Dataset Information

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.

SUBMITTER: Madelung M

PROVIDER: S-EPMC5404293 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Publications

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

Madelung Michelle M Kronborg Tina T Doktor Thomas Koed TK Struve Carsten C Krogfelt Karen Angeliki KA Møller-Jensen Jakob J

BMC microbiology 20170424 1

<h4>Background</h4>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 g ...[more]

PMID: 28438119

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Project description:Urinary tract infections (UTI) are among the most frequently encountered infections in clinical practice globally. Predominantly a burden among female adults and infants, UTIs primarily caused by uropathogenic Escherichia coli (UPEC) results in high morbidity and fiscal health strains. During pathogenesis, colonization of the urinary tract via fimbrial adhesion to mucosal cells is the most critical point in infection and has been linked to DNA methylation. Furthermore, with continuous exposure to antibiotics as the standard therapeutic strategy, UPEC has evolved to become highly adaptable in circumventing the effect of antimicrobial agents and host defenses. Hence, the need for alternative treatment strategies arises. Since differential DNA methylation is observed as a critical precursor to virulence in various pathogenic bacteria, this body of work sought to assess the influence of the DNA adenine methylase (dam) gene on gene expression and cellular adhesion in UPEC and its potential as a therapeutic target. To monitor the influence of dam on attachment and FQ resistance, selected UPEC dam mutants created via one-step allelic exchange were transformed with cloned qnrA and dam complement plasmid for comparative analysis of growth rate, antimicrobial susceptibility, biofilm formation, gene expression, and mammalian cell attachment. The absence of DNA methylation among dam mutants was apparent. Varying deficiencies in cell growth, antimicrobial resistance and biofilm formation, alongside low-level increases in gene expression (recA and papI), and adherence to HEK-293 and HTB-9 mammalian cells were also detected as a factor of SOS induction to result in increased mutability. Phenotypic characteristics of parental strains were restored in dam complement strains. Dam's vital role in DNA methylation and gene expression in local UPEC isolates was confirmed. Similarly to dam-deficient Enterohemorrhagic E. coli (EHEC), these findings suggest unsuccessful therapeutic use of Dam inhibitors against UPEC or dam-deficient UPEC strains as attenuated live vaccines. However, further investigations are necessary to determine the post-transcriptional influence of dam on the regulatory network of virulence genes central to pathogenesis.

Project description:Urinary tract infection (UTI) is among the most common infections treated worldwide each year and is caused primarily by uropathogenic Escherichia coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred a consideration of alternative treatment strategies, such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, namely, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6 to 8 h of coincubation. Whole-genome sequencing revealed that UPEC strains resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. Phage-resistant strains displayed several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells and increased biofilm formation in some isolates. Interestingly, these phage-resistant UPEC isolates demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation and were more sensitive to several outer membrane-targeting antibiotics than parental strains. Additionally, phage-resistant UPEC isolates were attenuated in bladder colonization in a murine UTI model. In total, our findings suggest that while resistance to phages, such as HP3 and ES17, may arise readily in the urinary environment, phage resistance is accompanied by fitness costs which may render UPEC more susceptible to host immunity or antibiotics. IMPORTANCE UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage resistance during bacterial infection. In our current study, we found that while phage-resistant bacteria quickly emerged in vitro, these bacteria were less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, of which each is evaded only at the cost of bacterial fitness.

Project description:BackgroundThe uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity.MethodRecombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni2+ chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study.ResultsWe revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni2+ chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity.ConclusionThis is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs.

Dataset Information

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

Background

Results

Conclusions

Publications

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

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