Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.
The ferrous iron transport system Feo is widely distributed among bacterial species, yet its physical structure and mechanism of iron transport are poorly understood. In Vibrio cholerae, the feo operon consists of three genes, feoABC. feoB encodes an 83-kDa protein with an amino-terminal GTPase domain and a carboxy-terminal domain predicted to be embedded in the inner membrane. While FeoB is believed to form the pore for iron transport, the roles of FeoA and FeoC are unknown. In this work, we show that FeoA and FeoC, as well as the more highly conserved FeoB, are all required for iron acquisition by V. cholerae Feo. An in-frame deletion of feoA, feoB, or feoC eliminated iron acquisition. The loss of transport activity in the feoA and feoC mutants was not due to reduced transcription of the feo operon, suggesting that these two small proteins are required for activity of the transporter. feoC was found to encode a protein that interacts with the cytoplasmic domain of FeoB, as determined using the BACTH bacterial two-hybrid system. Two conserved amino acids in FeoC were found to be necessary for the interaction with FeoB in the two-hybrid assay, and when either of these amino acids was mutated in the context of the entire feo operon, iron acquisition via Feo was reduced. No interaction of FeoA with FeoB or FeoC was detected in the BACTH two-hybrid assay.