AdhE spirosome length in enterohaemorrhagicEscherichia coliis correlated with enzymatic directionality and is perturbed by salicylidene acylhydrazide binding

Abstract

ABSTRACTAntibiotics are contraindicated for the treatment of infection enterohemorrhagicEscherichia coli(EHEC), a human pathogen that causes diarrhea or hemorrhagic colitis in humans which can progress to hemolytic uremic syndrome (HUS). As an alternative to the use of antibiotics, previous studies developed the salicylidene acylhydrazides (SA), a family of anti-virulence compounds capable of blocking expression of the type three secretion system (T3SS), thereby reducing bacterial infections. Here we validate AdhE as the anti-virulence protein target of the SA compound ME0054. AdhE is a bidirectional enzyme able to catalyse the conversion of acetyl-CoA to ethanol andvice versa. AdhE oligomerises forming helicoidal filaments heterogeneous in length called spirosomes. In this work, we show that it is possible to partially fractionate AdhE spirosomes using size exclusion chromatography (SEC) and to characterise the spirosome oligomers present in each fraction with biophysical techniques such as small angle X-ray scattering (SAXS) and sedimentation velocity analytical ultracentrifugation (SV-AUC). Also, we observe that short spirosomes are more efficient in the reverse reaction whereas the spirosome length has no impact on the forward reaction. Therefore, for first time, we reveal that AdhE spirosome formation is necessary to regulate the direction of its enzymatic reactions. In addition, we show that ME0054 disrupts AdhE spirosomes, thereby enhancing the conversion of ethanol to acetyl-CoA. Importantly, SV-AUC data show that ME0054 binds to the AdhE filaments. Finally, time-resolved (TR) SAXS allowed us to follow the kinetics of spirosome disruption produced by ME0054, confirming its effectiveness at biologically relevant temperatures and timescales.SIGNIFICANCE STATEMENTThere is an urgent need to develop alternative strategies to combat bacterial infections. Salicylidene acylhydrazides (SA) are able to reduce expression of the bacterial type three secretion system (T3SS), used by many pathogens to manipulate host eukaryotic cells, including our pathogen of interest: enterohaemorrhagicE. coli(EHEC). The mechanism underpinning these compounds is a mystery. Here we show how the SA compound ME0054, by disrupting AdhE spirosomes, enhances metabolic conversion of ethanol to acetyl-CoA. This finding is consistent with the phenotype observed in an EHEC AdhE mutant: alterations in acetate levels and changes in T3SS expression. Our work establishes a crucial mechanistic link between the binding of the SA compound to a key target protein and changes in bacterial metabolism.