Malpighian tubules, one closely related to C. burnetii and the other closely related to F. tularensis, appear to be of ancient origin and be widely distributed in ticks.     

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.     

A proteomic approach to the identification of salivary proteins from the argasid ticks Ornithodoros moubata and Ornithodoros erraticus

The saliva of ticks contains anti-haemostatic, anti-inflammatory and immunomodulatory molecules that allow these parasites to obtain a blood meal from the host and help tick-borne pathogens to infect the vertebrate host more efficiently. This makes the salivary molecules attractive targets to control ticks and tick-borne pathogens. Although Ornithodoros moubata and O. erraticus are important argasid ticks that transmit severe diseases, to date only a few of their salivary proteins have been identified. Here we report our initial studies using proteomic approaches to characterize the protein profiles of salivary gland extracts (SGE) from these two argasids. The present work describes the proteome of the SGEs of both tick species, their antigenic spots, and the identification of several of their proteins. The whole number of identifications was low despite the good general quality of the peptide mass maps obtained. In the O. moubata SGE, 18 isoforms of a protein similar to O. savignyi TSGP1 were identified. In the O. erraticus SGE we identified 6 novel proteins similar to unknown secreted protein DS-1 precursor, NADPH dehydrogenase subunit 5, proteasome alpha subunit, ATP synthase F0 subunit 6, lipocalin and alpha tubulin. Finally, the current drawbacks of proteomics when applied to the identification of acarine peptides and proteins are discussed.     

Immune responses of the argasid tick Ornithodoros moubata moubata induced by infection with the filarial worm Acanthocheilonema viteae

Investigations were undertaken to determine whether the tick Ornithodoros moubata moubata mounted a detectable immune response to primary and secondary infections with Acanthocheilonema viteae. Uninfected control tick survival rate was 70%, but only 45% in the primary infection group. Post-secondary infection survival rate (82%) was comparable to controls, indicating that these selected ticks had some protective advantage. Mean A. viteae infective larvae recovery from ticks with secondary infections was 31.4% lower than expected, suggesting the development of immunity. SDS-PAGE of haemolymph for proteins induced post-primary infection yielded a stronger signal at 45 kDa than controls, which was further elevated post-secondary infection. Proteins at 48, 22 and 16 to 18 kDa were detected in haemolymph from infected ticks but not seen from controls. The direct effect of haemolymph on microfilarial viability was examined using a novel in vitro assay; in these preliminary trials no differences were observed in parasite viability when exposed to haemolymph from infected or uninfected groups of ticks.     

Evidence for conservation of ferritin sequences among plants and animals and for a transit peptide in soybean

Ferritin is a large multisubunit protein that stores iron in plants, animals, and bacteria. In animals, the protein is mainly cytoplasmic and is highly conserved, while in plants ferritin is found in chloroplasts and other plastids. Ferritin is synthesized in plants as a larger precursor of the mature subunit. There is no sequence information for ferritin from plants, except an NH2-terminal peptide of 35 residues which shows little similarity to any known ferritin sequences or transit peptides (Laulhere, J. P., Laboure, A. M., and Briat, J. F. (1989) J. Biol. Chem. 264, 3629-3635). To understand the genetic origin and the location of ferritin synthesis in plant cells, as well as the structure of ferritin from plants, we have sequenced both CNBr peptides from pea seed ferritin and nucleotides of a soybean hypocotyl ferritin cDNA, identified using a frog ferritin cDNA as a probe. Comparison of pea and soybean sequences showed an identity of 89%. Alignment of the plant ferritin sequences with animal ferritins showed 55-65% sequence identity in the common regions. However, a peptide of 28 amino acids extended the NH2 terminus of the plant ferritins. Furthermore, the cDNA encoded additional amino acids which appear to be a transit peptide. None of the sequences in soybean ferritin were found in the tobacco chloroplast genome, suggesting, as does the transit peptide, a nuclear location of ferritin gene(s) in plants. Plant ferritin mRNA is 400-500 nucleotides longer than animal ferritin mRNAs, a difference accounted for in part by the extra peptides encoded. The size of soybean ferritin mRNA was constant in different tissues but expression varied in different tissues (leaf greater than hypocotyl). Thus, higher plants and animal ferritins display sequence homology and differential tissue expression. An ancient, common progenitor apparently gave rise to contemporary eukaryotic ferritins after specific modifications, e.g. transport to plasmids.     

Isolation and Expression Pattern Analysis of Two Ferritin Genes in Tobacco

For understanding of the ferritin gene expression pattern and the mechanism of iron homeostasis in tobacco (Nicotiana tabaccum L.) plants, two full-length ferritin cDNAs, NtFerl and NtFer2, were isolated from tobacco seedlings and characterized. These cDNAs are 1 214 and 1 125 bp nucleotides and encode 25 1 and 259 amino acid residues, respectively. The deduced amino acid sequences showed that two tobacco ferritins share the same characteristics as the plant ferritins from Arabidopsis, soybean, and maize.Southern blotting analysis indicated that both NtFerl and NtFer2 were probably multicopy genes in the tobacco genome. Northern blotting analysis indicated that iron loading of tobacco plantlets increased the ferritin mRNA abundance and that NtFerl expression was higher and more sensitive to iron than NtFer2expression. Furthermore, NtFerl was expressed in both leaves and roots, whereas NtFer2 was expressed mainly in leaves.     

Mouse hepatoma and liver ferritins. Comparative structural studies.

Pure ferritin from male mouse liver produces a single band of monomers (RF = 0.199) with electrophoresis in polyacrylamide gels at pH 9.0. The five sub-bands within this monomeric band appear to represent charge isomers having the same molecular size. Ferritin from BH3 transplantable mouse hepatoma shows two overlapping bands of monomers (RFA = 0.208 and RFB = 0.240); further electrophoretic studies show that these bands represent two subpopulations of molecules differing both in charge and size. Sub-bands are not found in this hepatoma ferritin. The larger tumor ferritin reaches the same end migration position as all liver isoferritins on gradient gels, signifying a very similar or identical molecular size; however, the absence of sub-bands indicates that this hepatoma ferritin differs in charge from the homologous liver proteins. Liver and hepatoma ferritins both produce a single prominent subunit band corresponding to nominal molecular weights of 22 250 and 21 700, with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. With electrophoresis on polyacrylamide gradient slabs containing sodium dodecyl sulfate and dithiothreitol, both liver and hepatoma ferritins now reveal two subunits bands situated at identical positions. The polypeptides of these two closely spaced bands have a nominal molecular weight difference of less than 1000. Neither the hepatoma nor the liver seems to produce the ferritins found in the other tissue. Nevertheless, all these ferritins are composed of the same two types of subunits, albeit in different relative amounts. Observed distinctions in the ferritins from these normal or neoplastic cells must reflect differences in assembly and processing, as well as in the regulated expression of the same ferritin genes.     

Evidence for the presence of ferritin in plant mitochondria.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.