Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly (Glossina) after bacterial challenge.

Proteins containing a glutamic acid-proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% (G. m. morsitans) and 46% (G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.

Identification of midgut proteins that are differentially expressed in trypanosome-susceptible and normal tsetse flies (Glossina morsitans morsitans).

Molecules in the midgut of tsetse flies (Diptera: Glossinidiae) are thought to play important roles in the life cycle of African trypanosomes by influencing initial parasite establishment and subsequent differentiation events that ultimately lead to maturation of mammal-infective trypanosomes. The molecular composition of the tsetse midgut is, therefore, of critical importance to disease transmission by these medically important vectors. In this study we compared protein expression profiles of midguts of the salmon mutant and wild type Glossina morsitans morsitans Westwood that display marked differences in their susceptibility to infection by African trypanosomes. Isotope coded affinity tag (ICAT) technology was used to identify 207 proteins including 17 that were up regulated and nine that were down regulated in the salmon mutants. Several of the up regulated molecules were previously described as tsetse midgut or salivary gland proteins. Of particular interest was the up regulation in the salmon flies of tsetse midgut EP protein, a recently described molecule with lectin-like activity that was also found to be induced in tsetse by bacterial challenge. The up regulation of the EP protein in midguts of salmon mutants was confirmed by two-dimensional gel electrophoresis and tandem mass spectrometry.

Proteome analysis of abundantly expressed proteins from unfed larvae of the cattle tick, Boophilus microplus.

Protein expression in unfed larvae of the cattle tick, Boophilus microplus, was characterized using gel electrophoresis and mass spectrometry in an effort to assemble a database of proteins produced at this stage of development. Soluble and insoluble proteins were extracted and resolved by two-dimensional (2D) gel electrophoresis. Twenty abundantly expressed larval proteins were selected for peptide mass mapping and for peptide sequencing by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) and quadrupole time-of-flight (Q-ToF) tandem mass spectrometry (MS), respectively. Only one protein, tropomyosin, was unequivocally identified from its peptide mass map. Ten proteins were assigned putative identities based on BLAST searching of heterologous databases with peptide sequences. These included a cytoskeletal protein (troponin I), multiple cuticular proteins, a glycine-rich salivary gland-associated protein and proteins with a presumed housekeeping role (arginine kinase, a high-mobility group protein and a small heat shock protein). Eight additional proteins were identified by searching translated open reading frames of a B. microplus EST database (unpublished): putative fatty-acid binding protein, thioredoxin, glycine-rich salivary gland protein and additional cuticular proteins. One remaining protein was not identifiable, suggesting it may be a novel molecule. The ongoing assembly of this database contributes to our understanding of proteins expressed by the tick and provides a resource that can be mined for molecules that play a role in tick-host interactions.

Immunodetection of UCP1 in rat thymocytes.

Thymi were dissected from rats and connective tissue was removed. Mitochondria were purified from isolated thymocytes and immunoblot analysis was performed using an antibody specific for uncoupling protein 1, which detected a 32.5 kDa protein associated with mitochondria from the thymocytes. This implies that rat thymocytes contain uncoupling protein 1.

The relationship between flesh quality and numbers of Kudoa thyrsites plasmodia and spores in farmed Atlantic salmon, Salmo salar L.

Atlantic salmon, Salmo salar L., were exposed to Kudoa thyrsites (Myxozoa, Myxosporea)-containing sea water for 15 months, and then harvested and assessed for parasite burden and fillet quality. At harvest, parasites were enumerated in muscle samples from a variety of somatic and opercular sites, and mean counts were determined for each fish. After 6 days storage at 4 degrees C, fillet quality was determined by visual assessment and by analysis of muscle firmness using a texture analyzer. Fillet quality could best be predicted by determining mean parasite numbers and spore counts in all eight tissue samples (somatic and opercular) or in four fillet samples, as the counts from opercular samples alone showed greater variability and thus decreased reliability. The variability in both plasmodia and spore numbers between tissue samples taken from an individual fish indicated that the parasites were not uniformly distributed in the somatic musculature. Therefore, to best predict the probable level of fillet degradation caused by K. thyrsites infections, multiple samples must be taken from each fish. If this is performed, a mean plasmodia count of 0.3 mm(-2) or a mean spore count of 4.0 x 10(5) g(-1) of tissue are the levels where the probability of severe myoliquefaction becomes a significant risk.

Identification of major soluble salivary gland proteins in teneral Glossina morsitans morsitans.

Salivary glands of tsetse flies (Diptera: Glossinidiae) contain molecules that are involved in preventing blood clotting during feeding as well as molecules thought to be intimately associated with trypanosome development and maturation. Here we present a protein microchemical analysis of the major soluble proteins of the salivary glands of Glossina morsitans morsitans, an important vector of African trypanosomes. Differential solubilization of salivary proteins was followed by reverse-phase, high-performance liquid chromatography (HPLC) and analysis of fractions by 1-D gel electrophoresis to reveal four major proteins. Each protein was subjected to amino acid microanalysis and N-terminal microsequencing. A protein chemical approach using high-resolution 2-D gel electrophoresis and mass spectrometry was also used to identify the salivary proteins. Matrix-assisted, laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass mapping and sequencing, respectively. Sequence information and peptide mass maps queried against the NCBI non-redundant database confirmed the identity of the first protein as tsetse salivary gland growth factor-1 (TSGF-1). Two proteins with no known function were identified as tsetse salivary gland protein 1 (Tsal 1) and tsetse salivary gland protein 2 (Tsal 2). The fourth protein was identified as Tsetse antigen-5 (TAg-5), which is a member of a large family of anti-haemostatic proteins. The results show that these four proteins are the most abundant soluble gene products present in salivary glands of teneral G. m. morsitans. We discuss the possible functions of these major proteins in cyclical transmission of African trypanosomes.

Trypanosoma simiae and Trypanosoma congolense: surface glycoconjugates of procyclic forms-the same coats on different hangers?

Organic solvent extraction, reverse-phase high performance liquid chromatography and enzyme-linked immunosorbent assay with surface binding monoclonal antibodies were used to isolate membrane molecules of procyclic culture forms of Trypanosoma simiae and Trypanosoma congolense. Gel electrophoresis of the purified molecules revealed two predominant molecular species from each parasite that were broadly similar yet showed different apparent molecular masses and staining characteristics. The molecules were shown to be glycosylphosphatidylinositol-lipid anchored glycoconjugates, rich in carbohydrates. Each moiety displayed surface-disposed carbohydrate epitopes that were recognized on the surface of both species of trypanosomes by monoclonal antibodies specific for procyclic parasites of the subgenus Nannomonas. The epitopes were previously shown to be displayed on the glutamic acid-alanine rich protein of T. congolense yet neither this protein, nor its encoding gene is present in T. simiae. The results indicate that although T. congolense and T. simiae share common carbohydrate surface epitopes, these are displayed on biochemically different molecules. We speculate that the surface disposed carbohydrate structures are involved in parasite-tsetse interactions since these species have the same developmental cycles in the insect vector.

The major protein in the midgut of teneral Glossina morsitans morsitans is a molecular chaperone from the endosymbiotic bacterium Wigglesworthia glossinidia.

Molecules in the midgut of the tsetse fly (Diptera: Glossinidiae) are thought to play an important role in the life cycle of African trypanosomes by influencing their initial establishment in the midgut and subsequent differentiation events that ultimately affect parasite transmission. It is thus important to determine the molecular composition of the tsetse midgut to aid in understanding disease transmission by these medically important insect vectors. Here, we report that the most abundant protein in the midguts of teneral (unfed) Glossina morsitans morsitans is a 60 kDa molecular chaperone of bacterial origin. Two species of symbiotic bacteria reside in the tsetse midgut, Sodalis glossinidius and Wigglesworthia glossinidia. To determine the exact origin of the 60 kDa molecule, a protein microchemical approach involving two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. Peptide mass maps were compared to virtual peptide maps predicted for S. glossinidius and W. glossinidia 60 kDa chaperone sequences. Four signature peptides were identified, revealing that the source of the chaperone was W. glossinidia. Comparative 2-D gel electrophoresis and immunoblotting further revealed that this protein was localized to the bacteriome and not the distal portion of the tsetse midgut. The possible function of this highly abundant endosymbiont chaperone in the tsetse midgut is discussed.

Procyclins, proteases and proteomics: dissecting trypanosomes in the tsetse fly.

The forms of African trypanosomes that live in tsetse fly vectors are coated with lipid-anchored proteins and glycoproteins known collectively as procyclins. Procyclins are expressed during development in the fly in a multiplicity of isoforms yet their functions remain unknown. Recent studies involving a multidisciplinary synthesis of tsetse biology, immunochemistry, biological chemistry and mass spectrometry have yielded much new information about procyclins, which could now provide an unparalleled view of the dynamic molecular interactions between this parasite and its insect vector.

Analysis of Kudoa thyrsites (Myxozoa: Myxosporea) spore antigens using monoclonal antibodies.

A method employing Percoll gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites, K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to > 220 kDa, whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses, depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated, intact, permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting, indicating that these mAbs have potential for use in developing a field-based diagnostic test.

The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro.

Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypanosomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms but not in BSF after incubation with concanavalin A (Con A), wheat germ agglutinin and Ricinus communis agglutinin. These lectins caused the trypanosomes to cease division, become round and increase dramatically in size, the latter being partially attributable to the formation of what appeared to be a large 'vacuole-like structure' or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extracted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramatically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were multinucleate whereas the kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and kinetoplast replication. Immunofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP-11), showed that the Con A-treated parasites had begun to duplicate the flagellum but that this had only proceeded along part of the length of the cells, suggesting that the cell division process was initiated but that cytokinesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosomes and mutant trypanosomes expressing both high levels of non-glycosylated procyclins and procyclin isoforms with truncated N-linked sugars were resistant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was supported by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homologous recombination. The knockout mutants showed reduced binding of fluorescein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces multinucleation, a disequilibrium between nuclear and kinetoplast replication and a unique form of cell death in procyclic African trypanosomes and that the ligands for Con A binding are carbohydrates on the EP forms of procyclin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed.

Phosphorylation of a major GPI-anchored surface protein of Trypanosoma brucei during transport to the plasma membrane.

The surface coat of procyclic forms of Trypanosoma brucei consists of related, internally repetitive glycoproteins known as EP and GPEET procyclins. Previously we showed that the extracellular domain of GPEET is phosphorylated. We now show that phosphorylation of this glycosylphosphatidylinositol-anchored surface protein can be induced in vitro using a procyclic membrane extract. Using antibodies that recognize either the phosphorylated or unphosphorylated form of GPEET, we analyzed their expression during differentiation of bloodstream forms to procyclic forms. Unphosphorylated GPEET, together with EP, was detected in cell lysates 2-4 hours after initiating differentiation whereas phosphorylated GPEET only appeared after 24 hours. Surface expression of EP and both forms of GPEET occurred after 24-48 hours and correlated with the detection of phosphorylated GPEET on immuno-blots. Electron micrographs showed that unphosphorylated GPEET was predominantly in the flagellar pocket whereas the phosphorylated form was distributed over the cell surface. In contrast, expression of a membrane-bound human placental alkaline phosphatase in procyclic forms caused the accumulation of dephosphorylated GPEET on the cell surface, while the phosphorylated form was restricted to the flagellar pocket. A GPEET-Fc fusion protein, which was retained intracellularly, was not phosphorylated. We propose that unphosphorylated GPEET procyclin is transported to a location close to or at the cell surface, most probably the flagellar pocket, where it becomes phosphorylated. To the best of our knowledge, this study represents the first localization of phosphorylated and unphosphorylated forms of a GPI-anchored protein within a cell.

Molecular characterization of the kinetoplastid membrane protein-11 from African trypanosomes.

The kinetoplastid membrane protein-11 molecule was purified from Trypanosoma brucei rhodesiense and an internal peptide sequence was obtained. This sequence information was used with cosmid library screening and polymerase chain reaction amplifications of both genomic DNA and cDNA to obtain the entire DNA sequence of the encoding gene and the corresponding translated amino acid sequence of 92 residues. The sequence showed 18% divergence from the corresponding molecule of the related kinetoplastid Leishmania donovani, including one key amino acid at position 45 which may be of functional relevance. The protein had a calculated molecular mass of 11078 Da, a pI of 6.0 and an overall net charge of -2 at physiological pH. The secondary structure of the molecule was predicted to consist of two amphipathic helices connected by a random-coil segment, and suggests that it would interact with lipid bilayers in the trypanosome cell membrane. Northern and Southern blot analyses showed that the trypanosome kinetoplastid membrane protein-11 molecule was translated from a single transcript and was transcribed from a single gene copy, thus making this molecule an attractive target for knockout mutagenesis.

Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro.

Apoptosis is a phenomenon previously associated exclusively with metazoan organisms. We show here that procyclic insect form Trypanosoma brucei rhodesiense, a protozoan parasite, when treated in vitro with concanavalin A displayed several features normally associated with apoptosis in metazoan cells. Lectin treatment induced cleavage of nuclear DNA into oligonucleosomal fragments, suggesting activation of an endogenous nuclease in the parasite. Treated trypanosomes, although agglutinated and non-motile, exhibited fluorescence after treatment with the vital stain fluorescein diacetate and retained (3)H-uridine indicating that their cell membranes remained intact during the period of DNA fragmentation. Electron micrographs showed characteristic morphology of cells undergoing apoptosis, including surface membrane vesiculation and migration of chromatin to the periphery of the nuclear membrane while mitochondria remained intact. These results suggest that treatment with concanavalin A triggers a cell death mechanism in T. b. rhodesiense similar to the process of apoptosis described in metazoa.

Molecular characterization of glycosomal NAD(+)-dependent glycerol 3-phosphate dehydrogenase from Trypanosoma brucei rhodesiense.

The primary structure of a 38-kDa protein isolated from membrane preparations of African trypanosomes was determined by protein and DNA sequencing. Searching of the protein database with the trypanosome translated amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from various prokaryotic and eukaryotic organisms as the optimal scoring protein. Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence identity with the corresponding enzyme from the prokaryote Escherichia coli. The trypanosome molecule was expressed in Escherichia coli and found to be enzymatically active, thus confirming the identity of the molecule as an NAD(+)-dependent glycerol 3-phosphate dehydrogenase. A monoclonal antibody specific for the 38-kDa protein was used to localize the enzyme to glycosomes. Immunoblotting showed that the monoclonal antibody bound to a 38-kDa protein in African trypanosomes but not in T. cruzi, Leishmania or Crithidia. The enzyme has a pI of 9.1, a net charge of +17 and contains the peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of glycosomal enzymes. Amino acids predicted to be involved in the NAD(+)-dependent glycerol 3-phosphate dehydrogenase active site have diverged from those of the mammalian enzyme. Kinetic analyses of the trypanosome GPD and GPD from rabbit muscle showed that the Km values of the two enzymes are different. The data suggest that the trypanosome protein may be a candidate target for rational drug design.

Characterization of monoclonal antibodies to the alpha IIb beta 3 integrin on bovine platelets.

A set of monoclonal antibodies (MoAbs) to leucocyte antigens is an essential tool to identify different cell types and functional membrane molecules involved in immune responses. Since no MoAbs existed to bovine integrins, except against the beta 2 subfamily, we generated MoAbs to beta 3 integrin after the immunization of mice with bovine platelets. Two MoAbs, IL-A164 (IgG2a) and IL-A166 (IgG1), were selected that reacted specifically with bovine platelets and detected the same membrane molecule. The antigen was a heterodimer of two polypeptide chains of 122 kDa and 95 kDa as resolved by SDS-PAGE under reducing conditions. Although the Mr of the smaller subunit is identical to that of beta 2 integrin, preabsorption with an antibody to beta 2 (or CD18) did not remove the bovine antigen. Comparing the molecular masses of the two subunits in reduced and non-reduced forms showed a pattern that was similar to that of human GPIIb/IIIa (also called alpha IIb beta 3 or CD41a). Reduction of the bovine molecule increased the apparent Mr of the light chain from 76 kDa to 95 kDa, while the heavy subunit changed from 136 kDa to 122 kDa. As with human GPIIb, the decrease in Mr of the alpha-subunit is probably a result of a small disulphide-linked polypeptide, although no additional evidence for this was detected for the bovine integrin. Sequencing of the N-terminal amino acids of both bovine polypeptides showed identity of the bovine integrin with human GPIIb/IIIa.

Identification of a 44 kDa protein localized within the endoplasmic reticulum of Trypanosoma brucei brucei.

Immunoaffinity chromatography and gel electrophoresis were used to isolate a 44 kDa protein that was bound to a 72 kDa chaperone in Trypanosoma brucei brucei. A polyclonal antiserum to the 44 kDa protein was raised in rats and employed in conjunction with chromatography using DEAE-cellulose, Sephacryl S-300, and hydroxyapatite to purify the protein from membranes of bloodstream forms of the trypanosomes. Immunoblot analysis using this antiserum revealed a protein doublet of 44/45 kDa in T. b. brucei and a single protein band of 53 kDa in almost equivalent amounts throughout the life-cycle stages of T. congolense. Indirect immunofluorescence using affinity-purified antibodies specific for the 44 kDa protein showed labelling of the perinuclear area and reticular system extending throughout the parasites, suggesting that this protein was located in the endoplasmic reticulum. Localization of the 44 kDa molecule in the endoplasmic reticulum was confirmed by immunoelectron microscopy. Protease protection experiments demonstrated that the epitopes bound by antibody were buried within the membrane or towards the lumenal face of the endoplasmic reticulum. Ruthenium Red overlay of nitrocellulose blots containing the 44/45 kDa doublet suggested that the molecules have the potential to bind calcium. The N-terminal amino acid sequence of the 44 kDa protein showed no sequence similarity to any proteins in the database.