RpoS acts as a global repressor of virulence gene expression in E. coli O104:H4 and enteroaggregative E. coli.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.

A Robust One-Step Recombineering System for Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3-4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.

The Superior Adherence Phenotype of E. coli O104:H4 is Directly Mediated by the Aggregative Adherence Fimbriae Type I.

Whereas the O104:H4 enterohemorrhagic Escherichia coli (EHEC) outbreak strain from 2011 expresses aggregative adherence fimbriae of subtype I (AAF/I), its close relative, the O104:H4 enteroaggregative Escherichia coli (EAEC) strain 55989, encodes AAF of subtype III. Tight adherence mediated by AAF/I in combination with Shiga toxin 2 production has been suggested to result in the outbreak strain's exceptional pathogenicity. Furthermore, the O104:H4 outbreak strain adheres significantly better to cultured epithelial cells than archetypal EAEC strains expressing different AAF subtypes. To test whether AAF/I expression is associated with the different virulence phenotypes of the outbreak strain, we heterologously expressed AAF subtypes I, III, IV, and V in an AAF-negative EAEC 55989 mutant and compared AAF-mediated phenotypes, incl. autoaggregation, biofilm formation, as well as bacterial adherence to HEp-2 cells. We observed that the expression of all four AAF subtypes promoted bacterial autoaggregation, though with different kinetics. Disturbance of AAF interaction on the bacterial surface via addition of α-AAF antibodies impeded autoaggregation. Biofilm formation was enhanced upon heterologous expression of AAF variants and inversely correlated with the autoaggregation phenotype. Co-cultivation of bacteria expressing different AAF subtypes resulted in mixed bacterial aggregates. Interestingly, bacteria expressing AAF/I formed the largest bacterial clusters on HEp-2 cells, indicating a stronger host cell adherence similar to the EHEC O104:H4 outbreak strain. Our findings show that, compared to the closely related O104:H4 EAEC strain 55989, not only the acquisition of the Shiga toxin phage, but also the acquisition of the AAF/I subtype might have contributed to the increased EHEC O104:H4 pathogenicity.

IHF stabilizes pathogenicity island I of uropathogenic Escherichia coli strain 536 by attenuating integrase I promoter activity.

Pathogenicity islands (PAIs) represent horizontally acquired chromosomal regions and encode their cognate integrase, which mediates chromosomal integration and excision of the island. These site-specific recombination reactions have to be tightly controlled to maintain genomic stability, and their directionality depends on accessory proteins. The integration host factor (IHF) and the factor for inversion stimulation (Fis) are often involved in recombinogenic complex formation and controlling the directionality of the recombination reaction. We investigated the role of the accessory host factors IHF and Fis in controlling the stability of six PAIs in uropathogenic Escherichia coli strain 536. By comparing the loss of individual PAIs in the presence or absence of IHF or Fis, we showed that IHF specifically stabilized PAI I536 and that in particular the IHFB subunit seems to be important for this function. We employed complex genetic studies to address the role of IHF in PAI I536-encoded integrase (IntI) expression. Based on different YFP-reporter constructs and electrophoretic mobility shift assays we demonstrated that IntI acts a strong repressor of its own synthesis, and that IHF binding to the intI promoter region reduces the probability of intI promoter activation. Our results extend the current knowledge of the role of IHF in controlling directionality of site specific recombination reactions and thus PAI stability.

RAB5A and TRAPPC6B are novel targets for Shiga toxin 2a inactivation in kidney epithelial cells.

The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown. To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells. Though both lines exhibited the Stx receptor globotriaosylceramide, RNAseq revealed strikingly different transcriptomic responses to an Stx challenge. Using RNAi to silence factors involved in ACHN cells' Stx response, the greatest protection occurred when silencing RAB5A and TRAPPC6B, two host factors that we newly link to Stx trafficking. Silencing these factors alongside YKT6 fully prevented the cytotoxic Stx effect. Overall, our approach reveals novel subcellular targets for potential therapies against Stx-mediated kidney failure.

Transcriptional and Translational Inhibitors Block SOS Response and Shiga Toxin Expression in Enterohemorrhagic Escherichia coli.

Shiga toxins (Stx) induce the symptoms of the life-threatening hemolytic uremic syndrome (HUS) and are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC). The bacterial SOS response is the essential signal for high level production and release of Stx1/2. To assess the potential effectiveness of different antibiotics in blocking SOS response and Stx1/2 production, we constructed a reporter gene based test system that allows for the time-resolved, simultaneous read-out of the SOS response (recAP-cfp) and Stx1 production (stx1::yfp) in EHEC O157:H7 EDL933. We find that cells exposed to inhibitory or subinhibitory concentrations of ciprofloxacin did induce the SOS response, but not when the cells were exposed to rifaximine, azithromycin, tetracycline, gentamicin or ampicillin. Cell lysis and the peak in Stx1 production were substantially delayed with respect to the peak of the SOS response. We used this feature to show that adding transcriptional or translational inhibitors can block Stx1 production even after the SOS response is fully induced. RT-qPCR based tests with other clinically relevant EHEC isolates showed similar results for both Stx1 and Stx2. These observations suggest that transcriptional and translational inhibitors may be of value in treating EHEC infections.

Carriage of Shiga toxin phage profoundly affects Escherichia coli gene expression and carbon source utilization.

BACKGROUND: Enterohemorrhagic Escherichia coli (E. coli) are intestinal pathogenic bacteria that cause life-threatening disease in humans. Their cardinal virulence factor is Shiga toxin (Stx), which is encoded on lambdoid phages integrated in the chromosome. Stx phages can infect and lysogenize susceptible bacteria, thus either increasing the virulence of already pathogenic bacterial hosts or transforming commensal strains into potential pathogens. There is increasing evidence that Stx phage-encoded factors adaptively regulate bacterial host gene expression. Here, we investigated the effects of Stx phage carriage in E. coli K-12 strain MG1655. We compared the transcriptome and phenotype of naive MG1655 and two lysogens carrying closely related Stx2a phages: ϕO104 from the exceptionally pathogenic 2011 E. coli O104:H4 outbreak strain and ϕPA8 from an E. coli O157:H7 isolate. RESULTS: Analysis of quantitative RNA sequencing results showed that, in comparison to naive MG1655, genes involved in mixed acid fermentation were upregulated, while genes encoding NADH dehydrogenase I, TCA cycle enzymes and proteins involved in the transport and assimilation of carbon sources were downregulated in MG1655::ϕO104 and MG1655::ϕPA8. The majority of the changes in gene expression were found associated with the corresponding phenotypes. Notably, the Stx2a phage lysogens displayed moderate to severe growth defects in minimal medium supplemented with single carbon sources, e.g. galactose, ribose, L-lactate. In addition, in phenotype microarray assays, the Stx2a phage lysogens were characterized by a significant decrease in the cell respiration with gluconeogenic substrates such as amino acids, nucleosides, carboxylic and dicarboxylic acids. In contrast, MG1655::ϕO104 and MG1655::ϕPA8 displayed enhanced respiration with several sugar components of the intestinal mucus, e.g. arabinose, fucose, N-acetyl-D-glucosamine. We also found that prophage-encoded factors distinct from CI and Cro were responsible for the carbon utilization phenotypes of the Stx2a phage lysogens. CONCLUSIONS: Our study reveals a profound impact of the Stx phage carriage on E. coli carbon source utilization. The Stx2a prophage appears to reprogram the carbon metabolism of its bacterial host by turning down aerobic metabolism in favour of mixed acid fermentation.

In vivo competition and horizontal gene transfer among distinct Staphylococcus aureus lineages as major drivers for adaptational changes during long-term persistence in humans.

BACKGROUND: The airways of the majority of adolescent cystic fibrosis (CF) patients are persistently colonized or infected by Staphylococcus aureus. Using whole genome sequencing, we studied the evolutionary traits within a S. aureus population in the airways of a CF patient hypothesizing that horizontal gene transfer (HGT) and inter-bacterial interaction play a major role in adaptation during long-term persistence. RESULTS: Whole genome sequencing of 21 S. aureus isolates spanning 13 years resulted in seven lineages defined by the spa types t012, t021, t331, t338, t364, t056, and t2351. Of these, the successfully persisting lineages t012 and t021 were closely related suggesting the evolution of t021 from t012, which was further corroborated by a nearly identical, syntenic set of mobile genetic elements. During transformation from t012 to t021, an increase of genomic changes including HGT from other S. aureus lineages was detected. CONCLUSIONS: In summary, our in vivo data enabled us to conceptualize an evolutionary model showing the impact of HGT and inter-bacterial interaction on bacterial long-term adaptation to the human host during CF.

The 2011 German Enterohemorrhagic Escherichia Coli O104:H4 Outbreak-The Danger Is Still Out There.

Enterohemorrhagic Escherichia coli (EHEC) are Shiga toxin (Stx) producing bacteria causing a disease characterized by bloody (or non-bloody) diarrhea, which might progress to hemolytic uremic syndrome (HUS). EHEC O104:H4 caused the largest ever recorded EHEC outbreak in Germany in 2011, which in addition showed the so far highest incidence rate of EHEC-related HUS worldwide. The aggressive outbreak strain carries an unusual combination of virulence traits characteristic to both EHEC-a chromosomally integrated Stx-encoding bacteriophage, and enteroaggregative Escherichia coli-pAA plasmid-encoded aggregative adherence fimbriae mediating its tight adhesion to epithelia cells. There are currently still open questions regarding the 2011 EHEC outbreak, e.g., with respect to the exact molecular mechanisms resulting in the hypervirulence of the strain, the natural reservoir of EHEC O104:H4, and suitable therapeutic strategies. Nevertheless, our knowledge on these issues has substantially expanded since 2011. Here, we present an overview of the epidemiological, clinical, microbiological, and molecular biological data available on the 2011 German EHEC O104:H4 outbreak.

Core elements of the vegetative replication control of the Inc1 plasmid pO104_90 of Escherichia coli O104:H4 also regulate its transfer frequency.

The highly virulent Escherichia coli O104:H4 isolate that caused a large outbreak in 2011 carries three plasmids. Out of these, only one, the IncI plasmid pO104_90 that encodes two extended spectrum beta-lactamases, can transfer itself by conjugation. Considering its potential contribution to the emergence and virulence of the outbreak strain, we aimed to get a closer insight into pO104_90 transfer efficiency and control. We tested the host spectrum of the plasmid and observed transmission into Enterobactericeae including clinically relevant enterobacterial pathogens like Salmonella typhimurium and Shigella flexneri. However, we found that this plasmid did not transfer into E. coli strains that kill the donor strain due to bacteriocin production, e.g. the probiotic E. coli Nissle 1917. Under the same conditions, the highly transmittable control plasmid RP4 was efficiently transferred into all these recipients. Therefore we hypothesized that the failure of transfer of pO104_90 was simply due to the generally much lower transmission rates of this IncI plasmid and we decided to screen for factors that negatively affect the transfer of the plasmid by an in vivo deletion analysis. Our attempts to delete larger regions of the plasmid resulted in cells containing both a truncated plasmid (Δ50 kb and Δ75 kb) and a wild type copy of pO104_90. When used as donors in conjugation experiments, these cells transferred the wild type plasmid at dramatically increased rates. This indicated that the relatively limited region shared by both plasmids contained an activator of transfer. We therefore analyzed its transcriptional organization, dissected the candidate region by subcloning and showed that additional copies of repY/INC were sufficient to increase the transfer frequency of pO104_90 to the observed level. To our knowledge, this is the first evidence for a direct regulatory cross talk between core control elements of the vegetative replication and the transfer functions of an IncI1 plasmid.

Comparative virulence characterization of the Shiga toxin phage-cured Escherichia coli O104:H4 and enteroaggregative Escherichia coli.

Escherichia coli O104:H4 (E. coli O104:H4), which caused in 2011 a massive foodborne outbreak in Germany, is characterized by an unusual combination of virulence traits. E. coli O104:H4 contains a prophage-encoded Shiga toxin (Stx) gene, which is the cardinal virulence factor of enterohemorrhagic E. coli (EHEC). However, the outbreak strain shares highest DNA sequence similarity with enteroaggregative E. coli (EAEC) and displays the EAEC-characteristic tight adherence to epithelial cells. The virulence potential of the underlying EAEC background has not been investigated and it is therefore not clear whether E. coli O104:H4 displays distinct virulence characteristics in comparison to prototypical EAEC. In this study, we performed a detailed comparative phenotypic characterization of the Stx phage-cured E. coli O104:H4 strain C227-11φcu, the closely related EAEC strain 55989 and two other well-characterized EAEC strains 042 and 17-2 with focus on virulence traits. C227-11φcu displayed superior aggregative adherence phenotype to cultured HCT-8 epithelial cells, adhering with 3-6 times more bacteria per epithelial cells than the tested EAEC strains. Otherwise, C227-11φcu showed similar virulence characteristics to its closest relative 55989, i.e. strong acid resistance, good biofilm formation and cytotoxic culture supernatants. Furthermore, C227-11φcu was characterized by significantly weaker motility and pro-inflammatory properties than 55989 and 042, nevertheless stronger than 17-2. Taken together, C227-11φcu displayed mostly robust, but not outstanding virulence characteristics in comparison to the tested EAEC. Therefore, it appears likely that the combination of Stx production and EAEC characteristics in general, rather than an exceptionally potent EAEC background resulted in the unusual virulence of the E. coli O104:H4. Thus, the emergence of such hypervirulent strains in the future might be more likely than previously anticipated.

The primary transcriptome of the Escherichia coli O104:H4 pAA plasmid and novel insights into its virulence gene expression and regulation.

Escherichia coli O104:H4 (E. coli O104:H4), which caused a massive outbreak of acute gastroenteritis and hemolytic uremic syndrome in 2011, carries an aggregative adherence fimbriae I (AAF/I) encoding virulence plasmid, pAA. The importance of pAA in host-pathogen interaction and disease severity has been demonstrated, however, not much is known about its transcriptional organization and gene regulation. Here, we analyzed the pAA primary transcriptome using differential RNA sequencing, which allows for the high-throughput mapping of transcription start site (TSS) and non-coding RNA candidates. We identified 248 TSS candidates in the 74-kb pAA and only 21% of them could be assigned as TSS of annotated genes. We detected TSS for the majority of pAA-encoded virulence factors. Interestingly, we mapped TSS, which could allow for the transcriptional uncoupling of the AAF/I operon, and potentially regulatory antisense RNA candidates against the genes encoding dispersin and the serine protease SepA. Moreover, a computational search for transcription factor binding sites suggested for AggR-mediated activation of SepA expression, which was additionally experimentally validated. This work advances our understanding of the molecular basis of E. coli O104:H4 pathogenicity and provides a valuable resource for further characterization of pAA virulence gene regulation.

Genes on a Wire: The Nucleoid-Associated Protein HU Insulates Transcription Units in Escherichia coli.

The extent to which chromosomal gene position in prokaryotes affects local gene expression remains an open question. Several studies have shown that chromosomal re-positioning of bacterial transcription units does not alter their expression pattern, except for a general decrease in gene expression levels from chromosomal origin to terminus proximal positions, which is believed to result from gene dosage effects. Surprisingly, the question as to whether this chromosomal context independence is a cis encoded property of a bacterial transcription unit, or if position independence is a property conferred by factors acting in trans, has not been addressed so far. For this purpose, we established a genetic test system assessing the chromosomal positioning effects by means of identical promoter-fluorescent reporter gene fusions inserted equidistantly from OriC into both chromosomal replichores of Escherichia coli K-12. Our investigations of the reporter activities in mutant cells lacking the conserved nucleoid associated protein HU uncovered various drastic chromosomal positional effects on gene transcription. In addition we present evidence that these positional effects are caused by transcriptional activity nearby the insertion site of our reporter modules. We therefore suggest that the nucleoid-associated protein HU is functionally insulating transcription units, most likely by constraining transcription induced DNA supercoiling.