Analysis of the proteome of intracellular Shigella flexneri reveals pathways important for intracellular growth

Abstract

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.

Fig 1

Gel display for comparison of extracellular and intracellular bacterial proteomes during coculture with Henle cells. (A) Extracellular S. flexneri cells; (B) intracellular S. flexneri cells. The depicted Coomassie brilliant blue-stained gel images (20 by 25 by 0.15 cm) are representative for their groups (extracellular n = 11; intracellular n = 5). The spots are denoted in the gels with their short protein names, some of which are also listed in Tables 2, 3, 5, and 6. The molecular mass scale is denoted on the ordinate, and the isoelectric point scale (pI) is on the abscissa. Protein spots in red were increased in abundance in the respective gel, and protein spots in black were decreased compared to the other sample. Protein spots in blue were not altered to a statistically significant extent in the differential displays. The figure does not include all of the statistically significant changes (P < 0.05) with ratios of <0.66 or >1.5. More comprehensive data are provided in Data Sets S1 and S2 in the supplemental material.

Fig 2

Growth of mixed-acid fermentation pathway mutants in vitro. (A) Simplified schematic of the mixed-acid fermentation pathways. (B and C) Wild-type strain 2457T, the pflB mutant CFS212, and the adhE pflB ldhA triple mutant CFS224 (APL) were grown overnight in LB and diluted 1:50 in fresh LB. Once the cultures reached logarithmic-phase growth, they were diluted to an A₆₅₀ of 0.05 and grown at 37°C with shaking (B) or statically (C). Growth was monitored by measuring the A₆₅₀.