Using Salmonella Pathogenesis and Cell Biology as a Discovery Tool

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SUMMARY The inner membrane of pathogenic Gram-negative bacteria contains lipids and proteins that are distinct from those of mammalian cell membranes. Inner membranes therefore could be attacked by small molecules during infection, when soluble host innate immunity damages the protective outer membrane barrier, increasing access to chemicals. Exploration of molecules that a) interrupt pathogenesis in cell culture with minimal host cell damage, and b) perturb inner membranes under broth conditions that permeabilize the outer membrane has the potential to reveal whether bacterial virulence can be short-circuited by compounds that disturb inner membranes but do not inhibit bacterial growth under standard broth conditions. An in-macrophage cell biology- based method called SAFIRE appears to identify compounds that negatively affect bacterial inner membranes while sparing host cell membranes. Within this application we propose to study a SAFIRE-identified compound, D66, which appears to damage bacterial inner membranes without lysing them. Published and unpublished preliminary data together indicate that D66 accesses bacterial inner membranes when the LPS layer is damaged or efflux pumps are compromised, conditions consistent with the macrophage phagosome. However, D66 disrupts inner membrane voltage rapidly and at concentrations that do not destroy the inner membrane lipid bilayer, indicating that the compound is not causing bacterial lysis but is disturbing the membrane more subtly. We hypothesize that D66 damages the bacterial cell membrane in a process that triggers stress response pathways but does not cause rapid physical disruption of the lipid bilayer. We propose cell biological and animal infection experiments to test this hypothesis.