Immunogenicity of a Staphylococcus aureus-cholera toxin A2/B vaccine for bovine mastitis.

Staphylococcus aureus causes a chronic, contagious disease of the udder, or mastitis, in dairy cows. This infection is often refractory to antibiotic treatment, and has a significant economic impact on milk production worldwide. An effective vaccine to prevent S. aureus mastitis would improve animal health, reduce antibiotic dependence and inform human vaccine approaches. The iron-regulated surface determinant A (IsdA) and clumping factor A (ClfA) are conserved S. aureus extracellular-matrix adhesins and target vaccine antigens. Here we report the results of two bovine immunogenicity trials using purified IsdA and ClfA-cholera toxin A2/B chimeras (IsdA-CTA2/B and ClfA-CTA2/B). Cows were intranasally inoculated with IsdA-CTA2/B + ClfA-CTA2/B at dry off and followed for 70 days. Trial 1 utilized three groups with one or two booster doses at a total concentration of 600 or 900 µg. Trial 2 utilized two groups with one booster at a total concentration of 1200 µg. Humoral immune responses in serum and milk were examined by ELISA. Responses in serum were significant between groups and provide evidence of antigen-specific IgG induction after vaccination in both trials. Cellular proliferation was detected by flow cytometry using antigen-stimulated PBMCs from day 60 of Trial 2 and revealed an increase in CD4+ T cells from vaccinated cows. IsdA and ClfA stimulation induced IL-4 expression, but not IFN-γ or IL-17, in PBMCs from day 60 as determined by cytokine expression analysis. Opsonophagocytosis of S. aureus confirmed the functional in vitro activity of anti-IsdA antibodies from Trial 2 serum and milk. The vaccine was well tolerated and safe, and results support the potential of mucosally-delivered CTA2/B chimeras to protect cows from mastitis caused by S. aureus.

Immunoproteomics to identify Staphylococcus aureus antigens expressed in bovine milk during mastitis.

Staphylococcus aureus is an opportunistic pathogen affecting both human and animal species. An effective vaccine to prevent S. aureus bovine disease and transmission would have positive effects on animal well-being, food production, and human health. The objective of this study was to identify multiple antigens that are immunoreactive during udder colonization and disease for exploration as vaccine antigens to prevent bovine mastitis. Staphylococcus aureus produces several cell wall-anchored and surface-associated virulence factors that play key roles in the pathogenesis of mastitis. Many of these proteins are conserved between different strains of S. aureus and represent promising vaccine candidates. We used an immunoproteomics approach to identify antigenic proteins from the surface of S. aureus. The expression of cell wall and surface proteins from S. aureus was induced under low iron conditions, followed by trypsin extraction and separation by 2-dimensional electrophoresis. The separated proteins were blotted with antibodies from mastitic bovine milk and identified by liquid chromatography-mass spectrometry. Thirty-eight unique proteins were identified, of which 8 were predicted to be surface exposed and involved in S. aureus virulence. Two surface proteins, iron-regulated surface determinant protein C (IsdC) and ESAT-6 secretion system extracellular protein (EsxA), were cloned, expressed, and purified from Escherichia coli for confirmation of immune reactivity by ELISA. A PCR of 37 bovine S. aureus isolates indicated that the presence of esxA and isdC is conserved, and amino acid alignments revealed that IsdC and EsxA sequences are highly conserved. The immunoproteomics technique used in this study generated reproducible results and identified surface exposed and reactive antigens for further characterization.

Control of FimY translation and type 1 fimbrial production by the arginine tRNA encoded by fimU in Salmonella enterica serovar Typhimurium.

Expression of type 1 fimbriae in Salmonella enterica serovar Typhimurium undergoes phase variation or alteration between a fimbriate and a non-fimbriate phenotype. This variation is known to be dependent upon environmental conditions in vitro and is thought to be a complex process involving regulation by a number of proteins. The regulatory genes located within the fim cluster include fimZ, fimY and fimW. A fourth gene of the cluster, fimU, encodes a tRNA molecule specific for rare arginine codons. We have shown previously that fimU affects the expression of S. typhimurium type 1 fimbriae, and that fimU is functionally related to the Escherichia coli gene argU. A high frequency of rare arginine codons was found within the three fim regulatory genes, and five of these codons were clustered within fimY alone. To investigate the affects of fimU on FimY production, a FimY fusion with the E. coli maltose-binding protein was constructed and expressed in an E. coli argU background. Western blots of extracts from the argU mutant and parental strain indicated that production of FimY was significantly reduced in the absence of a functional tRNAArg(UCU). FimY production in this mutant could be restored to high levels when fimU was introduced on a plasmid, and also when three rare arginine codons, located within the first 14 positions within fimY, were exchanged for major arginine codons. A Tn10 insertion from a Salmonella enteritidis fimU mutant was transduced into S. typhimurium, and this strain was analysed for the expression of type 1 fimbriae. The resulting S. typhimurium fimU mutant was found to be non-fimbriate under all conditions tested and could be complemented by the introduction of fimU alone on a plasmid. In addition, this mutant could be complemented by transformation with fimY altered in the first three rare arginine codons. Reverse transcriptase-polymerase chain reaction confirmed that the fimY transcript was present at similar levels in the fimU mutant and parental strain. These results indicated that the observed inhibition of protein expression was not occurring at the transcriptional level. Analysis of expression of the malEfimY fusion in the S. typhimurium fimU mutant and parental strain confirmed the data observed in E. coli. In contrast, a FimW fusion was found to be produced at similar levels in both the fimU mutant and the parental strain. Together, these data indicate that the absence of a functional fimU results in the inhibition of efficient FimY translation, and thus type 1 fimbrial production in S. typhimurium.

FimW is a negative regulator affecting type 1 fimbrial expression in Salmonella enterica serovar typhimurium.

Type 1 fimbriae are proteinaceous surface appendages that carry adhesins specific for mannosylated glycoproteins. These fimbriae are found on most members of the family Enterobacteriaceae and are known to facilitate binding to a variety of eukaryotic cells, including those found on the mucosal surfaces of the alimentary tract. We have shown that the regulation of type 1 fimbrial expression in Salmonella enterica serovar Typhimurium is controlled, in part, by the products of four genes found within the fim gene cluster: fimZ, fimY, fimW, and fimU. To better understand the specific role of FimW in fimbrial expression, a mutation was constructed in this gene by the insertion of a kanamycin resistance DNA cassette into the chromosome. The resulting fimW mutation was characterized by mannose-sensitive hemagglutination and agglutination with fimbria-specific antiserum. Assays suggested that this mutant was more strongly fimbriate than the parental strain, exhibiting a four- to eightfold increase in fimbrial production. The fimW mutation was introduced into a second strain of Salmonella enterica serovar Typhimurium, and this mutant was also found to be strongly fimbriate compared to the parental strain. Consistent with the role of this protein as a negative regulator, fimA-lacZ expression in serovar Typhimurium, as well as in Escherichia coli, was increased twofold in the absence of functional FimW. Primer extension analysis determined that fimW transcription is initiated from its own promoter 31 bp upstream of the translation start site. Analysis using a fimW-lacZ reporter indicated that fimW expression in serovar Typhimurium was increased under conditions that select for poorly fimbriate bacteria and low fimA expression. FimW also appears to act as an autoregulator, since expression from the fimW-lacZ reporter was increased in a fimW mutant. FimW was partially purified by fusion with the E. coli maltose-binding protein. Use of this FimW protein extract, as well as others, in DNA-binding assays was unable to identify a specific binding site for FimW in the fimA, fimZ, fimY, or fimW promoter regions. To analyze protein-protein interactions, FimW was expressed in a LexA-based two-hybrid system in E. coli. A significant interaction between FimW and the DNA-binding activator protein, FimZ, was detected using this system. These results indicate that FimW is a negative regulator of serovar Typhimurium type 1 fimbrial expression and may function by interfering with FimZ-mediated activation of fimA expression.

Characterization of FimY as a coactivator of type 1 fimbrial expression in Salmonella enterica serovar Typhimurium.

Type 1 fimbriae of Salmonella enterica serovar Typhimurium are surface appendages that carry adhesins specific for mannosylated host glycoconjugates. Regulation of the major fimbrial subunit is thought to be controlled by a number of ancillary fim genes, including fimZ, fimY, fimW, and fimU. Previous studies using a FimZ mutant have indicated that this protein is necessary for fimA expression, and in vitro DNA binding assays determined that FimZ is a transcriptional activator that binds directly to the fimA promoter. To determine the role of FimY as a potential regulator of fimbrial expression, a fimY mutant of serovar Typhimurium was generated by allelic exchange. This mutant was found to be phenotypically nonfimbriate. No transcription from the fimA promoter was detected in a fimY mutant containing a fimA-lacZ reporter construct located on the chromosome. In addition, transcription from the cloned fimY promoter was not detected in Escherichia coli unless both FimZ and FimY were present, indicating that these proteins also act as coactivators of fimY expression. Consistent with these results, there is no transcription from a fimY-lacZ reporter construct within a serovar Typhimurium fimY or fimZ mutant. Studies using the fimY-lacZ construct reveal that expression of this gene varies with environmental conditions in a manner similar to fimA expression. Extensive in vitro DNA binding assays using extracts from E. coli that overexpress FimY, as well as partially purified FimY, were unable to identify a specific interaction between FimY and the fimA or fimY promoter. The results indicate that FimY is a positive regulator of fimbrial expression and that this protein acts in cooperation with FimZ to regulate the expression of Salmonella type 1 fimbrial appendages.