RESUMEN
Host nitric oxide (NOâ ) production is important for controlling intracellular bacterial pathogens, including Salmonella enterica serovar Typhimurium, but the underlying mechanisms are incompletely understood. S. Typhmurium 14028s is prototrophic for all amino acids but cannot synthesize methionine (M) or lysine (K) during nitrosative stress. Here, we show that NOâ -induced MK auxotrophy results from reduced succinyl-CoA availability as a consequence of NOâ targeting of lipoamide-dependent lipoamide dehydrogenase (LpdA) activity. LpdA is an essential component of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additional effects of NOâ on gene regulation prevent compensatory pathways of succinyl-CoA production. Microarray analysis indicates that over 50% of the transcriptional response of S. Typhimurium to nitrosative stress is attributable to LpdA inhibition. Bacterial methionine transport is essential for virulence in NOâ -producing mice, demonstrating that NOâ -induced MK auxotrophy occurs in vivo. These observations underscore the importance of metabolic targets for antimicrobial actions of NOâ .
Asunto(s)
Ciclo del Ácido Cítrico , Óxido Nítrico/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Acilcoenzima A/metabolismo , Animales , Transporte Biológico , Medios de Cultivo , Dihidrolipoamida Deshidrogenasa/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Lisina/metabolismo , Lisina/farmacología , Metionina/metabolismo , Metionina/farmacología , Ratones , Ratones Endogámicos C3H , Óxido Nítrico/farmacología , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/efectos de los fármacos , Estrés Fisiológico , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismoRESUMEN
Intracellular pathogens must withstand nitric oxide (NO.) generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS) and possesses multiple systems to detoxify NO.. Consequently, the level of NO. stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER) system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP) sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO. sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO. fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO.-dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO.. These observations demonstrate that host-derived NO. damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity.
Asunto(s)
Daño del ADN , Reparación del ADN/fisiología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/inmunología , Salmonella typhimurium/genética , Animales , ADN Glicosilasas/metabolismo , Interacciones Huésped-Patógeno , Ratones , Ratones Endogámicos , Fagocitos/inmunología , Fagocitos/metabolismo , Salmonelosis Animal , Salmonella typhimurium/patogenicidadRESUMEN
Isocitrate lyase is required for fatty acid utilization via the glyoxylate shunt. Although isocitrate lyase is essential for Salmonella persistence during chronic infection, it is dispensable for acute lethal infection in mice. Substrate availability in the phagosome appears to evolve over time, with increasing fatty acid dependence during chronic infection.