Cytotoxic activity of coagulase-negative staphylococci in bovine mastitis

Abstract

Secreted toxins play important roles in the pathogenesis of bacterial infections. In this study, we examined the presence of secreted cytotoxic factors of coagulase-negative staphylococci (CoNS) from bovine clinical and subclinical mastitis. A 34- to 36-kDa protein with cell-rounding cytotoxic activity was found in many CoNS strains, especially in Staphylococcus chromogenes strains. The protein caused cell detachment and cell rounding in several cell lines, including HEp-2, Int 407, CHO-K1, and Y-1 cells. Native protein recovered from nondenatured polyacrylamide gel electrophoresis showed both cytotoxic activity and casein hydrolysis activity. The purified protein had a pH optimal at 7.2 to 7.5 and a pI of 5.1 and was heat labile. The proteolytic activity could be inhibited by zinc and metal specific inhibitors such as 1, 10-phenanthroline and EDTA, indicating that it is a metalloprotease. Protein mass analysis and peptide sequencing indicated that the protein is a novel metalloprotease. Different bacterial strains expressed variable levels of 34- to 36-kDa protease, which may provide an indication of strain virulence.

FIG. 1

Cytotoxic effects of bacterial supernatant (S. chromogenes 690LR) on HEp-2 cells. B-broth and bacterial cultural supernatants were diluted in MEM, loaded on a HEp-2 cell monolayer, and incubated for 20 h at 37°C in 95% air and 5% CO₂. (A) B-broth at a 1:2.5 dilution in MEM caused no cytopathic effects on the cultured HEp-2 cells. (B) Bacterial supernatant at a 1:5 dilution in MEM caused detachment and rounding of HEp-2 cells. (C) Bacterial supernatants at a 1:2.5 dilution in MEM caused rounding of all HEp-2 cells.

FIG. 2

Cytotoxic effects of bacterial supernatant (S. chromogenes 690LR) on Int 407 cells. B-broth and bacterial cultural supernatants were diluted in BEM, loaded onto a Int 407 cell monolayer, and incubated for 20 h at 37°C in 95% air and 5% CO₂. (A) B-broth at a 1:2.5 dilution in BEM caused no cytopathic effects on cultured Int 407 cells. (B) Bacterial supernatants at a 1:2.5 dilution in BEM caused rounding and clumping of Int 407 cells.

FIG. 3

Cytotoxic effects of bacterial supernatant (S. chromogenes 690LR) on CHO-K1 cells. B-broth and bacterial cultural supernatants were diluted in Ham F-12 medium, loaded onto an Int 407 cell monolayer, and incubated for 20 h at 37°C in 95% air and 5% CO₂. (A) B-broth at a 1:2.5 dilution in Ham F-12 medium caused no cytopathic effects on the cultured CHO-K1 cells. (B) Bacterial supernatants at 1:2.5 and 1:5 dilutions in Ham F-12 medium caused the rounding of CHO-K1 cells.

FIG. 4

Cytotoxic effects of bacterial supernatant (S. chromogenes 690LR) on Y-1 cells. B-broth and bacterial cultural supernatants were diluted in Ham F-12 medium, loaded onto a Y-1 cell monolayer, and incubated for 20 h at 37°C in 95% air and 5% CO₂. (A) B-broth at a 1:2.5 dilution in Ham F-12 medium caused no cytopathic effects on cultured Y-1 cells. (B) Bacterial supernatants at 1:2.5 and 1:5 dilutions in Ham F-12 medium caused rounding of Y-1 cells.

FIG. 5

Bacterial supernatants were concentrated 15 times with a 10-kDa molecular mass cutoff Centricon Plus-20 (Amicon). The concentrated supernatants (20 μl of each sample) were loaded onto SDS–12% PAGE gels. MW, low-range protein molecular mass marker (in kilodaltons) (Bio-Rad). Lanes 1 and 2, supernatants of two S. simulans strains; lanes 3 and 4, supernatants of two cytotoxin-negative S. chromogenes strains; lanes 5 to 9, supernatants of five cytotoxin-positive S. chromogenes strains. A 34- to 36-kDa protein band was only observed on cytotoxin-positive S. chromogenes strains.

FIG. 6

The 34- to 36-kDa protein was heat labile. The same amounts of concentrated supernatants were used for loading treated and untreated wells. For heat treatment, the supernatants were heated at 65°C for 30 min. (A) SDS-PAGE. MW, low-range protein molecular mass marker (in kilodaltons) (Bio-Rad). Lanes 1, 3, 5, and 7, untreated supernatants from S. chromogenes strains 1LR, 690LR, 302RR, and 726LR; lanes 2, 4, 6, and 8, the corresponding heat-treated supernatants. (B) Western blots. MW, low-range protein molecular mass marker (in kilodaltons) (Bio-Rad). Lanes 1, 3, 5, and 7, untreated supernatants from S. chromogenes strains 1LR, 690LR, 302RR, and 726LR; lanes 2, 4, 6, and 8, the corresponding heat-treated supernatants. The polyclonal antibody was raised in rabbits against the 34- to 36-kDa protein purified from S. chromogenes 690LR.

FIG. 7

Purification of native 34- to 36-kDa protein from nondenatured PAGE. (A) Supernatant of S. chromogenes 690LR was concentrated 300-fold and separated on 8% nondenatured PAGE gels. The dominant band was easy to recognize. (B) The dominant band was cut and eluted from the gel, and the purity of the eluted protein was checked by SDS–12% PAGE.

FIG. 8

Western blots showed a specific reaction of antisera with the 34- to 36-kDa protein among different species and strains. The polyclonal antibody was raised in rabbits against the 34- to 36-kDa protein purified from S. chromogenes 690LR. Lane 1, S. chromogenes 690LR; lanes 2, 3, and 6, field isolates of S. chromogenes; lane 7, S. chromogenes ATCC 10530; lane 4, a field isolate of Staphylococcus hominis (CoNS) strain (cytotoxin positive); lanes 5 and 8, field isolates of Staphylococcus simulans (CoNS) strains (cytotoxin negative).

FIG. 9

Casein hydrolysis activity of the 34- to 36-kDa protein from S. chromogenes 302RR. The white spot represents hydrolysis of casein. The crude supernatant was separated by nondenatured PAGE. After 10 min of washing in Tris-HCl buffer with 2 mM CaCl₂, the gel was laid over a casein plate. The result was recorded after 2 h of incubation at 37°C.

FIG. 10

The optimal pH (A), optimal temperature (B), and thermostability (C) of the protease activity of the 34- to 36-kDa protein were studied by using the protease assay (see Materials and Methods). The same amount of crude purified protein was used for all the reactions. 1LR and 690LR were field S. chromogenes strains. (A) The protease activity was measured in 100 mM Tris-HCl buffer with different pHs, ranging from pH 6.9 to 9. (B) The protease activity was measured after 45 min of incubation at different temperatures, from 20 to 60°C. (C) The crude purified protein was first incubated at different temperatures, from 20 to 65°C, for 20 min, and the protein activity was then measured.

FIG. 11

The 34- to 36-kDa protein expression was associated with the bacterial growth phase. Quantitative analysis of the 34- to 36-kDa protein was measured by the protease activity, and the bacterial growth was monitored by reading the optical density (OD) at 578 nm.