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

Abstract

Escherichia coli is a typical constituent of the enteric tract in many animals, including humans. However, specialized extraintestinal pathogenic E. colistrains (ExPEC) may transition from benign occupation of the enteric and vaginal tracts to sterile sites such as the urinary tract, bloodstream, and central nervous system. ExPEC isolates of urinary tract origin express type 1 pili as a critical virulence determinant mediating adherence to and invasion into urinary tract tissues. Type 1 pili expression is under epigenetic regulation by a family of site-specific recombinases, including FimX, which is encoded from a genomic islet called PAI-X for Pathogenicity Islet of FimX. A goal of this study was to determine the prevalence of the type 1 pili epigenetic regulator genes (fimB, fimE, fimX, ipuA, ipuB) and associated PAI-X genes (hyxR, hyxA, hyxB) present among an extended, diverse collection of pathogenic and commensal E. coli isolates. Using a new multiplex PCR, fimX and the additional PAI-X genes were found to be highly associated with ExPEC (83.2%) and more prevalent in ExPEC of lower urinary tract origin (87.5%) than upper urinary tract origin (73.6%) or human commensal isolates (20.6%; p < 0.05, all comparisons). Fim-like recombinase genes ipuA and ipuB also had a significant association with ExPEC compared to commensal isolates, but had a low overall prevalence (23.8% vs. 11.1%; p < 0.05). PAI-X also showed a strong positive correlation with the presence of virulence genes in the genomes of pathogenic isolates. Combined, our molecular epidemiology studies indicate PAI-X is highly associated with ExPEC isolates, and its high prevalence suggests a potential role in the ExPEC lifestyle. Further investigation into the regulation of PAI-X factors showed that FimX is also an epigenetic regulator of a LuxR-like response regulator HyxR, encoded on PAI-X. In multiple clinical ExPEC isolates, FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the inversion sites of the type 1 pili promoter and independent of integration host factor IHF. Additional studies into the role of HyxR during ExPEC pathogenesis uncovered that HyxR is involved in regulation of the nitrosative stress response. In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNI), primarily through derepression of hmpA, encoding a nitric oxide detoxifying flavohemoglobin. However, in the macrophage, HyxR expression 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. ExPEC reside in the enteric tract as commensal reservoirs, 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. This study demonstrates the functional versatility of the FimX recombinase and identifies novel epigenetic and transcriptional regulatory controls for ExPEC tolerance to RNI challenge and survival during intracellular macrophage infection. Further investigation of these pathways may shed light on the regulatory cues and programming that provoke the commensal to pathogen transition.