Identification of chromosomal Shigella flexneri genes induced by the eukaryotic intracellular environment

Abstract

Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium Shigella flexneri is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly. To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the S. flexneri chromosome fused to a promoterless green fluorescent protein gene (gfp). Bacteria containing promoter fusions that had a higher level of gfp expression when S. flexneri was intracellular (in Henle cells) than when S. flexneri was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting. Nine different genes with increased expression in Henle cells were identified. Several genes (uhpT, bioA, and lysA) were involved in metabolic processes. The uhpT gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro. Two of the intracellularly induced genes (pstS and phoA) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro. Additionally, three iron-regulated genes (sufA, sitA, and fhuA) were identified. The sufA promoter was derepressed in iron-limiting media and was also induced by oxidative stress. To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the S. flexneri uhpT and pstS genes by allelic exchange. The uhpT mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers. The pstS mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain. Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line.

FIG. 1.

Induction of the uhpT-gfp, pst-gfp, and phoA-gfp fusions in Henle cells. Henle cells were infected with SA100 carrying either pSIIG33 (uhpT-gfp) (A), pLR66 (pstS-gfp) (B), or pLR83 (phoA-gfp) (C) for 3.5 h. Bacteria were released from Henle cells by deoxycholate treatment, and the relative fluorescence was quantitated by FACS. Solid lines on the histograms indicate gfp expression in bacteria grown in Henle cells, and lighter, dashed lines indicate gfp expression in bacteria from LB broth cultures used for invasions of Henle cells. The x axis shows the geometric mean fluorescence, and the y axis depicts the number of bacterial cells. M1 and M2 delineate uninduced and induced cell populations, respectively. Ten thousand bacterial cells were assayed for each experimental condition. The experiments were performed three times, and results from a representative experiment are shown.

FIG. 2.

Induction of the uhpT promoter by G6P. SA100 carrying pSIIG33 (uhpT-gfp) was grown in ISM containing 0.4% G6P, and the fluorescence was quantitated by FACS at the indicated times. Ten thousand bacterial cells were assayed for each experimental condition. The data presented are the means from three experiments, and the standard deviations of the means are indicated. The mean fluorescence for a SA100/pSIIG33 fusion grown in ISM containing 0.4% glucose was always less than 10 U and thus is not shown.

FIG. 3.

Induction of the S. flexneri pstS and phoA promoters by phosphate limitation. SA100 carrying either pLR66 (pstS-gfp) (A) or pLR83 (phoA-gfp) (B) was grown in T medium supplemented with potassium phosphate at the indicated levels, and the fluorescence was quantitated by FACS after 5 to 6 h. Ten thousand bacterial cells were assayed for each experimental condition. The experiments were performed two times, and results from a representative experiment are shown.

FIG. 4.

Induction of the S. flexneri sufA promoter. (A) SA100 carrying pLR67 (sufA-gfp) was grown in LB broth to mid-log phase and treated with hydrogen peroxide for 1.5 h as indicated, and the relative fluorescence was quantitated by FACS. (B) SA100 carrying pLR67 (sufA-gfp) was grown in LB broth containing EDDA as indicated, and the fluorescence was quantitated by FACS after 5 h. Ten thousand bacterial cells were assayed for each experimental condition. The data presented are the means from three experiments, and the standard deviations of the means are indicated.

FIG. 5.

The S. flexneri uhpT mutant cannot use G6P as a carbon source. Overnight cultures of the parental strain SM100 (solid bars) and the uhpT mutant SM165 (stippled bars) were subcultured into ISM with either 0.4% glucose or G6P as the sole carbon source. The optical density at 650 nm (OD₆₅₀) of the cultures was measured after 20 h. The data presented are the means from three experiments, and the standard deviations of the means are indicated.

FIG. 6.

The S. flexneri pstS mutant forms small plaques on Henle cell monolayers. Confluent Henle cell monolayers were infected with 10⁴ bacteria per 35-mm-diameter plate and the plaques were photographed after 4 days. SM100 containing the vector control pLAFR1 formed plaques of the same size as those formed by SM100.