Intranasal vaccination with protein bodies elicit strong protection against Streptococcus pneumoniae colonization.

Protein bodies (PBs) are particles consisting of insoluble, aggregated proteins with potential as a vaccine formulation. PBs can contain high concentrations of antigen, are stable and relatively resistant to proteases, release antigen slowly and are cost-effective to manufacture. Yet, the capacity of PBs to provoke immune responses and protection in the upper respiratory tract, a major entry route of respiratory pathogens, is largely unknown. In this study, we vaccinated mice intranasally with PBs comprising antigens from Streptococcus pneumoniae and evaluated the level of protection against nasopharyngeal colonization. PBs composed of the α-helical domain of pneumococcal surface protein A (PspAα) provided superior protection against colonization with S. pneumoniae compared to soluble PspAα. Immunization with soluble protein or PBs induced differences in antibody binding to pneumococci as well as a highly distinct antigen-specific nasal cytokine profile upon in vivo stimulation with inactivated S. pneumoniae. Moreover, immunization with PBs composed of conserved putative pneumococcal antigens reduced colonization by S. pneumoniae in mice, both as a single- and as a multi-antigen formulation. In conclusion, PBs represent a vaccine formulation that elicits strong mucosal immune responses and protection. The versatility of this platform offers opportunities for development of next-generation vaccine formulations.

Candidate gene analysis of anthocyanin pigmentation loci in the Solanaceae.

Crop species in the Solanaceae, which includes tomato ( Lycopersicon esculentum), potato ( Solanum tuberosum), pepper ( Capsicum spp.), and eggplant ( S. melongena), exhibit natural variation in the types, levels, and tissue-specific expression patterns of anthocyanin pigments. While the identities of the genes underpinning natural variation in anthocyanin traits in these crops are largely unknown, many structural genes and regulators of anthocyanin biosynthesis have been isolated from the solanaceous ornamental species Petunia. To identify candidate genes that may correspond to loci controlling natural variation in the four crops, 13 anthocyanin-related genes were localized on a tomato F(2) genetic map. Gene map positions were then compared to mapped mutants in tomato and through comparative genetic maps to natural variants in potato, eggplant, and pepper. Similar map positions suggest that the tomato mutants anthocyaninless, entirely anthocyaninless, and anthocyanin gainer correspond to flavonoid 3'5'-hydroxylase ( f3'5'h), anthocyanidin synthase, and the Petunia Myb domain trancriptional regulatory gene an2, respectively. Similarly potato R, required for the production of red pelargonidin-based pigments, P, required for production of purple delphinidin-based pigments, and I, required for tissue-specific expression in tuber skin, appear to correspond to dihydroflavonol 4-reductase, f3'5'h and an2, respectively. The map location of an2 also overlaps pepper A and eggplant fap10.1, lla10.1, lra10.1, sa10.1, pa10.1 and ca10.1, suggesting that a homologous regulatory locus has been subjected to parallel selection in the domestication of many solanaceous crops. To test the hypothesis that tomato anthocyaninless corresponds to f3'5'h, a portion of the gene was sequenced. A premature stop codon was observed in an anthocyaninless mutant, but not in wild-type.

A fluorogenic 5' nuclease (TaqMan) assay to assess dosage of a marker tightly linked to red skin color in autotetraploid potato.

We have recently identified an allele of dihydroflavonol 4-reductase ( dfr) that cosegregates with the ability of potato ( Solanum tuberosum L) to produce red pelargonidin-based anthocyanin pigments. A rapid assay to assess dosage of this allele in cultivated potato, an autotetraploid, would be useful for breeding programs that develop red-skinned cultivars. To identify regions of dfr that are conserved between alleles, as well as regions that are variable, a portion of the gene was sequenced from several cultivated and wild potato clones. In one region the sequence of the 'red' dfr allele differed at two nucleotide positions from the three other sequence classes observed. A fluorogenic oligonucleotide probe labeled with 6-FAM was designed to anneal specifically to the red allele in this region, while a second probe labeled with VIC was designed to anneal to the 'not-red' dfr alleles. PCR primers that annealed to conserved sequences flanking the variable region were also developed. When subjected to a fluorogenic 5' nuclease (TaqMan) allelic discrimination assay all diploid clones tested clustered into three distinct groups based on the relative amounts of FAM and VIC released. These three groups represented clones homozygous for the red allele, heterozygous for the red allele, and homozygous for the not-red allele(s). When tetraploid clones were tested they separated into five distinct clusters, three of which were shared with diploid clones. The five clusters were interpreted to represent clones quadruplex, triplex, duplex, simplex and nulliplex for the red dfr allele. This interpretation was supported by monitoring the segregation of red allele dosage in several tetraploid crosses. To the best of our knowledge this is the first report of a fluorogenic 5' nuclease assay being used for allelic discrimination in an autopolyploid.

An allele of dihydroflavonol 4-reductase associated with the ability to produce red anthocyanin pigments in potato (Solanum tuberosum L.).

The potato R locus is necessary for the production of red pelargonidin-based anthocyanin pigments in any tissue of the plant, including tuber skin and flower petals. The production of pelargonidins in plants requires the activity of dihydroflavonol 4-reductase (DFR) to catalyze the reduction of dihydrokaempferol into leucopelargonidin. To test the hypothesis that potato R encodes DFR, portions of both dfr alleles were sequenced from a diploid potato clone known to be heterozygous Rr. Sequence comparison revealed a polymorphic BamHI restriction site. The presence or absence of this site was monitored in three diploid populations that segregated for R, as well as in a wide range of tetraploid breeding clones and cultivars, by amplifying a fragment of dfr and digesting the products with BamHI. An identically sized dfr restriction fragment lacking the BamHI site was present in all potato clones that produced red anthocyanin pigments, while the same fragment was absent in many potato clones with white tuber skin and flowers. An independent RFLP test using DraI to detect sequence polymorphism was performed on a subset of the potato clones. This test also revealed dfr-derived bands that were present in all red-colored potatoes and absent in several white clones. The presence of shared restriction fragments in all red-colored potatoes provides strong evidence that R does indeed code for DFR. The data are also consistent with a 48 year-old hypothesis by Dodds and Long, that R was selected just once during the domestication of potato. A cDNA clone corresponding to the red allele of dfr was sequenced and compared to two other alleles. The red allele is predicted to encode a 382 amino acid protein that differs at ten amino acid positions from the gene products of the two alternative alleles. Several of these differences map in a region known to influence DFR substrate specificity in Gerbera.

Capsular polysaccharide typing of domestic mastitis-causing Staphylococcus aureus strains and its potential exploration of bovine mastitis vaccine development. I. Capsular polysaccharide typing, isolation and purification of the strains.

One hundred seven isolates of Staphylococcus aureus from bovine mastitis were investigated for colony morphology in serum-soft agar (SSA), autoagglutination in salt, and capsular serotype. Capsular polysaccharide (CP) was purified and quantified from the extracts of clinical isolates. Overall, 89 isolates (83.2%) were diffuse in the SSA, without any difference in the proportion of diffuse colony between type 5 and type 8 strains. Some strains exhibited compact colonies in the SSA and expressed CP as determined by an enzyme-linked immunosorbent assay, indicating that compact morphology does not exclude encapsulation. The majority of the strains (11/12) showed autoagglutination in the salt aggregation test. The serotype 336 accounted for 46.7% of the isolates followed by serotype 5 (12.1%) and serotype 8 (12.1%). Particularly, twenty-six (24.3%) isolates reacted with two serotypes; 7 for type 8/336 and 19 for type 5/336. Five isolates (4.7%) were nontypeable with monoclonal antibodies specific for CP serotype 5, 8, or 336. The CP concentration in culture supernatants varied with the serotypes, and the total amount of CP produced by cells grown in a liquid medium was much less than that produced by cells grown on a solid medium. The Western blotting indicated that the CP bands of S. aureus serotype 5 and 8 were ranged in the molecular mass of 58-84 kilodalton (kDa), with additional bands in the region of approximately >/= 48 or