A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid.

The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K-12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K-12, the 4.88 Mbp Fec10 genome is characterized by distinct single-nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two-component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpGGI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand-alone disaggregation activity, but observed distinct biochemical characteristics of ClpGGI-Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpGGI-SG17M. Furthermore, we noted a unique contribution of ClpGGI-Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food-borne heat-resistant strains in the human gut.

ClbR Is the Key Transcriptional Activator of Colibactin Gene Expression in Escherichia coli.

Colibactin is a nonribosomal peptide/polyketide hybrid natural product expressed by different members of the Enterobacteriaceae which can be correlated with induction of DNA double-strand breaks and interference with cell cycle progression in eukaryotes. Regulatory features of colibactin expression are only incompletely understood. We used Escherichia coli strain M1/5 as a model to investigate regulation of expression of the colibactin determinant at the transcriptional level and to characterize regulatory elements located within the colibactin pathogenicity island itself. We measured clbR transcription in vitro and observed that cultivation in defined minimal media led to increased colibactin expression relative to rich media. Transcription of clbR directly responds to iron availability. We also characterized structural DNA elements inside the colibactin determinant involved in ClbR-dependent regulation, i.e., ClbR binding sites and a variable number of tandem repeats located upstream of clbR We investigated the impact of clbR overexpression or deletion at the transcriptome and proteome levels. Moreover, we compared global gene regulation under these conditions with that occurring upon overexpression or deletion of clbQ, which affects the flux of colibactin production. Combining the results of the transcriptome and proteome analyses with indirect measurements of colibactin levels by cell culture assays and an approximate quantification of colibactin via the second product of colibactin cleavage from precolibactin, N-myristoyl-d-asparagine, we demonstrate that the variable number of tandem repeats plays a significant regulatory role in colibactin expression. We identify ClbR as the only transcriptional activator known so far that is specific and essential for efficient regulation of colibactin production.IMPORTANCE The nonribosomal peptide/polyketide hybrid colibactin can be considered a bacterial virulence factor involved in extraintestinal infection and also a procarcinogen. Nevertheless, and despite its genotoxic effect, colibactin expression can also inhibit bacterial or tumor growth and correlates with probiotic anti-inflammatory and analgesic properties. Although the biological function of this natural compound has been studied extensively, our understanding of the regulation of colibactin expression is still far from complete. We investigated in detail the role of regulatory elements involved in colibactin expression and in the growth conditions that promote colibactin expression. In this way, our data shed light on the regulatory mechanisms involved in colibactin expression and may support the expression and purification of this interesting nonribosomal peptide/polyketide hybrid for further molecular characterization.

Spontaneous onset of TNFα-triggered colonic inflammation depends on functional T lymphocytes, S100A8/A9 alarmins, and MHC H-2 haplotype.

Recently, we established a doxycycline-inducible human tumor necrosis factor alpha (TNFα)-transgenic mouse line, ihTNFtg. Non-induced young and elderly mice showed low but constitutive expression of hTNFα due to promoter leakiness. The persistently present hTNFα stimulated endogenous pro-inflammatory mouse mS100A8/A9 alarmins. Secreted mS100A8/A9 in turn induced the expression and release of mouse mTNFα. The continuous upregulation of pro-inflammatory mTNFα and mS100A8/A9 proteins, due to their mutual expression dependency, gradually led to increased levels in colon tissue and blood. This finally exceeded the threshold levels tolerated by the healthy organism, leading to the onset of intestinal inflammation. Here, recombinant hTNFα functioned as an initial trigger for the development of chronic inflammation. Crossing ihTNFtg mice with S100A9KO mice lacking active S100A8/A9 alarmins or with Rag1KO mice lacking T and B lymphocytes completely abrogated the development of colonic inflammation, despite the still leaky hTNFα promoter. Furthermore, both the intensity of the immune response and the strength of immunosuppressive Treg induction was found to depend on the major histocompatibility complex (MHC) genetic composition. In summary, the onset of intestinal inflammation in elderly mice depends on at least four factors that have to be present simultaneously: TNFα upregulation, S100A8/A9 protein expression, functional T lymphocytes and genetic composition, with the MHC haplotype being of central importance. Only joint action of these factors leads to chronic intestinal inflammation, while absence of any of these determinants abrogates the development of the autoimmune disorder. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.