Abstract
Enterohemorrhagic Escherichia coli (EHEC) is dependent on acid resistance for gastric passage and low oral infectious dose, and the locus of enterocyte effacement (LEE) for intestinal colonization. Mutation of rpoN, encoding sigma factor N (σN), dramatically alters the growth-phase dependent regulation of both acid resistance and the LEE. This study reports on the determinants of σN-directed acid resistance and LEE expression, and the underlying mechanism attributable to this phenotype. Glutamate-dependent acid resistance (GDAR) in TW14359ΔrpoN correlated with increased expression of the gadX-gadW regulatory circuit during exponential growth, whereas upregulation of arginine-dependent acid resistance (ADAR) genes adiA and adiC in TW14359ΔrpoN did not confer acid resistance by the ADAR mechanism. LEE regulatory (ler), structural (espA and cesT) and effector (tir) genes were downregulated in TW14359ΔrpoN, and mutation of rpoS encoding sigma factor 38 (σS) in TW14359ΔrpoN restored acid resistance and LEE genes to WT levels. Stability, but not the absolute level, of σS was increased in TW14359ΔrpoN; however, increased stability was not solely attributable to the GDAR and LEE expression phenotype. Complementation of TW14359ΔrpoN with a σN allele that binds RNA polymerase (RNAP) but not DNA, did not restore WT levels of σS stability, gadE, ler or GDAR, indicating a dependence on transcription from a σN promoter(s) and not RNAP competition for the phenotype. Among a library of σN enhancer binding protein mutants, only TW14359ΔntrC, inactivated for nitrogen regulatory protein NtrC, phenocopied TW14359ΔrpoN for σS stability, GDAR and ler expression. The results of this study suggest that during exponential growth, NtrC-σN regulate GDAR and LEE expression through downregulation of σS at the post-translational level; likely by altering σS stability or activity. The regulatory interplay between NtrC, other EBPs, and σN–σS, represents a mechanism by which EHEC can coordinate GDAR, LEE expression and other cellular functions, with nitrogen availability and physiologic stimuli.
Highlights
Enterohemorrhagic Escherichia coli (EHEC) is an enteric pathogen commonly implicated in food-borne outbreaks of hemorrhagic colitis, and in the life-threatening illness hemolytic uremic syndrome [1,2,3]
Alternative sigma factor 38 is a global regulator that plays an important role in coordinating acid resistance and locus of enterocyte effacement (LEE) expression with growth phase. sS is a protein of low abundance during exponential growth, but accumulates during transition into stationary phase [29]
SS stability was increased in both enhancerbinding proteins (EBP) mutant backgrounds to the level of stability observed in TW14359DrpoN (Fig. 7). These results reveal that mutation of fhlA and ntrC influence sS stability, yet only ntrC mutation phenocopies glutamate-dependent acid resistance (GDAR) and LEE expression observed in TW14359DrpoN
Summary
Enterohemorrhagic Escherichia coli (EHEC) is an enteric pathogen commonly implicated in food-borne outbreaks of hemorrhagic colitis, and in the life-threatening illness hemolytic uremic syndrome [1,2,3]. To cause disease in humans, EHEC must overcome two formidable innate barriers to infection: the acidity of the stomach, and competition for intestinal colonization sites For the former, EHEC (and other E. coli) has evolved multiple discrete acid resistance mechanisms [4], which allow for survival in highly acidic environments such as the stomach, and which determine a low oral infectious dose [5,6]. During exponential growth acid resistance is largely repressed, but is activated as cultures transition into stationary phase [13]; for the LEE, the inverse is true [18] This pattern of expression may reflect the importance of colonization and replication when resources are abundant, and that of stress durability when they are scarce. RpoS mutants are impaired in their ability to survive passage in both murine and bovine models of infection [33]. sS is tightly regulated at multiple levels of control [34], and the factors that dictate rpoS/sS expression indirectly influence acid resistance, the LEE, and EHEC pathogenesis
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