Staphylococcus aureus capsular polysaccharide types 5 and 8 reduce killing by bovine neutrophils in vitro

Abstract

Isogenic variants of Staphylococcus aureus strain Reynolds expressing either no capsule or capsular polysaccharide (CP) type 5 (CP5) or type 8 (CP8) were used to assess the effect of CP on bacterial killing and the respiratory burst of bovine neutrophils. The effects of antisera specific for CP5 and CP8 were also evaluated. The killing of live bacteria by isolated neutrophils was quantified in a bactericidal assay, while the respiratory burst after stimulation with live bacteria in whole blood was measured by flow cytometry. The expression of a CP5 or CP8 capsule protected the bacteria from being killed by bovine neutrophils in vitro (P <0.001), and the capsule-expressing variants did not stimulate respiratory burst activity in calf whole blood. The addition of serotype-specific antisera increased the killing of the capsule-expressing bacteria and enhanced their stimulating effect in the respiratory burst assay (P <0.01). When the S. aureus variants were grown under conditions known not to promote capsule expression, there were no significant differences between them. The present study demonstrates that the expression of S. aureus CP5 or CP8 confers resistance to opsonophagocytic killing and prevents the bacteria from inducing respiratory burst of bovine neutrophils in vitro and that these effects can be reversed by the addition of serotype-specific antisera.

FIG. 1.

Percentages of noncapsulated, CP5-producing, and CP8-producing S. aureus organisms killed by cow and calf neutrophils in the MTT bactericidal assay with heat-inactivated pooled bovine serum, anti-CP5 serum, or anti-CP8 serum. The results are means (SEMs) for five to eight animals.

FIG. 2.

Increase in the fluorescence (GMFI) of calf whole-blood samples stimulated with noncapsulated (Non-capsul.), CP5-producing, or CP8-producing S. aureus without in vitro serum addition or with anti-CP5 serum or anti-CP8 serum supplementation in the respiratory burst assay. The results are means (SEMs) for six or seven animals.

FIG. 3.

(A) Histogram showing green fluorescence of a calf whole-blood sample stimulated with noncapsulated, CP5-producing, or CP8-producing S. aureus in the flow cytometric respiratory burst assay (grey) plotted against the fluorescence of a nonstimulated sample (transparent). (B and C) Histograms showing the green fluorescence of a calf whole-blood sample stimulated with noncapsulated, CP5-producing, or CP8-producing S. aureus with the addition of anti-CP5 serum (grey) or preimmunization serum (transparent) (B) and with the addition of anti-CP8 serum (grey) or preimmunization serum (transparent) (C).

FIG. 4.

Increase in the fluorescence (GMFI) with twofold dilutions of anti-CP5 serum (A) or anti-CP8 serum (B) added to calf whole blood stimulated with CP5- or CP8-producing S. aureus in the respiratory burst assay, respectively. The results are means (SEMs) for four animals.