Transcriptomic and proteomic analysis of the virulence inducing effect of ciprofloxacin on enterohemorrhagic Escherichia coli

Abstract

Enterohemorrhagic E. coli (EHEC) is considered to be the most dangerous pathotype of E. coli, as it causes severe conditions such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Antibiotic treatment of EHEC infections is generally not recommended since it may promote the production of the Shiga toxin (Stx) and lead to worsened symptoms. This study explores how exposure to the fluoroquinolone ciprofloxacin reorganizes the transcriptome and proteome of EHEC O157:H7 strain EDL933, with special emphasis on virulence-associated factors. As expected, exposure to ciprofloxacin caused an extensive upregulation of SOS-response- and Stx-phage proteins, including Stx. A range of other virulence-associated factors were also upregulated, including many genes encoded by the LEE-pathogenicity island, the enterohemolysin gene (ehxA), as well as several genes and proteins involved in LPS production. However, a large proportion of the genes and proteins (17 and 8%, respectively) whose expression was upregulated upon ciprofloxacin exposure (17 and 8%, respectively) are not functionally assigned. This indicates a knowledge gap in our understanding of mechanisms involved in EHECs response to antibiotic-induced stress. Altogether, the results contribute to better understanding of how exposure to ciprofloxacin influences the virulome of EHEC and generates a knowledge base for further studies on how EHEC responds to antibiotic-induced stress. A deeper understanding on how EHEC responds to antibiotics will facilitate development of novel and safer treatments for EHEC infections.

Fig 1. The effect of ciprofloxacin (0.06 μg/mL) on the growth of EDL933 as measured by OD₆₀₀.

Results are shown as means of three independent experiments with bars showing ± standard deviation.

Fig 2. A column chart showing the fold-changes in gene expression in ciprofloxacin-treated samples in comparison to untreated samples.

All 5,370 locus tags from EDL933_RS00005- EDL933_RS34060 are shown from left to the right. Regions that contain the locus tags for Stx1/2 phages (CP-933V and BP-933W) and the pO157 virulence plasmid has been marked with lines in that area.

Fig 3

A column chart showing the fold changes in protein abundance of ciprofloxacin-treated samples at 3 h (A) and 12 h (B) in comparison to unexposed samples. All 5,370 locus tags from EDL933_RS00005- EDL933_RS34060 are shown from left to right, and proteins that were not isolated were set to 1, which also means unchanged. Columns above 1 illustrates proteins upregulated by ciprofloxacin and columns below 1 illustrates proteins that are downregulated by ciprofloxacin.

Fig 4. The change in transcriptional pattern made by ciprofloxacin treatment of different functional categories of genes presented in a Voronoi tree map.

Red cells represent significantly upregulated genes (P-adj < 0.05), blue cells represent significantly downregulated genes (P-adj < 0.05), and gray cells represent either unchanged expression compared to uninduced control samples or DE but with P-adj > 0.05. The top panel show a general representation of the functional pathways that the genes are sorted by.

Fig 5

All detected proteins shown by Voronoi tree maps at 3 h (A) and 12 h (B). The fold change in protein abundance (ciprofloxacin/control) for individual proteins are indicated as follows: Sharp red = significantly increased abundance by ciprofloxacin treatment (p < 0.05), light red = increased abundance but not significant, sharp blue = significantly lower abundance (p < 0.05), light blue = lower abundance but not significant and white = proteins that were detected but found to be regulated (within parameters) P < 0.05 (Student’s t-test). In the Voronoi tree maps, clustering in categories indicate functional relationships in the same way as in the top panel of Fig 3.

Fig 6. A column charts showing the fold changes in gene expression in ciprofloxacin-treated samples in comparison to untreated samples in BP-933W (Stx2) and CP-933V (Stx1).

All shown data have a P-adj value < 0.05.

Fig 7. A column charts showing the fold changes of protein yield in ciprofloxacin-treated samples in comparison to untreated samples in the Stx2 (BP-933W) and Stx1 (CP-933V) phages.

(A) 3 h and (B) 12 h.