Characterisation of the gene encoding a protective antigen from Babesia microti identified it as η subunit of chaperonin containing T-complex protein 1

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT η subunit of B. microti and functions as a chaperonin.     

There are at least three genetically distinct small piroplasms from dogs

The 18S nuclear subunit ribosomal RNA (18S rRNA) gene of small piroplasms isolated from dogs from Okinawa (Japan), Oklahoma, North Carolina, Indiana, Missouri, and Alabama, was isolated and sequenced. Phylogenetic analysis of these sequences and comparisons with sequences from other Babesia, Cytauxzoon, and Theileria species revealed that all canine small babesial isolates, with the exception of isolates from California and Spain, were placed in a group containing the Babesia spp. sensu stricto. Within the Babesia spp. sensu stricto, there was support for separating the small canine piroplasms from the large canine piroplasm, Babesia canis. The isolate from California was in a distinct phylogenetic clade, closely related to babesial isolates from wildlife and humans from the Western US. The canine isolate from Spain was closely related to Babesia microti. These results suggest that there are multiple small piroplasm species in dogs. The isolates from the Midwestern and Eastern US and the one from Japan probably represent a single species with wide geographic distribution.     

Babesia bovis: Genome size, number of chromosomes and telomeric probe hybridisation

Pulsed field gel electrophoresis of intact chromosomes of Babesia bovis revealed four chromosomes in the haploid genome. A telomere probe, derived from Plasmodium berghei, hybridised to eight SfiI restriction fragments of genomic B. bovis DNA digests indicating the presence of four chromosomes. A small subunit (18S) ribosomal RNA gene probe hybridised to the third chromsome only. The genome size of B. bovis is estimated to be 9.4 million base pairs. The sizes of chromosomes 1, 2, 3 and 4 are estimated to be 1.4, 2.0, 2.8 and 3.2 million base pairs, respectively.     

Differences in the infectivity of Babesia incubated in plasma and serum and the role of glucose in prolonging viability

Babesia rodhaini was less infective to mice after incubation in rat serum than in rat plasma. This was explained by lower levels of glucose in serum than in plasma. Both serum and plasma were found to become metabolically depleted of glucose following prolonged contact with clotted and unclotted blood cells, respectively. When glucose concentrations in depleted samples were restored to those in freshly separated samples, infectivities of parasites were similar. This was shown for both B. rodhaini and B. argentina. Products of blood cell metabolism, produced when separation of plasma or serum was delayed for 24 h, were not shown to have any detrimental effects on the parasites. Average glucose values for plasma from rats and cattle were 153 mg/100 ml and 63 mg/100 ml, respectively, whereas serum and plasma remaining in contact with blood cells contained as little as 2 mg glucose/100 ml. Lactate values were correspondingly low in plasma and high in serum. Fibrinogen and platelets, factors involved in clotting, did not affect infectivity of B. rodhaini or B. argentina. The relevance of these findings to living babesial vaccines in which plasma- and serum-based diluents may be used is discussed.     

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice. International journal for Parasitology 3: 773–781. Babesia rodhaini infection was compared in irradiated, splenectomized and control mice. Although irradiation reduced the weight of the spleen by as much as 95 per cent, this reduction in size did not result in parasitaemia levels comparable to those seen in splenectomized mice, which were consistently higher. Parasitaemias were similar in irradiated and control mice, but the mean survival time in control mice was longer than that of irradiated or splenectomized mice, which were comparable. Splenectomy generally resulted in higher parasitaemias than those seen in non-splenectomized mice.

Since B. rodhaini has a predeliction for invading reticulocytes, the apparent failure of irradiated mice to develop parasitaemias comparable to those of splenectomized mice, may have been due to the selective destruction of these immature red cells by irradiation.     

Babesia bovis merozoites invade human, ovine, equine, porcine and caprine erythrocytes by a sialic acid-dependent mechanism followed by developmental arrest after a single round of cell fission

Babesia bovis infections have only been observed in bovine species in contrast to Babesia divergens that also can infect humans, sheep and rodents. Using an in vitro assay that assesses invasion of erythrocytes by free merozoites after a 1-h incubation period, it was shown that specificity is not determined by host-specific interactions associated with invasion. Human erythrocytes were invaded more efficiently than bovine erythrocytes whereas erythrocytes of sheep, pigs and horses were invaded only slightly less efficiently. In contrast, goat erythrocytes were refractory to efficient invasion. Significant differences in invasion efficiency into erythrocytes from different individuals of the same species were observed. Erythrocytes from all species, except for goats, supported intracellular development of newly invaded merozoites and high numbers of duplicated parasites, located in a morphologically normal accole position, were present. Only in bovine erythrocytes did subsequent rounds of invasion, leading to increased parasitaemia, take place. This suggests that host specificity is determined by factors operating late in the erythrocytic stage of the B. bovis life cycle. Incubation of erythrocytes with neuraminidase prior to invasion led to a decrease in invasion efficiency of 80%. This effect was observed for several species. The removal of either (2-3)-linked or (2-6)-linked sialic acid residues gave similar levels of reduction whereas simultaneous removal did not show an additive effect. Pre-incubation of merozoites with N-acetylneuraminyl-lactose decreased invasion efficiency by 45% whereas addition just prior to invasion had no significant effect. The results demonstrate that invasion is dependent on the presence of sialic-acid containing membrane receptors on erythrocytes that interact with merozoite ligands that are probably already accessible during pre-incubation prior to invasion.     

Seroprevalence of equine babesiosis in the Black Sea region of Turkey

The prevalence of Theileria equi and Babesia caballi was determined in equid blood samples in five provinces of the Black Sea region of Turkey by using the indirect fluorescent antibody test (IFAT). Of 153 samples, 53 (34.6%) and 33 (21.5%) were seropositive to B. caballi and T. equi, respectively. In addition, 8 (5.2%) of samples were seropositive to both T. equi and B. caballi. Anti T. equi and B. caballi antibodies were detected in all five regions. The prevalence of B. caballi was higher than T. equi in all counties. Antibodies to T. equi and B. caballi were detected in horses of all ages, and there were no significant differences among age groups. Out of 84 horses, 32 (38.0%) were positive for B. caballi infection and 20 (23.8%) were positive for T. equi infection. Five horses (5.6%) were found to be seropositive to both B. caballi and T. equi. Of 38 donkeys, 14 (36.8%) were found to be positive for B. caballi infection and 5 (13.1%) positive for T. equi infection. In addition, 2 (5.2%) samples were seropositive for both T. equi and B. caballi infections. Out of 31 mules, 8 (25.8%) were positive for B. caballi infection and 8 (25 8%) positive for T. equi infection. One (3.2%) sample was seropositive for both T. equi and B. caballi infections. Of all the animals in this study, only 3 horses were infected by Rhipicephalus turanicus and Hyalomma detritum, and no haemoparasites were detected by microscopic examination.     

Isolation, initial characterization and serological studies of glycolipid antigens from Babesia bovis-infected erythrocytes

Serological analysis of Babesia bovis-derived glycolipids by ELISA and the indirect fluorescent antibody technique demonstrated the existence of their antigenic and immunogenic activities not only in B. bovis but also in B. bigemina infections. This indicates that serological cross-reactivity of B. bovis and B. bigemina relates to glycolipids. The negative ELISA reaction obtained with Anaplasma marginale antisera suggested the specificity of the reaction to the genus Babesia. Fractionation of these glycolipids by Florisil Sep-Pak column chromatography with subsequent HPTLC immunostaining and Orcinol staining suggested the presence of carbohydrate antigenic determinants in B. bovis glycolipids.     

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.     

Ability of p53 and the adenovirus E1b 58-kilodalton protein to form a complex is determined by p53.

We have investigated p53-E1b 58-kilodalton (kDa) protein complex formation during permissive and semipermissive infections with adenovirus type 5 (Ad5) dl309. While metabolic labeling studies easily detected p53-E1b 58-kDa protein complexes in transformed rat cells (XhoI-C), the same methods have not revealed complexes during infection of either human osteosarcoma cells (permissive) or normal rat kidney cells (semipermissive). Complexes were not detectable at any stage during the replicative cycle of Ad5 dl309 in osteosarcoma cells, and they could not be stabilized by using an in vivo cross-linking agent. In addition, using the E4-defective mutant Ad5 dl355, no complexes were observed either. Thus, the lack of p53-E1b 58-kDa protein complex formation during infection is not due to competition from the E4 34-kDa protein. In vitro association experiments showed that in vitro-translated mouse and human p53 could form complexes with E1b 58-kDa antigen expressed during infection. Thus, such E1b proteins are competent to form complexes. The converse experiment, in which in vitro-translated E1b 58-kDa protein was mixed with lysates of osteosarcoma cells, showed little or no p53-E1b 58-kDa protein association, even though the in vitro E1b 58-kDa protein could associate stably with p53 from cells containing endogenous p53-E1b 58-kDa protein complex. These data suggest that competence to form p53-E1b 58-kDa protein complexes resides in some property of p53.     

Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro-complexes.

A collection of chaperonin containing TCP1 (CCT) micro-complexes that are comprised of subsets of the constitutively expressed CCT subunits have been identified. These CCT micro-complexes have mol. wts ranging from 120 to 250 kDa and are present in cells at lower abundance (<5%) as compared with intact CCT. Biochemical characterization of these microcomplexes has shown that several are comprised of two different types of CCT subunit. Furthermore, it was observed that each subunit associates with only one or two other different types of subunit, suggesting that each subunit has fixed partners. This observation, together with CCT gene counting being concordant with the 8-fold structural symmetry, is consistent with predictions derived from analysis of the primary structures of these subunits concerning inter-subunit interactions, and implies a unique topology of the subunits constituting the torodial ring in CCT. The series of subunit-subunit association patterns determined from CCT micro-complexes has provided information to infer, from the 5040 (7!factorial) combinatorial possibilities, one probable subunit orientation within the torodial ring.     

Bovine babesiosis: Partial purification and characterization of blood culture-derived Babesia bovis

Morphologic, biologic and immunologic properties of corpuscular and soluble fractions of Babesia bovis purified from in vitro blood cultures were studied. Supernatant fluids obtained during routine medium exchange were studied. Supernatant fluids obtained during routine medium exchange were submitted to differential centrifugation to separate soluble and corpuscular babesial antigens from erythrocyte stroma. Extracellular babesiae were sedimented with infected and uninfected erythrocyte ghosts. The majority of babesiae were found in erythrocyte ghosts. Clumps of extracellular parasites were sometimes formed in vitro and generally could not be separated from uninfected erythrocytes. Centrifugation over a discontinuous Ficoll density gradient did not improve separations. Parasites remained viable throughout the purification procedure but were killed by freezing and rapid thawing. Both corpuscular and soluble antigen fractions elicited the production of specific anti-babesial antibodies when injected into calves. Electron microscopy of corpuscular antigen revealed the presence of intra- and extraerythrocytic babesial merozoites. A surface coat was visible loosely adhering to the plasma membrane of the parasites. Parasite suspensions and cell-free supernatant fluids obtained from in vitro cultures of B. bovis should provide a variety of unique antigens for further in vitro and in vivo studies.     

Comparison between aseric and seric culture-derived exoantigens of Babesia divergens in their ability to induce immunoprotection in gerbils

Babesia divergens Rouen 1987 was cultivated with a high percentage of parasitized erythrocytes (30–40%) in either RPMI 1640 supplemented by 10% human serum or in a serum-free medium consisting of RPMI 1640 supplemented with 5 g/l Albumax I®. Analysis of serum and Albumax culture supernatants, using polyacrylamide gel electrophoresis, revealed the presence of at least 10 parasitic exoantigens of B. divergens with molecular weights ranging between 27 and 200 kDa. The gerbils were injected twice, at 3-week intervals, with Albumax culture supernatants or seric culture supernatants. The vaccine doses ranged from 3 μl to 1.5 ml. The highest immunofluorescent antibody titers in gerbils (in 42 days) were obtained using Albumax supernatant and Quil A saponin as adjuvant. Analysis of the gerbil humoral response by immunoprecipitation showed that only three exoantigens were immunodominant: 92 kDa, 50 kDa and 37 kDa proteins. The gerbils were challenged 3 weeks after the last vaccine injection and the maximum protection was observed with vaccine doses ranging from 30 μl to 1.5 ml of culture supernatant and Quil A adjuvant. Albumax medium-derived antigens potentiated better protection at lower dose rates than that of serous medium-derived antigens (for example the gerbil mortality was 0% when they are immunized with 30 μl of Albumax supernatant and 100% with 30 μl of seric supernatant).     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.     

Poly-γ-glutamate depolymerase of Bacillus subtilis: production, simple purification and substrate selectivity

The Bacillus subtilis pgdS gene, which is located at the immediate downstream of the pgs operon for poly-γ-glutamate (PGA) biosynthesis, encodes a PGA depolymerase. The pgdS gene product shows the structural feature of a membrane-associated protein. The mature form of the gene product, identified as a B. subtilis extracellular protein, was produced in Escherichia coli clone cells. Since the mature PGA depolymerase has been modified with the histidine-tag at its C-terminus, it could be simply purified by metal-chelating affinity chromatography. This purified enzyme digested PGAs from B. subtilis ( -glutamate content, 70%) and from Bacillus megaterium (30%) in an endopeptidase-like fashion. In contrast, PGA from Natrialba aegyptiaca, which consists only of -glutamate, was resistant to the enzyme, suggesting that, unlike fungal PGA endo-depolymerases, the bacterial enzyme recognizes the -glutamate unit in PGA.     

Testing the neutral fixation of hetero-oligomerism in the archaeal chaperonin CCT

The evolutionary transition from homo-oligomerism to hetero-oligomerism in multimeric proteins and its contribution to function innovation and organism complexity remain to be investigated. Here, we undertake the challenge of contributing to this theoretical ground by investigating the hetero-oligomerism in the molecular chaperonin cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) from archaea. CCT is amenable to this study because, in contrast to eukaryotic CCTs where sub-functionalization after gene duplication has been taken to completion, archaeal CCTs present no evidence for subunit functional specialization. Our analyses yield additional information to previous reports on archaeal CCT paralogy by identifying new duplication events. Analyses of selective constraints show that amino acid sites from 1 subunit have fixed slightly deleterious mutations at inter-subunit interfaces after gene duplication. These mutations have been followed by compensatory mutations in nearby regions of the same subunit and in the interface contact regions of its paralogous subunit. The strong selective constraints in these regions after speciation support the evolutionary entrapment of CCTs as hetero-oligomers. In addition, our results unveil different evolutionary dynamics depending on the degree of CCT hetero-oligomerism. Archaeal CCT protein complexes comprising 3 distinct classes of subunits present 2 evolutionary processes. First, slightly deleterious and compensatory mutations were fixed neutrally at inter-subunit regions. Second, sub-functionalization may have occurred at substrate-binding and adenosine triphosphate-binding regions after the 2nd gene duplication event took place. CCTs with 2 distinct types of subunits did not present evidence of sub-functionalization. Our results provide the 1st in silico evidence for the neutral fixation of hetero-oligomerism in archaeal CCTs and provide information on the evolution of hetero-oligomerism toward sub-functionalization in archaeal CCTs.     

Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein

DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.