A single‐component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress

Stephanie Paul,Paul McVeigh,Christopher J. Law,Kamela O. Alegre,James A. Brown,Scarlett R. Holdsworth,Sharon M. Kelly,Matthew Rice

doi:10.1111/mmi.12597

Stephanie Paul, Paul McVeigh + Show 6 more

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https://doi.org/10.1111/mmi.12597

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Abstract

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as E scherichia coli. Although the tripartite AcrAB–TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E . coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E . coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB–TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H+ antiport.

Highlights

The mammalian intestine is a complex, densely populated microbial ecosystem that is home to hundreds of different species and strains of bacteria (Ley et al, 2006; Qin et al, 2010; De Paepe et al, 2011)
Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli
We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm

Summary

Introduction

The mammalian intestine is a complex, densely populated microbial ecosystem that is home to hundreds of different species and strains of bacteria (Ley et al, 2006; Qin et al, 2010; De Paepe et al, 2011). Water-soluble, steroidal surfactants, synthesized in the liver from cholesterol and secreted into the bile, that aid emulsification and enzymatic digestion of dietary lipids in the small intestine (Maldonado-Valderrama et al, 2011). Bile acids are secreted from hepatocytes as salts, in a form conjugated, via an amide bond, to glycine or taurine (Maldonado-Valderrama et al, 2011). The majority of these bile salts are reabsorbed by the lining of the ileum for subsequent reuse, those that remain are deconjugated by bacterial enzymes to form free primary bile acids such as cholate (Hofmann, 1999; Ridlon et al, 2006). Again catalysed by intestinal flora enzymes, transform the primary bile acids into the secondary bile acids deoxycholate (DOC) and lithocholate (Hylemon et al, 1991; Hofmann, 1999; Ridlon et al, 2006); the latter is rapidly eliminated from the body and constitutes only trace amounts of the unconjugated biliary bile acids, the remainder being about equal amounts of cholate, chenodeoxycholate and deoxycholate (Hofmann, 1999)

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