Abstract
We show here that the nitric oxide (NO)-detoxifying Hmp flavohemoprotein increases by 3-fold the transcription of the Salmonella pathogenicity island 2 (SPI2) in macrophages expressing a functional inducible NO synthase (iNOS). However, Hmp does not prevent NO-related repression of SPI2 transcription in IFNgamma-primed phagocytes, despite preserving intracellular transcription of sdhA sdhB subunits of Salmonella succinate dehydrogenase within both control and IFNgamma-primed phagocytes. To shed light into the seemingly paradoxical role that Hmp plays in protecting intracellular SPI2 expression in various populations of macrophages, N(2)O(3) was quantified as an indicator of the nitrosative potential of Salmonella-infected phagocytes in different states of activation. Hmp was found to prevent the formation of 300nM N(2)O(3)/h/bacteria in IFNgamma-primed macrophages, accounting for about a 60% reduction of the nitrosative power of activated phagocytes. Utilization of the vacuolar ATPase inhibitor bafilomycin indicates that a fourth of the approximately 200nM N(2)O(3)/h sustained by IFNgamma-primed macrophages is generated in endosomal compartments via condensation of HNO(2). In sharp contrast, control macrophages infected with wild-type Salmonella produce as little N(2)O(3) as iNOS-deficient controls. Collectively, these findings indicate that the NO-metabolizing activity of Salmonella Hmp is functional in both control and IFNgamma-primed macrophages. Nonetheless, a substantial amount of the NO generated by IFNgamma-primed macrophages gives rise to N(2)O(3), a species that not only enhances the nitrosative potential of activated phagocytes but also avoids detoxification by Salmonella Hmp.