Localized Reactive Oxygen and Nitrogen Intermediates Inhibit Escape ofListeria monocytogenesfrom Vacuoles in Activated Macrophages

Abstract

Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm. Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm escaped from vacuoles of nonactivated macrophages within 30 min of infection. Macrophages activated with IFN-γ, LPS, IL-6, and a neutralizing Ab against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees. Measurements of Lm escape in macrophages from gp91phox−/− and NO synthase 2−/− mice showed that vacuolar retention required ROI and was augmented by RNI. Live cell imaging with the fluorogenic probe dihydro-2′,4,5,6,7,7′-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the localization of ROI and RNI to vacuoles, and by their combined actions in a small space

Stimulation of Inducible Nitric Oxide Synthase Expression by Beta Interferon Increases Necrotic Death of Macrophages uponListeria monocytogenesInfection

“…Strikingly, NO did not exert detectable antimicrobial functions in resting macrophages, as we saw no increase in bacterial number in iNOS-deficient cells. The degree to which RNS contribute to antilisterial defense of activated macrophages varies between different reports (32,34,44). iNOSdeficient mice were shown to be more sensitive to Listeria infection (28), but this has been questioned by others (11,12).…”

Pleiotropic Enhancement of Bacterial Pathogenesis Resulting from the Constitutive Activation of theListeria monocytogenesRegulatory Factor PrfA

“…It appears therefore that L. monocytogenes in some instances promotes its entry into selected cell types (such as epithelial cells) while delaying its entry into others (such as macrophages). Although macrophages are considered a significant portal of entry for L. monocytogenes into animal hosts (61), activated macrophages have been shown to effectively kill L. monocytogenes (45,50), thus they would be just the type of cell that L. monocytogenes would do well to avoid.…”

α-Galactosylceramide Promotes Killing ofListeria monocytogeneswithin the Macrophage Phagosome through Invariant NKT-Cell Activation

“…ROI are produced by M in response to L. monocytogenes infection, and they play a central role in listerial killing within phagosomes (52,53,59). To determine the role of ROI experimentally, the respiratory bursts in M from ␣-GalCer-and vehicle-treated C57BL/6 mice were compared.…”