The CsgA and Lpp proteins of an Escherichia coli O157:H7 strain affect HEp-2 cell invasion, motility, and biofilm formation

Abstract

In Escherichia coli O157:H7 strain ATCC 43895, a guanine-to-thymine transversion in the csgD promoter created strain 43895OR. Strain 43895OR produces an abundant extracellular matrix rich in curli fibers, forms biofilms on solid surfaces, invades cultured epithelial cells, and is more virulent in mice than strain 43895. In this study we compared the formic acid-soluble proteins expressed by strains 43895OR and 43895 using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and identified two differentially expressed proteins. A 17-kDa protein unique to strain 43895OR was identified from matrix-assisted laser desorption ionization-time of flight analysis combined with mass spectrometry (MS) and tandem MS (MS/MS) as the curli subunit encoded by csgA. A <10-kDa protein, more highly expressed in strain 43895, was identified as the Lpp lipoprotein. Mutants of strain 43895OR with disruption of lpp, csgA, or both lpp and csgA were created and tested for changes in phenotype and function. The results of this study show that both Lpp and CsgA contribute to the observed colony morphology, Congo red binding, motility, and biofilm formation. We also show that both CsgA and Lpp are required by strain 43895OR for the invasion of cultured HEp-2 cells. These studies suggest that in strain 43895OR, the murein lipoprotein Lpp indirectly regulates CsgA expression through the CpxAR system by a posttranscriptional mechanism.

FIG. 1.

A bis-Tris gel of formic acid extractions from strains 43895 (lane 2), 43895OR (lane 3), 43895OR-Lpp (lane 4), 43895OR-Lpp with pLPP322 (lane 5), 43895OR-CsgA (lane 6), and 43895OR-Lpp/CsgA (lane 7), shown with molecular weight marker proteins (lanes 1 and 8). The positions corresponding to CsgA and Lpp protein migrations are shown in bold outlines.

FIG. 2.

Comparison of the β-glucuronidase activities of strains 43895 and 43895OR containing an lpp::uidA fusion in plasmid pMLK117 and cultured at 25°C in YESCA broth for 24 h or on YESCA agar for 24 h or YESCA agar for 48 h. The enzymatic activity is reported as nanomoles of p-nitrophenol released/min/mg of total protein. Bars represent the averages of duplicate assays of three individual samples from one (24 h agar) or two (24 h broth and 48 h agar) independent trials and were analyzed by analysis of variance using a randomized complete block design.

FIG. 3.

Scanning electron micrographs of colony growth on YESCA agar after 48 h at 25°C for strains 43895 (A), 43895OR-Lpp (B), and 43895OR (C). The enlarged area of panel B demonstrates strain 43895OR-Lpp membrane blebs.

FIG. 4.

Biofilm assay results of strains 43895OR, 43895OR-Lpp, 43895OR-CsgA, 43895-C/L, and 43895 incubated with glass coupons in LB-NS broth for 48 h at 25°C. Each bar represents the mean optical density (OD) of eluted crystal violet dye measured at 590 nm ± the standard deviation from a single sample taken from each of three independent trials. Letters in brackets represent the results of a Bonferroni LSD means separation, and strains having the same letter are not statistically different from each other.

FIG. 5.

HEp-2 cell invasion assays comparing strains 43895OR, 43895OR-Lpp, 43895OR-CsgA, 43895-C/L, and 43895. Each bar represents the mean log₁₀ CFU/ml ± SD/√n of bacteria recovered from gentamicin-exposed HEp-2 cells. Single counts were taken on each of four independent samples from each strain per experiment. The results of three experiments performed on different days were compared by separating the run variability (blocks) from the error term. Letters in brackets represent the results of a Bonferroni LSD means separation, and strains having the same letter are not statistically different from each other.

FIG. 6.

Swimming motility of strains 43895OR, 43895, 43895OR-Lpp, 43895OR-CsgA, and 43895OR-C/L in 0.3% agar at 25°C. Points represent the mean diameters of the zones of migration measured at 16, 19, and 24 h during incubation at 25°C. Means were calculated for five independent biological samples of each strain at each time point, analyzed by analysis of variance, and separated using the Bonferroni LSD mean separation technique.

FIG. 7.

Surface spreading motility of strains 43895OR, 43895OR-Lpp, 43895OR-CsgA, 43895OR-C/L, and 43895 on 0.6% agar with or without 0.5% glucose following incubation for 48 h at 25°C. Bars represent the mean diameters of migration zones calculated for five independent biological samples of each strain and analyzed by analysis of variance with means separation by the Bonferroni LSD mean separation technique. Strains with the same letter in brackets or parentheses were not significantly different from each other in comparisons in agar with or without added glucose, respectively.