Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli

Abstract

Background It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells. Results We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane potential is presumably the same in each case). Conclusions Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of putatively broad and presently unknown specificity, and that the very large range between the ‘lowest’ and the ‘highest’ levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayer transport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo) . By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening. Electronic supplementary material The online version of this article (10.1186/s12866-019-1561-0) contains supplementary material, which is available to authorized users.

Why most transporter mutations that cause antibiotic resistance are to efflux pumps rather than to import transporters

“…Many efflux transporters are sufficiently active that even when the drug has relatively tight intracellular binding sites they can effectively remove almost all of it, as is the case with AcrAB/TolC and ethidium bromide 73, 74 . A recent experimental survey of several hundred gene knockouts in E. coli , using fluorescent probes as antibiotic surrogates showed that dozens of such efflux transporters could be active and thereby contribute to lowering the steady-state uptake 47 . There is also considerable redundancy and plasticity 75 .…”

“…it has the capability of raising the concentration of the drug of interest to a higher level inside than outside. Such transporters necessarily require a source of free energy; in prokaryotes this is mainly ATP 46, 47 . The effluxer is also taken to be ATP-driven.…”

Structural similarities between some common fluorophores used in biology and marketed drugs, endogenous metabolites, and natural products

“…[97][98][99]). As an illustr ation, and as a com plem ent to our detailed gene knockout studi es [17], Table 1 gives an indication of dyes w hose inter action w ith specific tr anspor ter s has been dem onstr ated dir ectly. In som e cases, thei r sur r ogacy as a substr ate for a tr anspor ter w ith a know n non-fluor escent substr ate is clear , and as m entioned in the intr oduction they ar e som etim es r efer r ed to as 'false fluor escent substr ates'.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]). Ou r sp ecific in ter est h er e is in th e tr a n sp or ter -m ed ia ted m ean s b y w h ich sm a ll flu or escen t m olecu les en ter livin g cells, a n d ou r in ter est h a s b een stim u la ted b y th e r ecogn ition th a t a given p r ob e m a y b e a su b str a te for a la r ge va r iety of b oth in flu x a n d efflu x tr a n sp or ter s [17]. Efflu x tr a n sp or ter s a r e often fa ir ly p r om iscu ou s, sin ce th eir job is la r gely to r id cells of u n w a n ted m olecu les th a t m a y h a ve en ter ed , a lth ou gh th ey ca n a n d d o h a ve oth er , im p or ta n t p h ysiologica l r oles (e.g.…”

An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines

“…As part of a wide-ranging study into the nature of transporter substrates (e.g. (Dobson and Kell, 2008;Jindal et al, 2019;Kell et al, 2013;Kell et al, 2011;Kell and Oliver, 2014;), we recognised that the approach of Gründemann and colleagues (Gründemann et al, 2005) could be an ideal strategy for implementation using modern, high-resolution metabolomics methods. We have previously developed low-resolution methods for determining the human serum metabolome reliably (Broadhurst and Kell, 2006) and over extended periods Dunn et al, 2011;Kenny et al, 2010;Zelena et al, 2009), including the extensive use of QA/QC samples.…”