Comparison of Staphylococcus aureus surface protein extraction methods and immunogenicity

Abstract

Staphylococcus aureus is the major contagious bovine mastitis pathogen and has no effective vaccine. Strain variation and limited knowledge of common immunogenic antigen/s are among major constraints for developing effective vaccines. S. aureus cell surface proteins that are exposed to the host immune system constitute good vaccine candidates. The objective of this study was to compare two novel S. aureus surface protein extraction methods with biotinylation method and evaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct S. aureus strains from cases of bovine mastitis. After extraction, bacterial cell integrity was examined by Gram staining and electron microscopy to determine if extraction methods caused damage to cells that may release non-surface proteins. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and evaluated for immune-reactivity using western blot. Results showed that all three extraction methods provided multiple protein bands on SDS-PAGE. Western blot result showed several immunoreactive surface proteins, in which some proteins strongly (well-resolved, thick, dark, and intense band) reacted across the nine strains tested. The three methods are valid for the extraction of surface proteins and hexadecane, and cholic acid methods are more feasible than biotinylation since both are easier, cheaper, and have minor effects on the bacterial cell. Strongly immune-reactive surface proteins may serve as potential candidates for a vaccine to control S. aureus mastitis in dairy cows.

Keywords: Bovine mastitis; Extraction; Immune-reactive; Immunology; Infectious disease; Microbiology; Staphylococcus aureus; Surface protein; Vaccines; Veterinary medicine.

Fig. 1

SDS-PAGE and western blot results of surface proteins extracted from nine genetically distinct strains of S. aureus using cholic acid (CA), biotinylation (BT) or hexadecane (HD). The three panels in the top row (A, B and C) indicated protein bands by 1D SDS-PAGE and the three panels in the bottom row (D, E and F) were western blot results of same proteins transferred to nitrocellulose membrane from the SDS-PAGE. M = Protein molecular weight marker, Capital letters B–I showed dominant strains (PFGE types). The letter ‘S’ was the surface protein from the S. aureus challenge strain. The arrows (No. 1–10) indicated immunoreactive protein bands and the five black arrows (# 1, 4, 6, 7 and 8) showed strongly immuno-reactive proteins.

Fig. 2

Gram-stained Staphylococcus aureus. A) Gram-stained S. aureus before surface proteins extraction, B) Gram-stained S. aureus after surface protein extraction by hexadecane and C) Gram-stained S. aureus after surface proteins extraction by cholic acid.

Fig. 3

Evaluation results of bacterial cell integrity by scanning and transmission electron microscopes before and after surface protein extractions. A) Scanning electron micromicrograph of untreated S. aureus before protein extraction, B) Scanning electron micrograph of S. aureus after surface protein extraction by hexadecane, C) Scanning electron micrograph of S. aureus after surface proteins extraction by cholic acid, D) Transmission electron micrograph of untreated S. aureus before protein extraction, E) Transmission electron micrograph of S. aureus after surface proteins extraction by hexadecane and F) Transmission electron micrograph of S. aureus after surface protein extraction by cholic acid. Bar = 200 nm.

supplemental figure 1

Nine genetically distinct S. aureus isolates from cases of bovine mastitis. Panel A: Pulsed field gel electrophoresis (PFGE) results of SmaI digested chromosomal DNA of S. aureus isolates (only 11 isolates and control strain shown). Lanes 1, 8 and 15 =indicated control strain (national collection of type cultures (NCTC) 8325), Lanes 2, 3, 4, 5, 6, 7 and 9, 10, 11, 12, 13 and 14= genetically different S. aureus strains (PFGE types) designated by letters F, H, S, E, G, S and B, C, I, D, I and I, respectively. The same letter indicated the same strain. Panel B: Dendrogram of PFGE bands analyzed by GelCompar II software that resulted in nine genetically distinct S. aureus isolates designated with capital letters (B through I). The strain designated by letter “S” was the experimental challenge strain of S. aureus (strain used to cause experimental intramammary infection in our previous studies). Serum from a cow previously vaccinated with S. aureus surface proteins (SASP) that was protected from clinical mastitis upon challenge (infection) was used as a primary antibody for western blot (data not published). The S. aureus strain NCTC 8325 was used as a control for the experimental variation between duplicate experiments in the PFGE analysis.