Escherichia coli O157:H7 survives within human macrophages: global gene expression profile and involvement of the Shiga toxins

Abstract

Escherichia coli O157:H7 is an important food-borne pathogen that specifically binds to the follicle-associated epithelium in the intestine, which rapidly brings this bacterial pathogen in contact with underlying human macrophages. Very little information is available about the interaction between E. coli O157:H7 and human macrophages. We evaluated the uptake and survival of strain EDL933 during infection of human macrophages. Surprisingly, EDL933 survived and multiplied in human macrophages at 24 h postinfection. The global gene expression profile of this pathogen during macrophage infection was determined. Inside human macrophages, upregulation of E. coli O157:H7 genes carried on O islands (such as pagC, the genes for both of the Shiga toxins, and the two iron transport system operons fit and chu) was observed. Genes involved in acid resistance and in the SOS response were upregulated. However, genes of the locus of enterocyte effacement or genes involved in peroxide resistance were not differentially expressed. Many genes with putative or unknown functions were upregulated inside human macrophages and may be newly discovered virulence factors. As the Shiga toxin genes were upregulated in macrophages, survival and cytotoxicity assays were performed with isogenic Shiga toxin mutants. The initial uptake of Shiga toxins mutants was higher than that of the wild type; however, the survival rates were significantly lower at 24 h postinfection. Thus, Shiga toxins are implicated in the interaction between E. coli O157:H7 and human macrophages. Understanding the molecular mechanisms used by E. coli to survive within macrophages may help in the identification of targets for new therapeutic agents.

FIG. 1.

Infection of THP-1 macrophages with EDL933. (A) Quantification of intracellular bacteria at different times postinfection, where 0 h corresponds to 20 min of phagocytosis. (B and C) Fluorescence microscopy of infected macrophages, with EDL933-GFP and human macrophages at 2 h (B) or 24 h (C) postinfection (magnification, ×1,000). (D) Cytometry analysis of EDL933-GFP in contact with human macrophages at 2 h (gray) and 24 h (black) postinfection. All assays were conducted in duplicate and repeated independently at least three times. Results are expressed as means ± standard deviations for the replicate experiments, and the Student t test was used for statistical analysis of the data.

FIG. 2.

Global analysis of differentially expressed genes. (A) Number of upregulated or downregulated genes at each intracellular time point of macrophage infection. (B) Venn diagram of the number of up- or downregulated genes at each intracellular time point. (C) Distribution of E. coli genes in functional categories (COG). Percentages of upregulated and downregulated genes for each functional class at 24 h postinfection are shown. Data are the means from four replicates.

FIG. 3.

Real-time qPCR and microarray results for a set of seven representative genes at 24 h postinfection compared to RPMI. Data are the means and standard deviations for RNA extracted from three different infections. See text for details.

FIG. 4.

Percentage of upregulated and downregulated genes for selected OI (OI containing more than six genes and showing a minimum of 15% of differentially expressed genes) at 24 h postinfection. Data are the means from four technical replicates.

FIG. 5.

Heat map of expression levels of predicted open reading frame selected from specific OI over time during macrophage infection. Expression values were determined from the ANOVA and are represented colorimetrically, with red representing upregulation (ratio of 3) and green representing downregulation (ratio of −3) on a log₂ scale. Data are the means from four technical replicates.

FIG. 6.

Heat map of gene expression from the SOS response, oxidative stress, and metal transport gene classes. Data are the means from four technical replicates.

FIG. 7.

Role of Stx during macrophages infection. (A) Bacterial uptake (percent compared to inoculum) and survival (percent compared to 0 h) of the stx mutants. (B) Cytotoxicity of strain EDL933 O157:H7 and isogenic mutants measured by LDH release after 24 h of infection. The figures show the means ± standard deviations from at least three independent experiments. An asterisk indicates a significant difference compared to EDL933 (P < 0.05).