Oroxylin A Inhibits Hemolysis via Hindering the Self-Assembly of α-Hemolysin Heptameric Transmembrane Pore

et al. (2013) Oroxylin A Inhibits Hemolysis via Hindering the Self-Assembly of a-Hemolysin Heptameric Transmembrane Pore. PLoS Comput Biol 9(1): e1002869. doi:10.1371/journal.pcbi.1002869 Oroxylin A Inhibits Hemolysis via Hindering the Self- Assembly of a-Hemolysin Heptameric Transmembrane Pore Jing Dong 0 Jiazhang Qiu 0 Yu Zhang 0 Chongjian Lu 0 Xiaohan Dai 0 Jianfeng Wang 0 Hongen Li 0 Xin Wang 0 Wei Tan 0 Mingjing Luo 0 Xiaodi Niu 0 Xuming Deng 0 Dennis R. Livesay, UNC Charlotte, United States of America 0 1 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University , Changchun , China , 2 Department of Food Quality and Safety, Jilin University , Changchun , China Alpha-hemolysin (a-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, a-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 a-HL. Molecular dynamics simulations, free energy calculations, and mutagenesis assays were performed to understand the formation of the a-HL-ORO complex. This combined approach revealed that the catalytic mechanism of inhibition involves the direct binding of ORO to a-HL, which blocks the conformational transition of the critical ''Loop'' region of the a-HL protein thereby inhibiting its hemolytic activity. This mechanism was confirmed by experimental data obtained from a deoxycholateinduced 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 a-HL-mediated injury. These findings indicate that ORO hinders the lytic activity of aHL through a novel mechanism, which should facilitate the design of new and more effective antibacterial agents against S. aureus. - Funding: This work was supported by the National Basic Research Program of China (No. 2013CB127205) and 2012 PhD interdisciplinary project of Jilin University (No. 2012JC 202). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. 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]. The virulence factor a-hemolysin (a-HL) is one of the most important factors produced by the majority of S. aureus strains and recent studies have demonstrated that it plays a major role in S. aureus pneumonia [4]. Previous studies using a mouse model of S. aureus pneumonia have shown that S. aureus strains that lack the hla gene (and thus 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, particularly erythrocytes and epithelial cells and serves primarily as a tool that converts host tissue into nutrients for any bacteria that expresses it [3]. In an effort to increase our understanding of the function of aHL, the structure of the heptameric pore was resolved by X-ray crystallography to a resolution of 0.19 nm [6]. Contained within the mushroom-shaped homo-oligomeric heptamer is a 10 nm long solvent-filled channel that runs along the seven-fold axis and ranges from 1.4 nm to 4.6 nm in diameter. The lytic transmembrane domain forms the lower half of a 14-strand antiparallel b barrel, to which each protomer contributes two 6.5 nm long b strands. Considering the essential nature of the heptameric crystal structure, Ragle et al. used a modified b-cyclodextrin compound, IB201, to prevent the a-HL-induced lysis of human alveolar epithelial cells (A549) [7]. This protective effect does not result from the ability of b-cyclodextrin to impair formation of the oligomeric a-HL on the cell surface, supporting a role for this molecule in the blockade of the lytic pore. Previous investigations had demonstrated the use of unsubstituted b-cyclodextrin as an adapter molecule that is capable of lodging within the central pore of a-HL and can thus facilitate the use of the toxin as a biosensor [8,9]. The investigation of b-cyclodextrin using IB201 revealed that it blocks ion conductance through the assembled hemolysin The mechanism controlling protein-ligand interactions is one of the most important processes in rational drug design. X-ray crystallography is a traditional tool used to investigate the interaction of ligands and proteins in a complex. However, protein crystallography is inefficient, and the development of crystal technology and research remains unequally distributed. Thus, it seems impractical to explore the structure of the a-hemolysin-ORO monomer complex by crystallography. Therefore, we used molecular dynamics simulations to investigate the receptor-ligand interaction in the a-HL-ORO monomer complex. In this study, 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. Through molecular docking and molecular dynamics simulations, we determined the potential binding mode of the protein-ligand interaction. 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 thus prevents the formation of the a-HL heptameric transmembrane pore, which ultimately inhibits the hemolytic activity of a-HL. This mechanism was confirmed by experimental data. Furthermore, we demonstrated that ORO could protect against a-HL-mediated injury in human alveolar epithelial (A549) cells. pore, which supports the finding that b-cyclodextrin inserts into the pore itself. Although the inhibitory effect of b-cyclodextrin on ion conductance and red blood cell hemolysis were both observed in the low micromolar concentration range, this treatment strategy is passive. It is clear that prior to inhibition by b-cyc (...truncated)