Staphylococcus aureus Beta-Toxin Induces Lung Injury through Syndecan-1

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 β-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 β-toxin compared with mice infected with S. aureus expressing β-toxin. In addition, intranasal administration of β-toxin evoked the characteristic features of lung injury in wild-type mice whereas neutropenic mice were protected from such injury. However, mutant β-toxin mice deficient in sphingomyelinase activity failed to trigger features of lung injury. Ablation of sphingomyelinase activity also interfered with the ability of β-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 β-toxin-induced lung injury relative to wild-type mice. These data indicate that S. aureus β-toxin is a critical virulence factor that induces neutrophil-mediated lung injury through both its sphingomyelinase activity and syndecan-1.Staphylococcus aureus pneumonia is a common complication among ventilated patients¹ and also a serious problem in individuals with cystic fibrosis,² patients at the extremes of ages with predisposing diseases,^3,4 and patients with immunosuppressive therapy or illness.⁵ Further, next to bacteremia, pneumonia is the second most prevalent condition during invasive methicillin-resistant S. aureus (MRSA) disease,⁶ and the incidence of severe pneumonia caused by MRSA strains is rising.^6,7 A characteristic feature of S. aureus pneumonia is the intense host inflammatory response with a rapid and excessive recruitment of neutrophils.^8,9,10,11 However, the underlying mechanisms of how S. aureus dysregulates the host inflammatory response and causes lung injury are incompletely understood.Recent animal studies have identified Panton-Valentine leukocidin,¹² α-toxin,¹¹ and protein A⁸ as virulence factors of S. aureus pneumonia. Gomez et al showed that protein A promotes lung injury and inflammation through activating tumor necrosis factor receptor-1 signaling and inducing tumor necrosis factor α-like responses.⁸ Both α-toxin and Panton-Valentine leukocidin are pore-forming toxins and they can exaggerate the host inflammatory response by inducing the expression of pro-inflammatory cytokines and lysing inflammatory cells to release additional pro-inflammatory mediators,^12,13,14,15 suggesting that these toxins possess both direct and indirect means of causing lung damage. Altogether, these observations suggest that the combined actions of several virulence factors may coordinately or synergistically enhance the characteristic neutrophil-mediated lung injury and edema seen in S. aureus pneumonia.In addition to α-toxin and Panton-Valentine leukocidin, S. aureus expresses several other exotoxins and enterotoxins that have the potential of causing tissue injury and inflammation, such as β-toxin, γ-toxin, staphylococcal enterotoxin A, and toxic shock syndrome toxin-1. However, little is known about the physiological significance of these toxins in S. aureus-induced lung injury. S. aureus β-toxin is a Mg²⁺-dependent neutral sphingomyelinase that hydrolyzes membrane sphingomyelin of host cells to generate phosphorylcholine and the bioactive secondary messenger, ceramide.^16,17,18 β-toxin does not lyse most types of host cells, but leaves them vulnerable to a number of other lytic agents, such as α-toxin and Panton-Valentine leukocidin. In fact, the cytotoxic effect of β-toxin is highly cell type- and species-specific, suggesting that its primary virulence activity is to modulate host processes that affect pathogenesis rather than to directly kill host cells.Among the S. aureus toxins, the least is known about the functions of β-toxin in vivo. This is partly because relative to other virulence factors, which are expressed by the majority of clinical isolates, β-toxin was found to be expressed in 13% of septicemia and 11% of nasal carrier isolates.¹⁹ However, there is epidemiological evidence that β-toxin expression is strongly associated with recurrent S. aureus furunculosis and chronic osteomyelitis despite its overall low occurrence in clinical isolates.²⁰ Animal studies have also established that β-toxin is a virulence factor for S. aureus keratitis²¹ and mastitis.²² Further, a recent study showed that the β-toxin gene (hlb) is present in the majority of MRSA strains²³ and suggested an association between hlb carriage and the capacity of S. aureus to infect the respiratory tract. MRSA strains USA200 and USA1100, belonging to the same clonal complex (CC30), possess highly similar virulence gene repertoires. However, the majority of USA200, but not USA1100, isolates tested positive for the genes for β-toxin, toxic shock syndrome toxin-1 and staphylococcal enterotoxin A and, more importantly, 30.4% of USA200, compared with only 5.3% of USA1100, were isolated from the respiratory tract.²³ These observations suggest that β-toxin, along with toxic shock syndrome toxin-1 and staphylococcal enterotoxin A, may impart a survival advantage for USA200 in the pulmonary environment.The present study was designed to examine the role of β-toxin in S. aureus-induced lung injury. Our data show that the major determinants of lung injury are significantly attenuated in mice infected intranasally with S. aureus deficient in β-toxin compared with those infected with S. aureus expressing β-toxin. Further, intranasal administration of β-toxin alone induces neutrophil influx into the lung and alveolar space, and increases vascular permeability and pulmonary edema. Our results also indicate that β-toxin achieves these pathological effects through syndecan-1, a major heparan sulfate proteoglycan of epithelial cells, because syndecan-1 null mice are significantly protected from β-toxin-induced lung injury. These data reveal a previously unknown in vivo function of β-toxin and establish that β-toxin is an important virulence factor in S. aureus-induced lung injury.