Prevention and treatment of Staphylococcus aureus pneumonia with a beta-cyclodextrin derivative

Abstract

Staphylococcus aureus pneumonia is a common, potentially life-threatening infection caused by this human pathogen. The only therapies available to treat S. aureus pneumonia are antibiotics, a modality that is jeopardized by the organism's remarkable ability to acquire antimicrobial resistance. S. aureus alpha-hemolysin is a pore-forming cytotoxin that is essential for the pathogenesis of pneumonia. Strains lacking this cytotoxin are avirulent in a murine model of pneumonia; likewise, vaccine-based strategies that antagonize the toxin afford protection against lethal disease. Disruption of the function of this toxin therefore provides a potent mechanism to prevent and/or treat S. aureus pneumonia. beta-Cyclodextrin derivatives are small molecules with a sevenfold symmetry that mirrors the heptameric alpha-hemolysin. These compounds block the assembled alpha-hemolysin pore, compromising toxin function. We report that a modified beta-cyclodextrin compound, IB201, prevents alpha-hemolysin-induced lysis of human alveolar epithelial cells. This protective effect does not result from the ability of the beta-cyclodextrin to impair formation of the oligomeric alpha-hemolysin on the cell surface, supporting a role for this molecule in blockade of the lytic pore. An examination of IB201 in murine S. aureus pneumonia demonstrated that administration of this compound prevents and treats disease, protecting against mortality. Consistent with the vital importance of alpha-hemolysin in pneumonia caused by methicillin-sensitive and highly virulent methicillin-resistant S. aureus strains, IB201 protects against lethal challenge with both types of isolates. These observations, coupled with a favorable safety profile of beta-cyclodextrin compounds, provide a novel strategy that may be developed to combat S. aureus pneumonia.

FIG. 1.

β-Cyclodextrin IB201 prevents alpha-toxin-induced hemolysis without disrupting formation of the fully assembled heptameric toxin. (A) Structure of β-cyclodextrin derivative IB201. (B) Hemolysis assays were performed with active [³⁵S]methionine-labeled toxin synthesized in vitro added to 12.5% rabbit red blood cells (rRBC) in K-PBSA/βME. The addition of 50 μM IB201 significantly reduced hemolysis as measured by the absorbance at 475 nm (Abs₄₇₅) of assay supernatants (P ≤ 1.7 × 10⁻⁷). Each condition was analyzed in triplicate, and the results shown are representative of two independent experiments. (C) Active [³⁵S]methionine-labeled toxin was synthesized in vitro and added to rRBC in the presence of PBS or 50 μM IB201 at 37°C, 50°C, or 90°C. Toxin oligomerization (designated Hla₇) was evident with both PBS and IB201. IB201 did not disrupt binding of the labeled toxin to rRBCs, indicated by the presence of the Hla monomer in all lanes. The results shown are representative of the results of two independent experiments.

FIG. 2.

β-Cyclodextrin derivative IB201 protects human alveolar epithelial cells from S. aureus injury. Live (green)/dead (red) staining of A549 alveolar epithelial cells was imaged by fluorescence microscopy 3.5 h after infection. (A to D) Cells were uninfected (A) or cocultured with S. aureus Newman in medium treated with PBS (B), PBS plus 0.1% DMSO (PBSD) (C), or 5 μM IB201 in PBSD (D). The percentage of dead cells for each experimental condition was calculated by scoring live versus dead cells in four independent fields and expressing the number of dead cells as a fraction of the total; these values are noted in the bottom right-hand corner of each panel. Images for each condition are representative of cells visualized in two independent experiments. Bars, 20 μm. (E) LDH release by A549 cells was observed by cells cocultured with S. aureus Newman in medium treated with the indicated concentrations of IB201; an asterisk indicates a significant reduction in LDH release for specific concentration (P < 0.05). (F) Optical density readings were taken at 600 nm (OD₆₀₀) to measure the growth of S. aureus strain Newman in the presence of either PBS plus 0.1% DMSO or 5 μM IB201. The values in panels E and F represent means ± standard deviations (SD) (error bars). The data shown are representative of the data in three independent experiments.

FIG. 3.

IB201 protects against S. aureus pneumonia. (A) IB201 was administered to mice via retro-orbital injection 2 h after infection, 12 h after infection, and then every 12 h thereafter. They were challenged with S. aureus Newman via the i.n. route, and mortality was recorded at 24, 48, and 72 hours postinfection (P < 0.039; 15 animals per group) (statistical significance is denoted by an asterisk). (B) CFU recovery from the right lung was determined and demonstrated a significant decrease in bacterial burden in mice that received IB201 (P = 0.008; 15 animals per group). The symbols show the values for individual mice, and the short horizontal lines indicate the mean bacterial load for that group. (C) Histopathology of S. aureus Newman-infected lung tissue from mice that were treated with either PBS or IB201. Scale bars on low-magnification images (left) represent 0.1 cm, while scale bars on high-magnification images (right) represent 20 μm. Experimental results shown in panels A, B, and C are representative of the results in two independent experiments.

FIG. 4.

Efficacy of a single dose of β-cyclodextrin derivative IB201 in S. aureus pneumonia. (A) IB201 (10 mg/kg) was administered as a single dose at the indicated time points before or after infection with S. aureus Newman. Mortality was then recorded at 24, 48, and 72 hours postinfection. Values were statistically significantly different (P < 0.035) for animals treated either 2 h before or 2 h after infection (15 animals per group) compared to the control (PBS-treated) animals (statistical significance is denoted by an asterisk). The experimental results shown are representative of the results of two independent experiments.

FIG. 5.

β-Cyclodextrin derivative IB201 protects against S. aureus pneumonia caused by highly virulent CA-MRSA isolate LAC/USA300. (A) IB201 (10, 5, 1, or 0.1 mg/kg) administered 2 h after infection and every 12 h thereafter conferred protection against mortality in mice infected via the i.n. route with S. aureus LAC/USA300 (10 animals per group). Values were statistically significantly different (P ≤ 0.02) from the values for the control (PBS-treated) animals (statistical significance is denoted by an asterisk). The experimental results shown are representative of the results of two independent experiments.