Identification of protective protein antigens for vaccination against Streptococcus dysgalactiae in cobia (Rachycentron canadum).

Streptococcus dysgalactiae is considered a causative agent of severe infection and economic loss for the cobia industry in Taiwan. In this study, protective antigens of this pathogenic bacterium were identified and screened in cobia (Rachycentron canadum). Outer surface proteins (OMPs) of this pathogen were extracted using mutanolysin digestion. Immunogenic targets were detected by western blot and then subjected to peptide sequencing using NanoLC-MS/MS. Two surface proteins, namely phosphoenolpyruvate protein phosphotransferase (PtsA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showed strong reactions with cobia antisera against S. dysgalactiae. Recombinant proteins were produced in Escherichia coli cells and their protective efficacies were investigated in cobia. Fish immunised with recombinant proteins, rPtsA + ISA (ISA 763 AVG) and rGAPDH + ISA, elicited higher levels of specific antibody responses against the recombinant proteins and had high levels of lysozyme activity. Notably, vaccinated fish were protected from lethal challenge with relative percentage of survival (RPS) values for rPtsA + ISA and rGAPDH + ISA groups being 91.67% and 83.33%, while 0% RPS value was found in both ISA injected and control groups. The results presented in the study demonstrate that the GAPDH and PtsA are promising vaccine candidates for preventing S. dysgalactiae disease in cobia.

Purification of two Bacillus subtilis proteins which cross-react with antibodies directed against eukaryotic protein kinase C, the His HPr kinase and trigger factor.

As in eukaryotes, phosphorylation of Ser residues in proteins appears to be common phenomenon in bacteria. Surprisingly, however, very few Ser/Thr protein kinases have been identified and in this study antibodies directed against mammalian protein kinase C (PKC) have been used in attempts to isolate conserved Ser/Thr protein kinases. Using the mAb M7 against rat brain PKC, a single 70 kDa band was identified in total cell extracts of Bacillus subtilis by Western blotting after SDS-PAGE, whilst using polyclonal antibody alpha-PKC1p against Saccharomyces cerevisiae PKC a single 67 kDa band was identified by the same procedure. The two proteins were purified independently on the basis of antibody recognition employing two-dimensional gel electrophoresis as a final step, which allowed subsequent microsequencing. The 70 kDa band was thus identified as the phosphoenolpyruvate-dependent His HPr kinase, Enzyme 1 of the phosphotransferase system. This identity was confirmed using a mutant deleted for ptsl, encoding Enzyme 1. The 67 kDa protein was identified as a previously unknown B. subtilis 'trigger factor', homologous to an Escherichia coli protein-folding enzyme, peptidylprolyl cis-trans-isomerase implicated in cell division.