Abstract
Alpha-hemolysin (α-HL) is a self-assembling, channel-forming toxin produced by most Staphylococcus aureus strains as a 33.2-kDa soluble monomer. Upon binding to a susceptible cell membrane, the monomer self-assembles to form a 232.4-kDa heptamer that ultimately causes host cell lysis and death. Consequently, α-HL plays a significant role in the pathogenesis of S. aureus infections, such as pneumonia, mastitis, keratitis and arthritis. In this paper, experimental studies show that oroxylin A (ORO), a natural compound without anti-S. aureus activity, can inhibit the hemolytic activity of α-HL. Molecular dynamics simulations, free energy calculations, and mutagenesis assays were performed to understand the formation of the α-HL-ORO complex. This combined approach revealed that the catalytic mechanism of inhibition involves the direct binding of ORO to α-HL, which blocks the conformational transition of the critical “Loop” region of the α-HL protein thereby inhibiting its hemolytic activity. This mechanism was confirmed by experimental data obtained from a deoxycholate-induced oligomerization assay. It was also found that, in a co-culture system with S. aureus and human alveolar epithelial (A549) cells, ORO could protect against α-HL-mediated injury. These findings indicate that ORO hinders the lytic activity of α-HL through a novel mechanism, which should facilitate the design of new and more effective antibacterial agents against S. aureus.
Highlights
Staphylococcus aureus is an opportunistic pathogen in humans and other mammals that causes many different types of infections, including superficial abscesses, septic arthritis, osteomyelitis, pneumonia, endocarditis, and sepsis [1,2]
We found that oroxylin A (ORO), a natural compound with little anti-S. aureus activity, can inhibit the hemolytic activity of a-HL at low concentrations
The data revealed that ORO directly binds to a-HL, an interaction that blacks the conformational transition of the critical ‘‘Loop’’ region in aHL and prevents the formation of the a-HL heptameric transmembrane pore, which inhibits the hemolytic activity of a-HL
Summary
Staphylococcus aureus is an opportunistic pathogen in humans and other mammals that causes many different types of infections, including superficial abscesses, septic arthritis, osteomyelitis, pneumonia, endocarditis, and sepsis [1,2]. The number of virulence factors secreted by S. aureus, including extracellular and cell wall-related proteins, determines its pathogenicity [3]. Previous studies using a mouse model of S. aureus pneumonia have shown that S. aureus strains that lack the hla gene (and do not secrete a-HL) cause less lung injury and inflammation than the hla positive strains [5]. The a-HL protein, isolated from the gram-positive pathogenic bacterium S. aureus, is a well-studied model that has been used to elucidate mechanisms of membrane insertion by soluble proteins. Studies have shown that a-HL can self-assemble on the lipid bilayers of the membranes of susceptible host cells to form a wide heptameric pore [6]. The protein is toxic for a wide range of mammalian cells, erythrocytes and epithelial cells and serves primarily as a tool that converts host tissue into nutrients for any bacteria that expresses it [3]
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