Haem-responsive gene transporter enables mobilization of host haem in ticks.

Ticks, notorious blood-feeders and disease-vectors, have lost a part of their genetic complement encoding haem biosynthetic enzymes and are, therefore, dependent on the acquisition and distribution of host haem. Solute carrier protein SLC48A1, aka haem-responsive gene 1 protein (HRG1), has been implicated in haem transport, regulating the availability of intracellular haem. HRG1 transporter has been identified in both free-living and parasitic organisms ranging from unicellular kinetoplastids, nematodes, up to vertebrates. However, an HRG1 homologue in the arthropod lineage has not yet been identified. We have identified a single HRG1 homologue in the midgut transcriptome of the tick Ixodes ricinus, denoted as IrHRG, and have elucidated its role as a haem transporter. Data from haem biosynthesis-deficient yeast growth assays, systemic RNA interference and the evaluation of gallium protoporphyrin IX-mediated toxicity through tick membrane feeding clearly show that IrHRG is the bona fide tetrapyrrole transporter. We argue that during evolution, ticks profited from retaining a functional hrg1 gene in the genome because its protein product facilitates host haem escort from intracellularly digested haemoglobin, rendering haem bioavailable for a haem-dependent network of enzymes.

Strategies for new and improved vaccines against ticks and tick-borne diseases.

Ticks infest a variety of animal species and transmit pathogens causing disease in both humans and animals worldwide. Tick-host-pathogen interactions have evolved through dynamic processes that accommodated the genetic traits of the hosts, pathogens transmitted and the vector tick species that mediate their development and survival. New approaches for tick control are dependent on defining molecular interactions between hosts, ticks and pathogens to allow for discovery of key molecules that could be tested in vaccines or new generation therapeutics for intervention of tick-pathogen cycles. Currently, tick vaccines constitute an effective and environmentally sound approach for the control of ticks and the transmission of the associated tick-borne diseases. New candidate protective antigens will most likely be identified by focusing on proteins with relevant biological function in the feeding, reproduction, development, immune response, subversion of host immunity of the tick vector and/or molecules vital for pathogen infection and transmission. This review addresses different approaches and strategies used for the discovery of protective antigens, including focusing on relevant tick biological functions and proteins, reverse genetics, vaccinomics and tick protein evolution and interactomics. New and improved tick vaccines will most likely contain multiple antigens to control tick infestations and pathogen infection and transmission.

Mortality caused by experimental infection with the yeast Candida haemulonii in the adults of Ornithodoros moubata (Acarina: Argasidae).

A relatively high rate of mortality among engorged females of Ornithodoros moubata (Murray, 1877) was observed in our laboratory colony. The general aim of the study was to identify the causative agent responsible for this mortality. The diagnostic tests were performed by Yeast Identification Service (CBS-Delft, Netherlands) and the pathogen was identified as the yeast Candida haemulonii (van Uden et Kolipinski, 1962) Meyer et Yarrovi, 1978. The artificial infection study was performed by intrahaemocoelic inoculation of yeast suspension, resulting in a mortality of 37%. The maximum mortality of ticks infected per os by contaminated blood meal was 13%. Re-isolated yeast cells from haemolymph of dead and paralysed ticks were apparently identical with primary yeast cells, without loosing reproductive abilities. An occasional formation of elongated chains of yeast cells (pseudomycelium) was recorded. The majority of ticks infected in both experiments mentioned above survived and displayed no evident symptoms of the infection. The presence of yeast cells in the haemolymph of surviving ticks was not detected. The in vitro phagocytosis assay performed with FITC-labelled yeast cells showed that about 4% of tick haemocytes were phagocytically active against the pathogenic yeast cells. Thus phagocytosis seems to be a potent defence reaction against spreading and multiplying of the yeast C. haemulonii within the tick haemocoel.

Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata.

A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl-D-hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl-D-glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks.

Characterization of an alpha-macroglobulin-like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata.

We report the identification of the first representative of the alpha-2-macroglobulin family identified in terrestrial invertebrates. An abundant acidic glycoprotein was isolated from the plasma of the soft tick Ornithodoros moubata. Its molecular mass is about 420 kDa in the native state, whereas in SDS/PAGE it migrates as one band of 190 kDa under nonreducing conditions and a band of 92 kDa when reduced. Chemical deglycosylation reveals that it is composed of two different subunits, designated A and B. The N-terminal amino-acid sequence of subunit A is similar to the N-terminus of invertebrate alpha-2-macroglobulin. Sequence analysis of several internal peptides confirms that the tick protein belongs to the alpha-2-macroglobulin family, and the protein is therefore referred to as tick alpha-macroglobulin (TAM). Functional analyses strengthen this assignment. TAM inhibits trypsin and thermolysin cleavage of the high-molecular-weight substrate azocoll in a manner similar to that of bovine alpha-2-macroglobulin. This effect is abolished by pre-treatment of TAM with methylamine. In the presence of TAM, trypsin is protected against active-site inhibition by soybean trypsin inhibitor. We cloned and sequenced a PCR product containing sequences from both subunits and spanning the N-terminus of subunit B and the putative 'bait region' (a segment of alpha-2-macroglobulin which serves as target for various proteases). This indicates that the two subunits are generated from a precursor polypeptide by post-translational processing.

Purification and characterization of the lysozyme from the gut of the soft tick Ornithodoros moubata.

The gut of the adult soft ticks Ornithodoros moubata displays high lytic activity against the bacteria Micrococcus luteus. The activity differed in the range of two orders of magnitude among individual animals and increased on average 4 fold during the first week following ingestion. In homogenates of first instar nymphs the activity was much lower increasing exponentially as nymphs neared the first molt. The protein responsible for this activity was purified out of gut contents of adult ticks by means of affinity adsorption on magnetic-chitin followed by two chromatography steps on cation exchange FPLC column MonoS. The homogeneous active protein has a mass of 14006 +/- 20 Daltons as determined by MALDI-TOF mass spectrometry. The N-terminal amino-acid sequence of this protein is K-V-Y-D-R-C-S-L-A-S-E-L-R with the highest similarity to the lysozyme from liver of rainbow trout and to lysozymes from digestive tracts of several mammals. The motif DRCSLA is specific for the digestive lysozymes of several dipteran insects. Based on this evidence, we have identified the protein as the tick gut lysozyme. The tick gut lysozyme has a pI near 9.7 and retains its full activity after treatment at 60 degrees C for 30 minutes. The pH optimum of the tick lysozyme was in the range from pH 5-7. Only marginal activity could be detected at pH > 8 which raises the question about the function of lysozyme in anti-bacterial defense in the environment of the tick gut.

Primary structure of apolipophorin-III from the greater wax moth, Galleria mellonella.

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth, Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.

Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella.

In this paper, we report the detection, purification and characterization of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI was purified from the hemolymph of last-instar larvae of Galleria mellonella by precipitation with trichloroacetic acid and heat followed by affinity chromatography on a thermolysin-Sepharose column and gel filtration or reverse-phase high-performance liquid chromatography. For the detection of inhibitor activity, a new azocoll assay was established. IMPI was only detectable in larvae that had been injected with bacterial or fungal provocators, suggesting that it is induced nonspecifically during the humoral immune response. Injection of larvae with IMPI rendered them resistant to thermolysin, in quantities that normally would be lethal for them. IMPI was shown to be specific for metalloproteases. The molecular mass of IMPI was determined by mass spectrometry to be 8360 Da. Purified IMPI was heterogeneous, owing to different degrees of glycosylation with hexose/hexosamine and deoxyhexose residues. Ten cysteine residues were found in the molecule, and these are presumed to form five disulfide bridges. The amino terminus was blocked, but a partial amino-acid sequence starting from the thermolysin cleavage site was determined; this sequence exhibited no similarity with other known proteins, suggesting that the IMPI represents a new type of protease inhibitor.

Isolated Apolipophorin III from Galleria mellonella Stimulates the Immune Reactions of This Insect.

Apolipophorin III (apoLp-III) was isolated from the haemolymph of last instar larvae of Galleria mellonella. The ultraviolet (u.v.) spectrum and the N-terminal amino acid sequence reveal high similarities with the apoLp-III from Manduca sexta. The protein is heat-stable. The molecular mass of apoLp-III was determined to be 18 077 Da using mass spectrometry. The heat treatment (90 degrees C, 30 min) resulted in a pI shift from 6.6 for the non-heated to 6.1 for the heat-treated apoLp-III without change in the molecular mass, indicating that a conformational change might have been caused by the heat treatment, rather than covalent alterations. Intrahaemocoelic injection of pure apoLp-III into last instar G. mellonella larvae is followed by a dose-dependent increase of antibacterial activity in cell-free haemolymph of treated larvae 24 h after injection. Furthermore, pure apoLp-III enhances the phagocytic activity of isolated haemocytes in vitro. The newly discovered role of apoLp-III in inducing immune-related functions in insects is discussed in regard to the known features of this molecule in lipid metabolism. Arylphorin, another heat-stable protein in G. mellonella haemolymph, was likewise isolated in this study. The protein was identified by N-terminal protein sequencing, the sequence obtained exactly matches the known sequence data for this protein. Copyright 1997 Elsevier Science Ltd. All rights reserved

The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme.

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. The molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel. In the presence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions. The PPO was shown to be a glycoprotein and its isoelectric point was 6.2. The amino-acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sexta. The activation of the PPO with chymotrypsin was investigated and two proteins of 67 and 50 kDa were found to be products of the proteolytic cleavage. The N-terminus of the G. mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited significant homology with highly conserved sequences found in arthopod hemocyanins and insect storage proteins, which indicates that the PPO belongs to this family.

Isolation and characterization of a hemagglutinin with affinity for lipopolysaccharides from plasma of the crayfish Pacifastacus leniusculus.

A hemagglutinin with a high specific activity against trypsinized rabbit erythrocytes was identified in plasma of the freshwater crayfish Pacifastacus leniusculus. The activity of this crayfish hemagglutinin could be inhibited by sialoglycoproteins such as porcine stomach mucin, bovine submaxillary mucin, fetuin, and ovalbumin. However, the involvement of sialic acid in its binding specificity could not be unambiguously proven. Furthermore, the hemagglutinating activity in the crayfish plasma could be specifically inhibited by lipopolysaccharide from E. coli K-235, which might indicate a recognition role for this hemagglutinin. This hemagglutinin, which accounts for less than 0.01% of the total plasma protein, was purified to near homogeneity using affinity chromatography on a Fetuin-Sepharose 4B column. The molecular mass of the unreduced protein as revealed by sodium dodecyl sulphate electrophoresis in polyacrylamide gel was found to be 420,000 Da. Upon reduction with dithiothreitol the hemagglutinin dissociated to several subunits with masses ranging from 65,000 to 80,000 Da. Affinoblotting with peroxidase labelled lectins indicated that the hemagglutinin was likely to be a glycoprotein.

Characterization of a clotting protein, isolated from plasma of the freshwater crayfish Pacifastacus leniusculus.

A protein responsible for clot formation was isolated from plasma of the crayfish Pacifastacus leniusculus, by repeated precipitation at low ionic strength, pH 6.0. The protein, here named clotting protein (CP), is a lipoglycoprotein, which consists of two 210-kDa subunits, covalently associated by disulfide bonds. Preparations of the CP can form stable clots in the presence of crayfish haemocyte lysate supernatant, which contains endogenous, Ca(2+)-dependent transglutaminase (TGase) activity. The covalent, TGase-mediated polymerization of CP could clearly be visualized in SDS/PAGE under reducing conditions, where the 210-kDa subunit is covalently cross-linked into dimeric, trimeric and higher polymeric forms. The CP was shown to be a substrate for transglutaminases, since two different fluorescent TGase substrates, namely dansylcadaverine and a dansylated glutamine-containing peptide, were incorporated into the CP subunit by active TGase. This indicates the presence of both glutamine and lysine residues in the CP, accessible for TGase cross-linking. The amino acid composition and the N-terminal amino acid sequence of the clotting protein was determined.

Antigenic characterization of human isolates of cryptosporidia.

Isolates of cryptosporidia from immunodeficient patients and from immunocompetent children suffering from diarrhoea were sources for preparation of antigens. Altogether, antigens from 21 isolates were obtained, 19 from children and 2 from AIDS patients. With one exception, all children were under 4 years old and most of them were between 1 and 2 years old. The probable source of infection was found in 11 cases. In 8 cases, as well as in both AIDS patients, the source of infection was not determined. The same groups of persons and the control group of healthy blood donors were examined serologically using ELISA with the antigen of bovine origin and positive sera were used for following experiments. Soluble and membrane-enriched antigens from oocysts were compared using polyacrylamide gel electrophoresis and electroimmunotransfer blots developed by human immune sera. While no differences were recorded in soluble antigens, two of the membrane-enriched antigens were missing (15.5 and 33 kD bands). While all isolates with a pattern typical for Cryptosporidium parvum were infective for neonatal mice, both isolates with missing bands were not infective for newborn mice in repeated experiments. The first of them was infective for chickens and was originally determined to be Cryptosporidium baileyi (Ditrich et al. 1991). However, the antigenic pattern differs from pattern of this species. The second isolate was infective for guinea pigs and its species classification remains uncertain.

Serological differentiation of human small fluke infections using Opisthorchis viverrini and Haplorchis taichui antigens.

Sera from 642 inhabitants of Vientiane Province (Laos) were examined by enzyme-linked immunosorbent assay (ELISA) using cytoplasmic and membranous antigens prepared from adult worms. Worms of Opisthorchis viverrini originated from liver of dissected cats, Haplorchis taichui were obtained from a stool specimen of a Laotian patient after praziquantel treatment. The sera were divided into five groups according to the intensity of infection expressed as egg count per gram of patients stool (EPG). Correlation between intensity of infection and the level of antibodies in serum was recorded. Reactions obtained using the cytoplasmic antigens were more sensitive and more specific compared to those with membranous antigens. Cross-reactions between antigens of both helminth species were found. Highly positive sera were examined using electroimmunotransfer blots (EITB) with cytoplasmic antigens of both species, which enabled the species differentiation. Antigens of both species yielded several shared fractions; however, differences between them were found: homologous sera reacted specifically with O. viverrini antigen in the area of 70 kDa and with H. taichui antigen in the area of 10 kDa. Thirty-one of 122 tested sera had specific antibodies against O. viverrini, 77 sera against H. taichui and 14 sera against both species. The results confirmed our assumption about predominant occurrence of heterophyid flukes in the human population living in studied area, compared with the occurrence of opisthorchid flukes. Hence, serology seems to be helpful tool for correct diagnosis of small fluke infections.

[Immunoblotting in the study of the antigenic structure of Leishmania]. / Immunoblotting v izuchenii antigennoi struktury leishmanii.

Using immunoblotting, the antigenic structure of 6 Leishmania strains has been studied: 1) MHOM/IN/80/DDS--Leishmania (Leishmania) donovani; 2) MHOM/SU/63/VL--L. sp. ZMA; 3) MHOM/SU/73/K-27--L. tropica; 4) MHOM/SU/73/5Ash--L. major; 5) MHOM/GE/84/H-132--L. mexicana amazonensis; 6) REPT/SU/83/3960-GC--L. (Sauroleishmania) gymnodactyli. Antigens of Leishmania surface membranes and rabbit antisera against them have been used. Among the agents of leishmaniasis in the Old World (1-4), the most intensive antigenic lines were found in the medium and high-molecular mass area (43-200 kD). In L. (L) mexican a amazonensis (5) species-specific lines have been identified in 10-22 kD area, which is indicative of considerable antigenic differences in Leishmania of the Old and New Worlds. The most marked antigenic differences from other types were noted in L. (S) gymnodactyli (6). It was characterized by the absence of antigenic bands in the medium and high molecular mass areas, the lines associated with species-specific determinants located in the low molecular mass area (18 and 25 kD). The results of cross-reacting c-ELISA using the same antigens and antisera correlated well with the above data of immunoblotting. Immunoblotting may be used for identification of species-specific antigenic structures which may be helpful in serological tests for more accurate leishmania identification and leishmaniasis diagnosis.

Entamoeba polecki: morphology, immunology, antigen study and clinic of the first infections in Czechoslovakia.

Entamoeba polecki Prowazek, 1912 was recorded for the first time in Czechoslovakia in two students from Kampuchea. Uninucleated cysts of diameter 14.2-15.7 micron with nuclei diameter 3.2-4.2 micron were found-repeatedly in stool samples taken from them. The nucleus accounted in average for 24.3% of the cyst diameter. The isolation and two following subinoculations on Dobell-Leidlaw medium were achieved, more abundant growth was recorded on medium with pig serum. Sera of both students were positive in serological tests using the E. histolytica antigen. No serious clinical symptoms were observed, both patients were cured successfully by metronidazole and ornidazole. Electroimmunotransfer blots were used to characterize E. polecki as a separate species. The antigenic structure of polyxenically grown E. polecki was compared with the antigenic structure of E. histolytica (axenically grown HK-9 strain and 3 polyxenically grown strains). Different blot patterns of both species were obtained, but common fractions of 30-40 kD probably responsible for serological cross-reactions were found.

Analysis of soluble Entamoeba histolytica antigen using enzyme-L inked electroimmunotransfer blots.

Soluble antigens of ten strains of E. histolytica were studied by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked electroimmunotransfer blots (EITB). No relations of immune replicas to virulence, geographical origin and method of cultivation (xenic or axenic culture) were found. Antigens of all ten strains tested precipitated with anti-E. histolytica human serum in the area of 30-43 kD. Antigen of HK-9 strain created in this area a characteristic pattern with all sera containing the specific anti-E. histolytica antibodies and, therefore, EITB can be used for excluding false positive results in ELISA.

Studies on the properties of malic enzyme and malate dehydrogenase from Taenia crassiceps (Zeder, 1800) cysticerci.

It has been studied what type (or types) of malic enzyme is present in the mitochondria of Taenia crassiceps cysticerci. Only NADP-dependent malic enzyme (EC 1.1.1.40) was demonstrated. The method of isoelectric focusing was used for the detection of isoenzymes of malic enzyme and malate dehydrogenase in the cytoplasm and mitochondria of T. crassiceps cysticerci. The mitochondria contain 5 isoenzymes of malic enzyme and 5 isoenzymes of malate dehydrogenase. The cytoplasm contains 7 isoenzymes of malic enzyme (3 of them are identical with those from the mitochondria) and 7 isoenzymes of malate dehydrogenase (5 of them are identical with those from the mitochondria). pI values of all isoenzymes were assessed.