Staphylococcus aureus beta-toxin induces lung injury through syndecan-1

Abstract

In pneumonia caused by the bacterium Staphylococcus aureus, the intense inflammatory response that is triggered by this infection can lead to the development of lung injury. Little is known, however, about the impact of specific virulence factors on this inflammatory disorder, which causes both significant mortality and morbidity. In this study, we examined the role of beta-toxin, a neutral sphingomyelinase, in S. aureus-induced lung injury. Our results showed that the central features of lung injury--specifically, increased neutrophilic inflammation, vascular leakage of serum proteins into the lung tissue, and exudation of proteins into the airway--are significantly attenuated in mice infected intranasally with S. aureus deficient in beta-toxin compared with mice infected with S. aureus expressing beta-toxin. In addition, intranasal administration of beta-toxin evoked the characteristic features of lung injury in wild-type mice whereas neutropenic mice were protected from such injury. However, mutant beta-toxin mice deficient in sphingomyelinase activity failed to trigger features of lung injury. Ablation of sphingomyelinase activity also interfered with the ability of beta-toxin to stimulate ectodomain shedding of syndecan-1, a major heparan sulfate proteoglycan found in epithelial cells. Moreover, syndecan-1-null mice were significantly protected from beta-toxin-induced lung injury relative to wild-type mice. These data indicate that S. aureus beta-toxin is a critical virulence factor that induces neutrophil-mediated lung injury through both its sphingomyelinase activity and syndecan-1.

Figure 1

β-toxin is essential in the induction of lung injury by S. aureus. Wild-type C57BL/6J mice were anesthetized and inoculated intranasally with 14 μl PBS or infected intranasally with PBS containing 1.2 × 10⁸ cfu of hlb+ or hlb− S. aureus and evaluated for determinants of lung injury at 6 hours postchallenge. A: Vascular leakage of serum proteins was assessed by measuring the accumulation of Evans blue dye in lung tissues as described in the Materials and Methods. Data shown are means of Evans blue dye (μg) per lung weight (g) ± SE, n = 9. B: Total BAL protein concentration was measured by Bradford assay (mean ± SE, n = 8 to 9). C: BAL cytospin slides were stained with Hema-3 and leukocytes were counted. Data shown are percent neutrophils of total BAL cells (mean ± SE, n = 5). *P < 0.05 between mice infected with hlb+ S. aureus and hlb−S. aureus, **P < 0.01 between mice inoculated with PBS and infected with hlb+ S. aureus. D: Representative pictures of Hema-3-stained cytospin slides (magnification = original ×100). Neutrophils are identifiable by their pale staining cytoplasm and segmented ring-shaped nuclei. E: Lung sections were stained with hematoxylin-eosin or immunostained with 281-2 anti-syndecan-1 monoclonal antibodies (magnification = original ×200).

Figure 2

Intranasal administration of β-toxin causes lung injury. Wild-type mice were inoculated intranasally with PBS or 15 μg β-toxin, and lung injury was assessed at 7 hours postinoculation. A: The accumulation of Evans blue dye in lung tissues is shown (n = 4 to 5). B: Total BAL protein concentration was measured by Bradford assay (n = 8). C: Leukocytes that have migrated into the alveolar space were enumerated by counting Hema-3-stained BAL cytospin slides. Data are shown as % BAL neutrophils (n = 4). (*P < 0.05; **P < 0.01) D: Representative pictures of BAL cytospin slides (magnification = original ×100). E: Lung sections were stained with H&E or immunostained with 281-2 anti-syndecan-1 monoclonal antibodies (magnification = original ×200).

Figure 3

Neutropenic mice are protected from β-toxin-induced lung injury. Wild-type mice were injected intraperitoneally with 30 μg/mouse anti-GR1 antibodies or PBS 24 hours before intranasal inoculation of 15 μg β-toxin, and lung injury was assessed at 7 hours post-β-toxin inoculation. A: Evans blue dye accumulation in lung tissues is shown (n = 3 to 5). B: Total BAL protein concentration was measured by Bradford assay (n = 5). C: % BAL neutrophils was enumerated by differentially counting leukocytes in Hema-3-stained BAL cytospin slides (n = 5). *P < 0.05; **P < 0.01. D: Representative pictures of BAL cytospin slides (magnification = original ×100). E: Lung sections were stained with hematoxylin-eosin or immunostained with anti-GR-1 antibodies directly conjugated to Alexa 594 (magnification = original ×200).

Figure 4

β-toxin induces syndecan-1 shedding in vivo through its sphingomyelinase activity. A: Wild-type mice were inoculated intranasally with PBS or β-toxin (15 μg/mouse) and BAL was collected 7 hours later. BAL syndecan-1 ectodomain levels were determined by dot immunoblotting (n = 7 to 8). B: Confluent NMuMG epithelial cells were incubated with wild-type or mutant β-toxin (both at 30 μg/ml) for 4 hours at 37°C, and the concentration of syndecan-1 ectodomains in the conditioned medium was quantified by dot immunoblotting (n = 6 to 8). C: Confluent A549 cells were incubated with wild-type or mutant β-toxin, and the concentration of syndecan-1 ectodomains in the conditioned medium was quantified (n = 6). D: Wild-type mice were inoculated intranasally with wild-type or mutant β-toxin (15 μg/mouse) and BAL was collected 7 hours later. BAL syndecan-1 ectodomain levels were determined by dot immunoblotting (n = 4 to 7). **P < 0.01.

Figure 5

¹⁸⁴His and ³²³His are critical in β-toxin-induced lung injury. Wild-type mice were administered intranasally with PBS or 15 μg wild-type, H184L or H323L β-toxin and the extent of lung injury was assessed at 7 hours postinoculation by measuring: (A) the accumulation of Evans blue dye in lung tissues (n = 4 to 5) and (B) total BAL protein concentration (n = 4 to 7); and (C) assessing neutrophil influx into the alveolar space by Hema-3 staining of BAL cytospin slides (magnification = original ×100). *P < 0.05; **P < 0.01.

Figure 6

β-toxin induces lung injury through syndecan-1. Wild-type and Sdc1−/− mice were inoculated intranasally with β-toxin and lung injury was assessed at 7 hours postinoculation. A: Evans blue dye accumulation in lung tissues (n = 10–11; *P < 0.05). B: Representative pictures of Hema-3-stained BAL cytospin slides (magnification = original ×100). C: H&E staining and anti-GR1 immunostaining of wild-type and Sdc1−/− lung sections (magnification = original ×200).