Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli

Abstract

Summary Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection, and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor IHF. In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNI), primarily through de-repression of hmpA, encoding a nitric oxide detoxifying flavohemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX.

A Kinetic Platform to Determine the Fate of Nitric Oxide in Escherichia coli

“…This effect originates from the strong induction of Hmp expression upon NO• exposure even under anaerobic conditions [41], [42], and the rapid O 2 -mediated deactivation of NorV, the alternative NO• detoxification system that has been previously identified as critical for resisting NO• stress under anaerobic conditions [22], [31]. These data demonstrate the flexibility of this method to different environmental conditions (microaerobic), and provide support for the role of Hmp as a virulence factor [43], [44], since O 2 concentrations at infections sites/in macrophages and neutrophils are typically hypoxic (less than 50 µM O 2 [4], [45], [46]). Interestingly, both NorV- and Hmp-type enzymes have been found to be virulence factors for numerous organisms [36], [47]–[50], and thus a quantitative understanding of the conditions under which each contributes to NO• clearance would be valuable for the study of their importance to virulence.…”

The type 1 pili regulator gene fimX and pathogenicity island PAI-X as molecular markers of uropathogenic Escherichia coli

“…Translation and BLAST analysis of hyxR suggest it is a putative helix-turn-helix, LuxR-like response regulator. Recently, our laboratory has also shown that FimX exclusively regulates the PAI-X gene hyxR through bidirectional promoter inversion, and that HyxR regulates the intracellular survival of E. coli during macrophage infection (Bateman & Seed, 2012). The additional two conserved ORFs found in this locus appear to code for a putative outer membrane autotransporter (HyxB) and a conserved hypothetical protein (HyxA), which has homologues only in the genomes of E. coli and S. enterica (protein BLAST E-score ,5610 25 ).…”

“…Among a small collection of E. coli isolates, we previously demonstrated that fimX was highly associated with UPEC isolates (Hannan et al, 2008). More recently, we showed that the recombinase FimX exclusively regulates the predicted LuxR-like response regulator HyxR that is encoded from the same pathogenicity island and is a negative regulator of the nitrosative stress response and intracellular macrophage survival (Bateman & Seed, 2012). Therefore, a goal of this study was to determine the prevalence of the type 1 pili recombinase regulator genes and PAI-X genes present in an extended, diverse collection of pathogenic and commensal E. coli isolates.…”

Intracellular Macrophage Infections with E. coli under Nitrosative Stress

“…To accomplish this, RAW 264.7 murine macrophage-like cells are pre-incubated with either L-arginine, an NO precursor, or IFNγ to yield a high nitric oxide (NO) physiological state, or L-NAME, an inducible NO synthase (iNOS)-specific inhibitor, to yield a low NO physiological state. This protocol has been successfully utilized to assess the contribution of a novel ExPEC regulator to intracellular survival and the nitrosative stress response during macrophage infections (Bateman and Seed, 2012), but can be adapted for use with a variety of E. coli strains or isogenic deletions. …”