Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane

Abstract

Tel: 0044 (0)207 594 2072 23 Fax: 0044 (0)207 594 3096 24 a.edwards@imperial.ac.uk 25 26 Keywords: Colistin / polymyxin / Pseudomonas / E. coli / Klebsiella / lipopolysaccharide / resistance / MCR-1 27 28 Running title: Colistin mechanism of action 29 30 31 32 33 2 Summary 34 Colistin is an antibiotic of last resort for infections caused by drug-resistant Gram-negative pathogens such 35as Pseudomonas aeruginosa and Escherichia coli. For this reason, high rates of treatment failure and 36 increasing resistance to this antibiotic are very concerning and attempts to resolve these issues are hampered 37 by a poor understanding of colistin's mode of action. Whilst it is well established that colistin binds to 38 lipopolysaccharide in the bacterial outer membrane, it was unclear how this led to bacterial killing. Here, we 39show that colistin also targets lipopolysaccharide in the cytoplasmic membrane and that this interaction is 40 essential for cytoplasmic membrane permeabilisation, cell lysis and the bactericidal activity of the antibiotic. 41We also found that MCR-1-mediated colistin resistance confers protection against the antibiotic via the 42 presence of modified lipopolysaccharide within the cytoplasmic membrane, rather than the outer 43 membrane. These findings reveal key details about the mechanism by which colistin kills bacteria, providing 44 the foundations for the development of new approaches to enhance therapeutic outcomes.

Effect of host-mimicking medium and biofilm growth on the ability of colistin to killPseudomonas aeruginosa

“…Glass vials were used for these experiments because colistin can bind plastic and we wished to minimise loss to binding of the culture vessel in order to measure the proportion of colistin entering the biofilms as accurately as possible. Addition of the BODIPY-tag has been shown to reduce bactericidal activity in planktonic culture (19), and a pilot experiment using PA14, SED6 and SED8 had confirmed that the tag significantly reduced activity in our biofilm assay ( Figure S3). , therefore fluorescence values from samples exposed to BODIPY-colistin were standardised by subtracting the mean fluorescence measured in homogenate/surrounding ASM from tissues infected by the same strain but exposed to SCFM with no BODIPYcolistin.…”

“…First, colistin is widely prescribed for P. aeruginosa infection in CF and is commonly administered by inhalation; this means that topical lab exposure of biofilms to colistin, by spiking the surrounding culture medium, likely mimics in vivo exposure better than in the case of antibiotics that are only administered orally or through IV. Second, fluorescently-labelled colistin was available from colleagues, opening up the possibility to assay colistin concentration in biofilm via fluorimetry (19). Finally, colistin is able to bind the P. aeruginosa exopolymer Psl (20) and extracellular lipopolysaccharide (21), and may adsorb to outer membrane vesicles (22) -all of which may be present in P.…”

“…This process is described in detail by Sabnis et al (19). Briefly, colistin was labelled by incubating with BODIPY® FL SE D2184 (Thermo Fisher Scientific) and sodium bicarbonate for 2 h at 37°C; unbound BODIPY was removed by dialysis; and successful labelling was confirmed by time-of-flight mass spectrometry.…”

“…Labelled colistin was a gift from Akshay Sabnis and Andrew Edwards (see (19) ) Tubes were incubated for 18h at 37°C. Glass vials were used for these experiments because colistin can bind plastic and we wished to minimise loss to binding of the culture vessel in order to measure the proportion of colistin entering the biofilms as accurately as possible.…”

Non-clonal emergence of colistin resistance associated with mutations in the BasRS two-component system in Escherichia coli bloodstream isolates

“…Colistin specifically targets Gram-negative bacteria by binding to the anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPS) through electrostatic interactions (7–9). Colistin destabilizes the outer membrane, but the subsequent disruption of the inner membrane ultimately leads to cell death (9, 10). Acquired colistin resistance has been reported in various Gram-negative bacteria that were isolated from clinical, veterinary, and environmental sources (11–13).…”