N2O3 enhances the nitrosative potential of IFNγ-primed macrophages in response to Salmonella

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 IFNγ-primed phagocytes, despite preserving intracellular transcription of sdhA sdhB subunits of Salmonella succinate dehydrogenase within both control and IFNγ-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 300 nM N 2 O 3 /h/bacteria in IFNγ-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 ~200 nM N 2 O 3 /h sustained by IFNγ-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 NOmetabolizing activity of Salmonella Hmp is functional in both control and IFNγ-primed macrophages. Nonetheless, a substantial amount of the NO generated by IFNγ-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.

Redox sensor SsrB Cys 203 enhances Salmonella fitness against nitric oxide generated in the host immune response to oral infection

“…Of note, the expression of sifA::lacZY and sspH2::lacZY was slightly higher in Salmonella bearing the ssrB C203S allele. Because of their wild-type nitrosative capacity but inability to kill Salmonella (8,14), macrophages lacking the gp91phox subunit of the NADPH oxidase were used to determine the contribution of the redox-active Cys 203 to the transcriptional activity of SsrB in Salmonella exposed to host-derived RNS. Macrophages deficient in gp91phox iNOS hemoproteins unable to sustain nitrosative stress were used as controls.…”

“…N 2 O 3 , which can arise independently from the condensation of acidified nitrite (NO 2 − ), oxidizes and nitrosylates redox-active thiol groups. Salmonella are likely to be exposed to N 2 O 3 in the context of IFNγ-primed macrophages or during transit through the stomach (12)(13)(14). In turn, microorganisms sense NO through the nitrosylation of either iron or sulfhydryl metal groups in response regulators, thereby engaging signaling pathways that promote the enzymatic and nonenzymatic detoxification of RNS.…”

“…Two molecules of HNO 2 in turn condensate to form the strong oxidative and nitrosative species N 2 O 3 . Acidified NO 2 − is a relevant source of nitrosative stress in our system because it adds to the RNS of Salmonella-infected macrophages (14). Strain AV07104 expressing an SsrB::3xFLAG was cultured for 6 h in EG medium, pH 5.5, containing 750 μM NO 2 − or NO 3 − .…”

Codependent and Independent Effects of Nitric Oxide-Mediated Suppression of PhoPQ andSalmonellaPathogenicity Island 2 on IntracellularSalmonella entericaSerovar Typhimurium Survival

“…In addition, RNS generated in acidic environments typical of gastric juice repress the PhoPQ-regulated acid tolerance response that protects rapidly growing S. Typhimurium bacteria against inorganic acid stress (5). S. Typhimurium is exposed to a similar repertoire of RNS within the gastric lumen and acidified phagosomes of gamma interferon (IFN-␥)-stimulated macrophages (36,40). NO itself, as well as nitrogen dioxide (NO 2 ⅐ ) and dinitrogen trioxide (N 2 O 3 ), arising from either the autooxidation of NO or the condensation of acidified nitrite (HNO 2 ), are similarly generated in the lumen of phagosomes and in the stomach (36,40).…”

“…S. Typhimurium is exposed to a similar repertoire of RNS within the gastric lumen and acidified phagosomes of gamma interferon (IFN-␥)-stimulated macrophages (36,40). NO itself, as well as nitrogen dioxide (NO 2 ⅐ ) and dinitrogen trioxide (N 2 O 3 ), arising from either the autooxidation of NO or the condensation of acidified nitrite (HNO 2 ), are similarly generated in the lumen of phagosomes and in the stomach (36,40). Because the PhoPQ signal transduction pathway is susceptible to inhibition by RNS produced under acidic conditions (5), we set out to investigate whether the PhoP regulon is functional in the context of the nitrosative stress engendered in the innate or IFN-␥-activated responses of professional phagocytes.…”