PH Modulation of Efflux Pump Activity of Multi-Drug Resistant Escherichia coli: Protection During Its Passage and Eventual Colonization of the Colon

Ana Martins,Jean Michel Bolla,Isabel Couto,Joseph Molnar,Marta Martins,Leonard Amaral,Séamus Fanning,Jorge Ramos,Jean-Marie Pagès,Liliana Rodrigues,Miguel Viveiros,Gabriella Spengler

doi:10.1371/journal.pone.0006656

Abstract

BackgroundResistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets. Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can also extrude antibiotics, obtain energy from the hydrolysis of ATP. Because E. coli must pass through two pH distinct environments of the gastrointestinal system of the host, it must be able to extrude toxic agents at very acidic and at near neutral pH (bile salts in duodenum and colon for example). The herein described study examines the effect of pH on the extrusion of ethidium bromide (EB).Methodology/Principal Findings E. coli AG100 and its tetracycline induced progeny AG100TET that over-expresses the acrAB efflux pump were evaluated for their ability to extrude EB at pH 5 and 8, by our recently developed semi-automated fluorometric method. At pH 5 the organism extrudes EB without the need for metabolic energy (glucose), whereas at pH 8 extrusion of EB is dependent upon metabolic energy. Phe-Arg β-naphtylamide (PAβN), a commonly assumed inhibitor of RND efflux pumps has no effect on the extrusion of EB as others claim. However, it does cause accumulation of EB. Competition between EB and PAβN was demonstrated and suggested that PAβN was preferentially extruded. A Km representing competition between PAβN and EB has been calculated.Conclusions/SignificanceThe results suggest that E. coli has two general efflux systems (not to be confused with a distinct efflux pump) that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ABC transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host.

Highlights

Bacteria that are orally ingested need to survive when exposed to noxious agents such as toxic bile salts that are present along the digestive tract
Conclusions/Significance: The results suggest that E. coli has two general efflux systems that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ATP-Binding Cassette (ABC) transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host
This difference is in accordance to the degree of AcrAB efflux pump expression, namely the AG100TET over-expresses this pump 6-fold over that of AG100, as shown by real-time quantitative reverse transcriptase-PCR [5]

Summary

Introduction

Bacteria that are orally ingested need to survive when exposed to noxious agents such as toxic bile salts that are present along the digestive tract (ex: duodenum and colon). The tripartite pump obtains the necessary energy to power extrusion of the agent directly from the trans-membrane proton gradient [9,10] This gradient provides the protons that are present in the periplasmic space and, when they enter the transporter at its PLoS ONE | www.plosone.org pH Modulation of Efflux plasma membrane base, they energize the pump and the agent which is believed to be concentrated within the outer leaflet of the inner membrane is in turn extruded [11]. Resistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can extrude antibiotics, obtain energy from the hydrolysis of ATP. The described study examines the effect of pH on the extrusion of ethidium bromide (EB)

Methods

Results

Conclusion