DFI-seq identification of environment-specific gene expression in uropathogenic Escherichia coli

Abstract

Background: During infection of the urinary tract, uropathogenic Escherichia coli (UPEC) are exposed to different environments, such as human urine and the intracellular environments of bladder epithelial cells. Each environment elicits a distinct bacterial environment-specific transcriptional response. We combined differential fluorescence induction (DFI) with next-generation sequencing, collectively termed DFI-seq, to identify differentially expressed genes in UPEC strain UTI89 during growth in human urine and bladder cells.

Results: DFI-seq eliminates the need for iterative cell sorting of the bacterial library and yields a genome-wide view of gene expression. By analysing the gene expression of UPEC in human urine we found that genes involved in amino acid biosynthesis were upregulated. Deletion mutants lacking genes involved in arginine biosynthesis were outcompeted by the wild type during growth in human urine and inhibited in their ability to invade or proliferate in the J82 bladder epithelial cell line. Furthermore, DFI-seq was used to identify genes involved in invasion of J82 bladder epithelial cells. 56 genes were identified to be differentially expressed of which almost 60% encoded hypothetical proteins. One such gene UTI89_C5139, displayed increased adhesion and invasion of J82 cells when deleted from UPEC strain UTI89.

Conclusions: We demonstrate the usefulness of DFI-seq for identification of genes required for optimal growth of UPEC in human urine, as well as potential virulence genes upregulated during infection of bladder cell culture. DFI-seq holds potential for the study of bacterial gene expression in live-animal infection systems. By linking fitness genes, such as those genes involved in amino acid biosynthesis, to virulence, this study contributes to our understanding of UPEC pathophysiology.

Keywords: Amino acid biosynthesis; DFI; NGS; UPEC; UTI; Virulence.

Fig. 1

A promoter trap library is created using a gfp gene as reporter. The library is transformed into the bacteria of interest. The pool of transformants is both used to infect a tissue culture model and grown in vitro. From both environments, green fluorescent bacteria are enriched using a fluorescence-activated cell sorting instrument and grown over night in vitro. From the two populations plasmids are purified and PCR is performed to amplify trapped promoters. Through next-generation sequencing, host-induced promoters can be identified by comparing promoter read counts between the two conditions

Fig. 2

RT-qPCR verification of differentially expressed genes identified by DFI. RT-qPCR was performed on bacterial RNA isolated from UTI89 grown in human urine and LB, respectively. a and b Expression ratios (log2 scale) of genes differentially expressed in human urine compared to LB, with P < 0.05. c Gene expression ratios (log2 scale) of uniformly expressed genes (P > 0.05). A pairwise fixed reallocation randomisation test was performed [45] and exact p-values can be found in Additional file 10: Table S1

Fig. 3

RT-qPCR verification of differentially expressed genes identified in the DFI-seq experiment. RT-qPCR was performed on bacterial RNA isolated from UTI89 grown in human urine and LB, respectively. a and b Expression ratios (log2 scale) of genes differentially expressed in human urine compared to LB, with P < 0.05. c Gene expression ratios (log2 scale) of uniformly expressed genes (P > 0.05). A pairwise fixed reallocation randomisation test was performed [45] and exact p-values can be found in Additional file 11: Table S2

Fig. 4

Area-proportional Venn-diagram demonstrating the overlap between DFI and DFI-seq results. Genes included were all confirmed by RT-qPCR

Fig. 5

Fitness and relative cell adhesion and invasion of select deletion mutants. a Competitive index of deletion mutants during growth in human urine. The standard error of the mean of two independent 24-h growth experiments is shown (P < 0.05), the exact p-value can be seen in Additional file 7: Additional file 7: Table S3. b Mutants with statistically significant (P < 0,05) adhesion defects to J82 cells, the exact p-value can be seen in Additional file 9: Table S4. c Mutants with statistically significant (P < 0,05) invasion defects the exact p-value can be seen in Additional file 8: Table S5. The standard error of the mean of three independent assays is shown. a, b and c Unpaired t tests with two-tailed p-values were performed using GraphPad Prism 5 software

Fig. 6

Cell adhesion and invasion of UTI89ΔC5139. The standard error of the mean of a minimum of three independent assays is shown for both adhesion and invasion. P-values are indicated above columns. An unpaired t test with two-tailed p-values was performed using GraphPad Prism 5 software