Shigella flexneri ArcA and Fnr regulate iron acquisition and contribute to plaque formation under anaerobic conditions



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Shigella flexneri is a Gram negative, intracellular pathogen responsible for bacillary dysentery in humans. To achieve infection of the human colonic epithelium, S. flexneri must adapt to varying environmental conditions, including fluctuations in pH, osmolarity, and nutrient and oxygen availabilities. The plaque assay is commonly used to measure the ability of S. flexneri to invade epithelial cells, grow intracellularly, and spread intercellularly to adjacent cells. However, as traditionally performed, this assay is of limited use in testing the virulence of Shigella in response to many of the conditions encountered in the host. I have modified the plaque assay to identify factors contributing to the virulence of S. flexneri under the anaerobic conditions present in the colon. This assay demonstrated that the Feo transport system that acquires anaerobically abundant ferrous iron, as well as the transcription factors ArcA, Fnr, and Fur, impact Shigella plaque formation in anoxic environments. Transcriptional analyses indicated that anaerobic conditions activated expression of feoABC in S. flexneri. Anaerobiosis also repressed genes encoding two other iron transport systems that allow plaque formation by S. flexneri in aerobic environments, the ABC transporter Sit and the Iuc/Iut aerobactin siderophore synthesis and acquisition system that binds ferric iron, the dominant form of iron under aerobic conditions. The anaerobic regulators ArcA and Fnr induced expression of feoABC. Additionally, ArcA represses transcription of iucABCDiutA and fur. fur encodes a transcriptional regulator that is activated in the presence of iron and is responsible for repression of genes encoding iron acquisition systems and the sRNA RyhB that affects synthesis of iron-storage proteins including many TCA cycle enzymes. ArcA is a redox regulator known for redirecting metabolism upon oxygen depletion by down-regulating TCA cycle and aerobic respiratory enzymes and inducing fermentation and anaerobic respiratory complexes. However, ArcA regulation of fur and its downstream targets offers S. flexneri a means to coordinate energy and carbon metabolism with that of iron availability in response to environmental redox conditions.