Staphylococcus aureus pneumonia is one of the most common invasive diseases caused by this human pathogen. S. aureus alpha-hemolysin, a pore-forming cytotoxin, is an essential virulence factor in the pathogenesis of pneumonia. Vaccine-based targeting of this toxin provides protection against lethal staphylococcal pneumonia in a murine model system, suggesting that a monoclonal antibody-based therapy may likewise prove to be efficacious for prevention and treatment of this disease. We report the generation of two distinct anti-alpha-hemolysin monoclonal antibodies that antagonize toxin activity, preventing human lung cell injury in vitro and protecting experimental animals against lethal S. aureus pneumonia. Each of these two monoclonal antibodies recognized an epitope within the first 50 amino acid residues of the mature toxin and blocked the formation of a stable alpha-hemolysin oligomer on the target cell surface. Active immunization with the first 50 amino acids of the toxin also conferred protection against S. aureus pneumonia. Together, these data reveal passive and active immunization strategies for prevention or therapy of staphylococcal pneumonia and highlight the potential role that a critical epitope may play in defining human susceptibility to this deadly disease.

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. ␤-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 ␤-cyclodextrin compound, IB201, prevents alpha-hemolysin-induced lysis of human alveolar epithelial cells. This protective effect does not result from the ability of the ␤-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 ␤-cyclodextrin compounds, provide a novel strategy that may be developed to combat S. aureus pneumonia.

In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygenavailability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory iscR gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.