SpA, ClfA, and FnbA genetic variations lead to Staphaurex test-negative phenotypes in bovine mastitis Staphylococcus aureus isolates

Abstract

Staphylococcus aureus encodes many proteins that act as virulence factors, leading to a variety of diseases, including mastitis in cows. Among these virulence factors, SpA, ClfA, ClfB, FnbA, and FnbB are important for the ability of S. aureus to adhere to and invade host cells as well as to evade host immune responses. The interaction between these S. aureus surface proteins and human immunoglobulin G and fibrinogen that are coupled to latex particles is utilized to induce latex agglutination reactions, which are used widely in diagnostic kits for confirmation of presumptive S. aureus isolates. In this study, the Staphaurex latex agglutination test was performed on a collection of confirmed bovine mastitis S. aureus isolates. Notably, 54% (43/79 isolates) of these isolates exhibited latex agglutination-negative phenotypes (Staphaurex-negative result). To gain insights into the reasons for the high frequency of Staphaurex-negative bovine mastitis S. aureus isolates, the spa, clfA, clfB, fnbA, and fnbB genes were examined. Specific genetic changes in spa, clfA, and fnbA, as well as a loss of fnbB, which may impair SpA, ClfA, FnbA, and FnbB functions in latex agglutination reactions, were detected in Staphaurex-negative S. aureus isolates. The genetic changes included a premature stop codon in the spa gene, leading to a truncated SpA protein that is unable to participate in S. aureus cell-mediated agglutination of latex particles. In addition, clfA and fnbA genetic polymorphisms were detected that were linked to ClfA and FnbA amino acid changes that may significantly reduce fibrinogen-binding activity. The genetic variations in these S. aureus isolates might also have implications for their bovine mastitis virulence capacity.

Fig. 1.

Box plots showing the spa, clfB, and clfA relative gene expression levels in Staphaurex-negative (n = 17) and -positive (n = 11) bovine mastitis S. aureus isolates. Staphaurex-negative isolates showed significantly (*, P < 0.05) lower expression of spa but significantly higher expression of clfA than did Staphaurex-positive isolates. No significant differences were detected in clfB relative gene expression levels between the two groups of S. aureus isolates.

Fig. 2.

Western blot analysis of cell wall fractions and supernatants of Staphaurex-positive and -negative bovine mastitis S. aureus isolates. Proteins derived from the cell wall fractions of stationary-phase BHI cultures and their concentrated supernatants were resolved by SDS-PAGE. The proteins were analyzed by Western immunoblotting using an HRP-conjugated goat anti-human IgA. Lane 1, molecular size marker; lanes 2 and 6, Staphaurex-positive S. aureus isolate 1887; lanes 3 and 7, Staphaurex-positive S. aureus isolate 1917; lanes 4 and 8, Staphaurex-negative S. aureus isolate 1910; lanes 5 and 9, Staphaurex-negative S. aureus isolate 2188. The expected SpA protein sizes are 57.2 (520 aa) and 50.1 (456 aa) kDa for Staphaurex-positive isolates 1887 and 1917, respectively. In the Staphaurex-negative isolates 1910 and 2188, the expected truncated SpA protein size would be 35.8 kDa (326 aa).