Molecular evolution of a central region containing B cell epitopes in the gene encoding the p67 sporozoite antigen within a field population of Theileria parva.

Protective immunity induced by the infective sporozoite stage of Theileria parva indicates a potential role for antibodies directed against conserved serologically reactive regions of the major sporozoite surface antigen p67 in vaccination to control the parasite. We have examined the allelic variation and determined the extent of B cell epitope polymorphism of the gene encoding p67 among field isolates originating from cattle exposed to infected ticks in the Marula area of the rift valley in central Kenya where the African cape buffalo (Syncerus caffer) and cattle co-graze. In the first of two closely juxtaposed epitope sequences in the central region of the p67 protein, an in-frame deletion of a seven-amino acid segment results in a truncation that was observed in parasites derived from cattle that co-grazed with buffalo. In contrast, the variation in the second epitope was primarily due to nonsynonymous substitutions, resulting in relatively low overall amino acid conservation in this segment of the protein. We also observed polymorphism in the region of the protein adjacent to the two defined epitopes, but this was not sufficient to provide statistically significant evidence for positive selection. The data indicates that B cell epitopes previously identified within the p67 gene are polymorphic within the Marula field isolates. Given the complete sequence identity of the p67 gene in all previously characterized T. parva isolates that are transmissible between cattle by ticks, the diversity observed in p67 from the Marula isolates in combination with the clinical reaction of the infected cattle is consistent with them originating from ticks that had acquired T. parva from buffalo.

High-resolution genotyping and mapping of recombination and gene conversion in the protozoan Theileria parva using whole genome sequencing.

BACKGROUND: Theileria parva is a tick-borne protozoan parasite, which causes East Coast Fever, a disease of cattle in sub-Saharan Africa. Like Plasmodium falciparum, the parasite undergoes a transient diploid life-cycle stage in the gut of the arthropod vector, which involves an obligate sexual cycle. As assessed using low-resolution VNTR markers, the crossover (CO) rate in T. parva is relatively high and has been reported to vary across different regions of the genome; non-crossovers (NCOs) and CO-associated gene conversions have not yet been characterised due to the lack of informative markers. To examine all recombination events at high marker resolution, we sequenced the haploid genomes of two parental strains, and two recombinant clones derived from ticks fed on cattle that had been simultaneously co-infected with two different parasite isolates. RESULTS: By comparing the genome sequences, we were able to genotype over 64 thousand SNP markers with an average spacing of 127 bp in the two progeny clones. Previously unrecognized COs in sub-telomeric regions were detected. About 50% of CO breakpoints were accompanied by gene conversion events. Such a high fraction of COs accompanied by gene conversions demonstrated the contributions of meiotic recombination to the diversity and evolutionary success of T. parva, as the process not only redistributed existing genetic variations, but also altered allelic frequencies. Compared to COs, NCOs were more frequently observed and more uniformly distributed across the genome. In both progeny clones, genomic regions with more SNP markers had a reduced frequency of COs or NCOs, suggesting that the sequence divergence between the parental strains was high enough to adversely affect recombination frequencies. Intra-species polymorphism analysis identified 81 loci as likely to be under selection in the sequenced genomes. CONCLUSIONS: Using whole genome sequencing of two recombinant clones and their parents, we generated maps of COs, NCOs, and CO-associated gene conversion events for T. parva. The data comprises one of the highest-resolution genome-wide analyses of the multiple outcomes of meiotic recombination for this pathogen. The study also demonstrates the usefulness of high throughput sequencing typing for detailed analysis of recombination in organisms in which conventional genetic analysis is technically difficult.

Multilocus genotyping of Theileria parva isolates associated with a live vaccination trial in Kenya provides evidence for transmission of immunizing parasites into local tick and cattle populations.

The live infection and treatment (ITM) vaccination procedure using the trivalent Muguga cocktail is increasingly being used to control East Coast fever, with potential implications for Theileria parva population genetic structure in the field. Transmission of the Kiambu V T. parva component to unvaccinated cattle has previously been described in Uganda. We monitored the T. parva carrier state in vaccinated and control animals on a farm in West Kenya where an ITM stabilate derived from the Kenyan T. parva Marikebuni stock was evaluated for field efficacy. A nested PCR-based Marikebuni-specific marker identified a carrier state in nine of ten vaccinated animals, detectable for a period of two years. We used 22 variable number tandem repeat (VNTR) markers to determine multilocus genotypes (MLGs) of 19 T. parva schizont-infected lymphocyte isolates derived from cattle and field ticks. Two isolates from unimmunized cattle were identical to the Marikebuni vaccination stock. Two cattle isolates were identical to a Muguga cocktail component Kiambu V. Seven isolates from ticks exhibited MLGs that were identical to the Serengeti/Muguga vaccine stocks. Six cattle and two tick-derived stocks exhibited unique MLGs. The data strongly suggest transmission of immunizing genotypes, from Marikebuni vaccine-induced carrier cattle to unimmunized cattle. It is possible that genotypes similar to those in the Muguga cocktail are present in the field in Western Kenya. An alternative hypothesis is that these parasites may have originated from vaccine trial sites in Eastern Uganda. If correct, this suggests that T. parva stocks used for immunization can potentially be disseminated 125 km beyond the immediate vaccination site. Regardless of their origin, the data provide evidence that genotypes similar to those in the Muguga cocktail are circulating in the field in East Africa, alleviating concerns about dissemination of 'alien' T. parva germplasm through live vaccination.

The ring of the rhodopsin chromophore in a hydrophobic activation switch within the binding pocket.

The current view that the beta-ionone ring of the rhodopsin chromophore vacates its binding pocket within the protein early in the photocascade has been adopted in efforts to provide structural models of photoreceptor activation. This event casts doubt on the ability of this covalently bonded ligand to participate directly in later stages involving activation of the photoreceptor and it is difficult to translate into predictions for the activation of related G protein-coupled receptors by diffusable ligands (e.g. neurotransmitters). The binding pocket fixes the formally equivalent pair of ring methyl groups (C16/C17) in different orientations that can be distinguished easily by (13)C NMR. Solid-state NMR observations on C16 and C17 are reported here that show instead that the ring is retained with strong selective interactions within the binding site into the activated state. We further show how increased steric interactions for this segment in the activated receptor can be explained by adjustment in the protein structure around the ring whilst it remains in its original location. This describes a plausible role for the ring in operating a hydrophobic switch from within the aromatic cluster of helix 6 of rhodopsin, which is coupled to electronic changes within the receptor through water-mediated, hydrogen-bonded networks between the conserved residues in G protein-coupled receptors.

Molecular characterization of live Theileria parva sporozoite vaccine stabilates reveals extensive genotypic diversity.

The current Infection and Treatment Method of vaccination against East Coast fever comprises an inoculation of live Theileria parva sporozoites and simultaneous administration of oxytetracycline. Immunization with a combination of parasite types has been shown to provide broader protection than inoculation of individual strains. In this study, we used a high-throughput capillary electrophoresis system to determine the genotypic composition of the Muguga Cocktail, a widely used vaccine stabilate derived from three seed stabilates-Muguga, Serengeti-transformed and Kiambu 5. Five satellite markers were used to genotype the vaccine and reference stabilates from two commercial-scale preparations of the vaccine. In addition, 224 cloned cell lines established by infection of bovine lymphocytes with T. parva parasites from the component stabilates were genotyped. The results indicate that, for the recently prepared batch, there are at least eight genotypes in each of the Muguga and the Serengeti-transformed stabilates, while parasites from the Kiambu 5 stabilate showed no diversity at the five loci. The Serengeti-transformed stabilate contained parasites of the Kiambu 5 genotype and of two genotypes present in the Muguga stabilate, whereas there were no genotypes common to the Muguga and Kiambu 5 stabilates. When stabilates from the two vaccine batches were compared, no allelic variations were identified between the Muguga and Kiambu 5 parasites, while lack of sufficient clones prevented a full comparison of the Serengeti-transformed stabilates. The findings will facilitate examination of the extent to which the vaccine strains become resident in areas under vaccination, the identification of 'breakthrough' strains and the establishment of the quality assurance protocols to detect variations in the production of the vaccine. The cloned cell lines will be useful for further understanding the antigenic diversity of parasites in the vaccine.

Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity.

BACKGROUND: Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection. METHODOLOGY/PRINCIPAL FINDINGS: Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. CONCLUSIONS/SIGNIFICANCE: The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.

Subunit vaccine based on the p67 major surface protein of Theileria parva sporozoites reduces severity of infection derived from field tick challenge.

Two recombinant vaccines against Theileriaparva, based on a near full-length version of the sporozoite surface antigen p67 (p67(635)), or an 80 amino acid C-terminal section (p67C), were evaluated by exposure of immunized cattle to natural tick challenge in two sites at the Kenya Coast and one in Central Kenya. Vaccination reduced severe ECF by 47% at the coast and by 52% in central Kenya from an average incidence of 0.53+/-0.07 (S.E.) in 50 non-immunised controls to an average of 0.27+/-0.05 in 83 immunised animals. The reduction in severe East Coast fever was similar to that observed in laboratory experiments with p67(635) and p67C. The p67 coding sequence from thirteen T. parva field isolates including seven from vaccinated cattle that were not protected, was 100% identical to the gene on which the recombinant vaccine is based, suggesting a predominantly homologous p67 antigenic challenge. The same parasite isolates were however genetically heterogeneous at several loci other than p67.

Relative orientation between the beta-ionone ring and the polyene chain for the chromophore of rhodopsin in native membranes.

Rotational resonance solid state nuclear magnetic resonance has been used to determine the relative orientation of the beta-ionone ring and the polyene chain of the chromophore 11-Z-retinylidene of rhodopsin in rod outer segment membranes from bovine retina. The bleached protein was regenerated with either 11-Z-[8,18-(13)C(2)]retinal or 11-Z-[8,16/17(13)C(2)]retinal, the latter having only one (13)C label at either of the chemically equivalent positions 16 and 17. Observation of (13)C selectively enriched in the ring methyl groups, C16/17, revealed alternative conformational states for the ring. Minor spectral components comprised around 26% of the chromophore. The major conformation (approximately 74%) has the chemical shift resolution required for measuring internuclear distances to (13)C in the retinal chain (C8) separately from each of these methyl groups. The resulting distance constraints, C8 to C16 and C17 (4.05 +/- 0.25 A) and from C8 to C18 (2.95 +/- 0.15 A), show that the major portion of retinylidene in rhodopsin has a twisted 6-s-cis conformation. The more precise distance measurement made here between C8 and C18 (2.95 A) predicts that the chain is twisted out-of-plane with respect to the ring by a modest amount (C5-C6-C7-C8 torsion angle = -28 +/- 7 degrees ).

Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate.

High-resolution solid-state NMR methods have been used to analyze the conformation of the chromophore in the late photointermediate metarhodopsin-I, from observation of (13)C nuclei introduced into the beta-ionone ring (at the C16, C17, and C18 methyl groups) and into the adjoining segment of the polyene chain (at C8). Bovine rhodopsin in its native membrane was also regenerated with retinal that was (13)C-labeled close to the 11-Z bond (C20 methyl group) to provide a reporter for optimizing and quantifying the photoconversion to metarhodopsin-I. Indirect photoconversion via the primary intermediate, bathorhodopin, was adopted as the preferred method since approximately 44% conversion to the metarhodopsin-I component could be achieved, with only low levels (approximately 18%) of ground-state rhodopsin remaining. The additional photoproduct, isorhodopsin, was resolved in (13)C spectra from C8 in the chain, at levels of approximately 38%, and was shown using rotational resonance NMR to adopt the 6-s-cis conformation between the ring and the polyene chain. The C8 resonance was not shifted in the metarhodopsin-I spectral component but was strongly broadened, revealing that the local conformation had become less well defined in this segment of the chain. This line broadening slowed rotational resonance exchange with the C17 and C18 ring methyl groups but was accounted for to show that, despite the chain being more relaxed in metarhodopsin-I, its average conformation with respect to the ring was similar to that in the ground state protein. Conformational restraints are also retained for the C16 and C17 methyl groups on photoactivation, which, together with the largely preserved conformation in the chain, argues convincingly that the ring remains with strong contacts in its binding pocket prior to activation of the receptor. Previous conclusions based on photocrosslinking studies are considered in view of the current findings.