Restoring catalase activity in Staphylococcus aureus subsp. anaerobius leads to loss of pathogenicity for lambs

Abstract

Staphylococcus aureus subsp. anaerobius, a microaerophilic and catalase-negative bacterium, is the etiological agent of abscess disease, a specific chronic condition of sheep and goats, which is characterized by formation of necrotic lesions that are located typically in superficial lymph nodes. We constructed an isogenic mutant of S. aureus subsp. anaerobius (RDKA84) that carried a repaired and functional catalase gene from S. aureus ATCC 12600, to investigate whether the lack of catalase in S. aureus subsp. anaerobius plays a role in its physiological and pathogenic characteristics. The catalase activity had no apparent influence on the in vitro growth characteristics of RDKA84, which, like the wild-type, did not grow on aerobically incubated agar plates. Restoration of catalase activity in RDKA84 substantially increased resistance to H2O2 when analyzed in a death assay. The intracellular survival rates of the catalase-positive mutant RDKA84 in polymorphonuclear neutrophils (PMN) isolated from adult sheep were significantly higher than those of the wild-type, while no differences were found with PMN isolated from lambs. RDKA84 showed significantly lower survival rates in murine macrophages (J774A.1 cells) than the wild-type strains did, whereas, in bovine mammary epithelial cells (MAC-T), no differences in intracellular survival were observed. Interestingly, the virulence for lambs, the natural host for abscess disease, of the catalase-positive mutant RDKA84 was reduced dramatically in comparison with wild-type S. aureus subsp. anaerobius in two experimental models of infection.

Figure 1.

H₂O₂ sensitivity of MVF-84 (wild-type S. aureus subsp. anaerobius) (●), RDKA84 (catalase-positive mutant of MVF-84) (○) and four strains of S. aureus isolated from clinical cases of mastitis (MN-42 (■), MN-45 (□), MN-73 (♦) and DGA-1 (◊)). These strains were exposed to various concentrations of H₂O₂. Each point represents the mean (± S.D.) of three experiments. Each experiment was performed in triplicate. Error bars indicate confidence level (95%); p values were determined using the Student’s t-test.

Figure 2.

Intracellular killing of S. aureus subsp. anaerobius strains MVF-84 (●) and RDKA84 (○) by PMN isolated from ewes (A) and lambs (B). Survival of MVF-84 and RDKA84 was determined using dilutional plate counts. Numbers of surviving bacteria are represented as the percentage of the original number of phagocytosed bacteria. Each point represents the mean (± S.D.) of three experiments. Each experiment was performed in triplicate. Error bars indicate confidence level (95%); p values were determined using the Student’s t-test.

Figure 3.

Intracellular survival of S. aureus subsp. anaerobius strains MVF-84 (●) and RDKA84 (○) in murine macrophage-like cell line J774A.1 (A) and bovine mammary epithelial cell line MAC-T (B). Survival of MVF-84 and RDKA84 was determined using dilutional plate counts. Data of viable intracellular bacteria at 4, 8 and 24 h are expressed as a percentage of internalized bacteria and represent the mean ± S.D. of three independent experiments running in triplicate. Error bars indicate confidence level (95%); p values were determined using the Student’s t-test.