Differential transcriptome analysis of enterohemorrhagic Escherichia coli strains reveals differences in response to plant-derived compounds

Abstract

Background: Several serious vegetable-associated outbreaks of enterohemorrhagic Escherichia coli (EHEC) infections have occurred during the last decades. In this context, vegetables have been suggested to function as secondary reservoirs for EHEC strains. Increased knowledge about the interaction of EHEC with plants including gene expression patterns in response to plant-derived compounds is required. In the current study, EHEC O157:H7 strain Sakai, EHEC O157:H^- strain 3072/96, and the EHEC/enteroaggregative E. coli (EAEC) hybrid O104:H4 strain C227-11φcu were grown in lamb's lettuce medium and in M9 minimal medium to study the differential transcriptional response of these strains to plant-derived compounds with RNA-Seq technology.

Results: Many genes involved in carbohydrate degradation and peptide utilization were similarly upregulated in all three strains, suggesting that the lamb's lettuce medium provides sufficient nutrients for proliferation. In particular, the genes galET and rbsAC involved in galactose metabolism and D-ribose catabolism, respectively, were uniformly upregulated in the investigated strains. The most prominent differences in shared genome transcript levels were observed for genes involved in the expression of flagella. Transcripts of all three classes of the flagellar hierarchy were highly abundant in strain C227-11φcu. Strain Sakai expressed only genes encoding the basal flagellar structure. In addition, both strains showed increased motility in presence of lamb's lettuce extract. Moreover, strain 3072/96 showed increased transcription activity for genes encoding the type III secretion system (T3SS) including effectors, and was identified as a powerful biofilm-producer in M9 minimal medium.

Conclusion: The current study provides clear evidence that EHEC and EHEC/EAEC strains are able to adjust their gene expression patterns towards metabolization of plant-derived compounds, demonstrating that they may proliferate well in a plant-associated environment. Moreover, we propose that flagella and other surface structures play a fundamental role in the interaction of EHEC and EHEC/EAEC with plants.

Keywords: Biofilm; Differential gene expression; EHEC; Energy metabolism; Flagella; Lettuce medium; Motility; O104:H4 strain C227–11φcu; O157:H7 strain Sakai; O157:H− strain 3072/96; Plant extract; Transcriptome.

Fig. 1

Growth of different E. coli strains. E. coli O157:H7 strain Sakai (squares), O157:H⁻ strain 3072/96 (circles) and O104:H4 strain C227–11φcu (triangles) were grown in lamb’s lettuce medium (a), and M9 minimal medium supplemented with 0.4% glucose (b) at room temperature and 180 rpm

Fig. 2

Analysis of differentially expressed genes and gene sets of the shared genome. a Principal component analysis plot of normalized expression values for the shared genome of the three different E. coli strains. The PCA-plot was calculated with the DESeq2 package, using TPM normalized gene counts (with the respective strain-specific gene lengths) for the shared genome [25]. Differences between the growth conditions in lamb’s lettuce medium are depicted as dots, and in M9 minimal medium as triangles. E. coli O157:H7 strain Sakai is shown in blue, E. coli O157:H⁻ strain 3072/96 in green, and E. coli O104:H4 strain C227–11φcu in red. b Venn diagram of differentially regulated shared genes shown for all three strains. Up- and downregulated genes resulted from transcriptional patterns of cells grown in lamb’s lettuce medium compared to cells grown in M9 minimal medium. Differentially regulated genes are shown in circles, intersections show overlapping genes of two or three strains. E. coli O157:H7 strain Sakai is marked in blue, E. coli O157:H⁻ strain 3072/96 in green and E. coli O104:H4 strain C227–11φcu in red. c Gene set enrichment analysis showing selected up- and downregulated gene sets derived from transcriptomic analyses of E. coli O157:H⁻ strain 3072/96 (black), E. coli O104:H4 strain C227–11φcu (shaded), and E. coli O157:H7 strain Sakai (grey) after growth in lamb’s lettuce medium and M9 minimal medium

Fig. 3

Differential gene expression of flagellar biosynthesis associated genes. Flagellar genes of the three strains were grouped into classes I to III according to Kalir et al. [28]. Log₂ fold-changes are shown for the different genes of the three classes for each strain with upregulated genes in red, downregulated genes in green and genes not differentially expressed (n. d. e.) in yellow

Fig. 4

Determination of the swimming motility of strains 3072/96 (black), C227–11φcu (shaded) and Sakai (grey) on MMS soft agar plates with and without lamb’s lettuce extract. Significant differences regarding the motility of the strains are indicated with *** (p < 0.001)

Fig. 5

Biofilm formation of the respective strains grown in M9 minimal medium (grey) and in lamb’s lettuce medium (black). Significant differences between the strains and growth conditions are marked with * (p < 0.05), ** (p < 0.01), and *** (p < 0.001)