Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. The infectious process depends on survival in the high bile-salt conditions encountered throughout the gastrointestinal tract, including the gallbladder. However, it is not clear how bile-salt resistance mechanisms are induced, especially under physiologically relevant conditions. This study sought to determine how the L. monocytogenes strains EGDe (serovar 1/2a), F2365 (serovar 4a) and HCC23 (serovar 4b) respond to bile salts under anaerobic conditions. Changes in the expressed proteome were analysed using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. In general, the response to bile salts among the strains tested involved significant alterations in the presence of cell-wallassociated proteins, DNA repair proteins, protein folding chaperones and oxidative stressresponse proteins. Strain viability correlated with an initial osmotic stress response, yet continued survival for EGDe and F2365 involved different mechanisms. Specifically, proteins associated with biofilm formation in EGDe and transmembrane efflux pumps in F2365 were expressed, suggesting that variations exist in how virulent strains respond and adapt to high bile-salt environments. These results indicate that the bile-salt response varies among these serovars and that further research is needed to elucidate how the response to bile salts correlates with colonization potential in vivo.

A key aspect underlying the severity of infections caused by Staphylococcus aureus is the abundance of virulence factors that the pathogen uses to thwart critical components of the human immune response. One such mechanism involves the destruction of host immune cells by cytolytic toxins secreted by S. aureus, including five bicomponent leukocidins: PVL, HlgAB, HlgCB, LukED, and LukAB. Purified leukocidins can lyse immune cells ex vivo, and systemic injections of purified LukED or HlgAB can acutely kill mice. Here, we describe the generation and characterization of centyrins that bind S. aureus leukocidins with high affinity and protect primary human immune cells from toxin-mediated cytolysis. Centyrins are small protein scaffolds derived from the fibronectin type III–binding domain of the human protein tenascin-C. Although centyrins are potent in tissue culture assays, their short serum half-lives limit their efficacies in vivo. By extending the serum half-lives of centyrins through their fusion to an albumin-binding consensus domain, we demonstrate the in vivo efficacy of these biologics in a murine intoxication model and in models of both prophylactic and therapeutic treatment of live S. aureus systemic infections. These biologics that target S. aureus virulence factors have potential for treating and preventing serious staphylococcal infections.

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