Article Figures & Data
Figures
- FIG 1
Aurodox inhibits secretion of T3SS-associated effector proteins in EPEC, EHEC, and Citrobacter rodentium without affecting bacterial growth. (A) Secreted protein fractions were prepared by culturing strains in T3S-inducing medium and precipitated from the supernatant using trichloroacetic acid. Whole-cell fractions were prepared by lysis of cell pellets. Samples were resolved using SDS-gel electrophoresis followed by Coomassie staining. Marked protein bands were excised and trypsin digested for mass spectrometry identification of proteins. (B) Aurodox does not inhibit the growth of EPEC, EHEC, or C. rodentium in T3S-inducing medium. EHEC was grown in MEM-HEPES, and EPEC and C. rodentium were grown in DMEM. Growth rates were determined spectrophotometrically (OD600; n = 3). Error bars plotted are SDs. Changes in growth were not statistically significant.
- FIG 2
Effect of Aurodox on EHEC infection of epithelial cells and A/E lesion formation. (A) Representative microscopy images from EHEC cell infection assay. Cells were infected with 107 EHEC cells transformed with prpsM-gfp (green) to facilitate quantification and imaging. HeLa cells were actin stained with phalloidin-Alexa Fluor 555 (red) and mounted in Vectashield with DAPI (blue). Scale bar represents 50 μm. Insets contain a ×4 magnification of the indicated area. (B) Colonization was quantitated by counting the numbers of cells possessing EHEC on their surface and expressing as a percentage of the total. (C) Following infection, HeLa cells were washed to remove nonadherent bacteria and subsequently lysed to release colonized bacteria. The CFU of EHEC in the lysate were enumerated, and colonization efficiency was calculated by expressing as a percentage of the inoculum. Significance was calculated by paired Student’s t test. *, P < 0.05; **, P < 0.01.
- FIG 3
Transcriptional changes induced by Aurodox in EHEC. (A) Venn diagram representing overlap between genes significantly downregulated by Aurodox (>1.5 fold; P < 0.05) and LEE genes. (B) Heat map representation of log2 fold change in gene expression after treatment with Aurodox. (C) Effect of Aurodox on expression of ler and the housekeeping gene rpsM. Aurodox was added at 5 µg/ml and expression measured after 4 h of growth. Expression of rpsM was not significantly altered in contrast to that of Ler (P = 0.009). Error bars plotted are equivalent to the SDs (n = 3). **, P < 0.005.
- FIG 4
Transcriptional repression of ler does not occur as a result of the interaction of Aurodox with the T3S needle. (A) Analysis of ler expression in EPEC mutants with deletions of genes encoding structural components of the T3SS. Mutant strains were transformed with pler-gfp. Optical density and fluorescence were measured at hourly intervals and ler expression was quantified as relative fluorescence units (fluorescence/OD600). (B) Relative ler expression during exponential phase in WT EPEC versus EPEC ΔescC treated with Aurodox. Expression of ler in treated samples is expressed as a percentage of untreated samples. (C) Effect of Ler overexpression on the Aurodox phenotype. Arabinose was added to induce expression, and the effect of Aurodox on Tir expression was determined by immunoblotting.
- FIG 5
Analysis of the effect of Aurodox on RecA-mediated Stx expression. (A) Analysis of in vitro recA-gfp expression in Aurodox-treated EHEC. Aurodox and ciprofloxacin were added at 6 µM. Error bars correspond to SDs (n = 4). P values for Aurodox-treated versus ciprofloxacin-treated bacteria were <0.01. *, P < 0.05; **, P < 0.005; ***, P < 0.001. (B) Immunoblot analysis of Stx expression in untreated, Aurodox-treated, ciprofloxacin-treated, and Aurodox- and ciprofloxacin-treated C. rodentium DBS100. Primary antibodies for Shiga toxin B subunit (6 kDa) were used.
Additional Files
Supplemental material
- Supplemental file 1 -
Table S1. Strains used in this study. Table S2. Plasmids used in this study. Table S3. MASCOT results for EPEC-secreted protein bands. Table S4. MASCOT results for EHEC-secreted protein bands. Table S5. MASCOT result for C. rodentium-secreted protein bands. Table S6. Chromosomal EHEC TUV93-0 genes downregulated in the presence of aurodox. Table S7. Genes upregulated in the presence of aurodox. Fig. S1. Effect of aurodox on ler expression in EPEC.
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- Supplemental file 1 -
