Temporal expression of enteropathogenic Escherichia coli virulence genes in an in vitro model of infection

Abstract

The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the ability of EPEC to cause attaching and effacing (A/E) lesions on intestinal epithelium. This event is reproducible in in vitro tissue culture models of infection. We used real-time PCR to measure transcription from several locus of enterocyte effacement (LEE) operons (LEE1 to LEE5) and from bfp during a 5-h infection of HEp-2 cells with EPEC. We found that after the initial formation of A/E lesions, which occurs as early as 5 min postinfection, EPEC continues to increase transcription from LEE3 to LEE5 as well as from bfp. These levels are maximized by 3 h postinfection and remain constant throughout the course of infection. This increase in transcription from LEE3 to LEE5 occurs when LEE1 (ler) transcription is decreasing. EspA, EspB, intimin, Tir, and bundle-forming pilus expression is detectable during the entire 5-h infection. These results indicate that the EPEC genes involved in localized and intimate adherence are continually expressed after the initial stages of A/E lesion formation on host cells.

FIG. 1.

EPEC virulence gene expression is increased by growth in DMEM. (a) Fold change in gene transcription in E2348/69 grown to mid-log phase in DMEM versus LB medium, as measured by real-time PCR. Primers used for each gene are shown in Table 2. Results shown are means and standard deviations from triplicate experiments. CT values were normalized to levels of 16S rRNA to correct for variations in bacterial numbers. Fold differences were calculated by using the relative comparison method. (b) Western blot showing the expression of intimin, Tir, EspB, and BfpA in E2348/69 grown to mid-log phase in LB medium (lane 1) and DMEM (lane 2). Bacterial cultures were adjusted to equivalent optical densities at 600 nm prior to loading.

FIG. 2.

Transcriptional profiles of LEE expression and bfp expression during a 5-h infection for EPEC on HEp-2 cells (a), for EPEC in the absence of HEp-2 cells (b), and for JAC719 (tir mutant) on HEp-2 cells (c). Relative expression represents the fold change in E2348/69 gene transcription from 10 min to 3 and 5 h postinfection, as measured by real-time PCR. Primers used for each gene are indicated in Table 2. Results shown are means and standard deviations from triplicate infections. CT values were normalized to levels of 16S rRNA to correct for variations in bacterial numbers. Fold differences from the 10-min time point were calculated by using the relative comparison method. Values that were significantly different from values at 10 min are indicated by an asterisk (P < 0.05).

FIG. 3.

EPEC protein production and translocation are evident 5 h postinfection. A Western blot of Triton X-100 fractions shows E2348/69 protein expression during 3-h and 5-h infections of HEp-2 cells. Triton X-100 total lysates (soluble and insoluble) were plated prior to fractionation to determine total CFU. Lanes of SDS-polyacrylamide gels were loaded with equivalent CFU. Uninfected lanes were loaded with the same volume as that used for the 5-h lysate. Transfer membranes were probed with anti-intimin, anti-Tir, anti-EspB, or anti-BfpA antisera. Sea-Blue 2 molecular size markers (in kilodaltons) are indicated at the left. The E2348/69 whole-cell lysate was prepared from bacteria grown to mid-log phase in DMEM. Protein bands in the Triton X-100-soluble fraction are indicated by solid arrowheads; protein bands in the Triton X-100-insoluble fraction are indicated by open arrowheads. PY, tyrosine phosphorylated.

FIG. 4.

Intimin and Tir are coexpressed at 3 and 5 h after HEp-2 cell infection with E2348/69. Intimin expression and Tir expression were demonstrated at 3 and 5 h postinfection by immunofluorescence microscopy and confocal microscopy. Intimin and Tir were not visualized in isogenic mutant strains at either time point (CVD206 and JAC719, respectively). Cells were triply labeled with either anti-Tir or anti-intimin antisera, for actin with Alexa-Fluor 488-phalloidin (green), and for bacterial and HEp-2 cell DNA with DAPI (blue). The primary antibody was visualized by using Alexa-Fluor 568-goat anti-rabbit secondary antibody (red). Bacteria were preactivated in DMEM prior to infection of HEp-2 cells at an approximate MOI of 100.

FIG. 5.

EspA filaments and the BFP are expressed at 3 and 5 h after HEp-2 cell infection with E2348/69. EspA expression and BFP expression were demonstrated by immunofluorescence microscopy and confocal microscopy. Isogenic mutants CVD452 and UMD901 did not express surface EspA and BFP, respectively. Cells were labeled with either anti-EspA or anti-BfpA antisera, for actin with Alexa-Fluor 488-phalloidin (green), and for bacterial and HEp-2 cell DNA with DAPI (blue). The primary antibody was visualized by using Alexa-Fluor 568-goat anti-rabbit secondary antibody (red). Bacteria were preactivated in DMEM prior to infection of HEp-2 cells at an approximate MOI of 100.