Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane

Abstract

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the cytoplasmic membrane of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic

“…Subsequently, the antibiotic interacts with LPS in the CM as it is being trafficked to the OM, resulting in CM permeabilisation [12]. It is this interaction with CM LPS that is key to the bactericidal action of colistin, since destabilisation of the CM leads to cell lysis and bacterial death [12,13].…”

“…In both MCR-mediated and mutation-mediated colistin resistance, LPS modification occurs in the outer leaflet of the CM [20,24,25]. This results in the presence of modified LPS in both the CM and OM, although not all LPS molecules are modified in either membrane [12,23]. Since both L-Ara4n and pEtN are positively charged, they reduce the anionic charge of lipid A, which is thought to reduce its affinity for the cationic peptide ring of colistin [17,24].…”

A Molecular Perspective on Colistin and Klebsiella pneumoniae: Mode of Action, Resistance Genetics, and Phenotypic Susceptibility

“…Sabnis et al (2020) postulated that, despite colistin bind to lipid A in OM, it might also target lipid A at the IM before it is transported to the OM. Supporting this theory, they showed the abundant presence of lipid A in the IM and also proved that the interaction between lipid A and colistin in the IM is required for colistin antibacterial activity ( Figure 2 C) [ 27 ]. This latter hypothesis provides a more solid base to explain the high colistin activity against Gram-negative bacteria and the absence of activity against Gram-positive microorganisms.…”

“…This electrostatic interaction, with the help of the hydrophobic regions of colistin, weakens the OM structure providing colistin access to periplasm ( 2 ). There, the way how colistin breaks IM is explained by the three hypotheses: the lysis mechanism ( A ), where colistin straddles the phospholipid bilayer decreasing the IM thickness and leading to cell lyses ( 3 ) [ 21 , 25 ]; a vesicle-vesicle contact pathway ( B ), where the colistin acyl tail induces the exchange of phospholipids between the outer leaflet of IM and inner leaflet of OM, resulting in the structure instability of theses membrane and cell death ( 4 ) [ 18 , 21 , 24 ]; fand, through inner membrane lipid A targeting ( C ), where colistin targets the lipid A molecules that are transiently in IM, after being translocated by MsbA from the cytoplasm (where they are synthesized) and before being transported to OM, which will induce cytoplasmic content leakage and consequently cell death ( 5 ) [ 26 , 27 ]. LPS—lipopolysaccharide; OM—outer membrane; IM—inner membrane.…”

Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae

“…Recently, colistin has also been found to target the inner cytoplasmic membrane. 44 Interestingly, we identified a highly mutated inner membrane ABC transporter permease 45 under strong positive selection (all detected mutations non-synonymous), named ynjC (21 UM, Uniprot, P76224). Proteins of this group utilize ATP to import many small molecules such as nutrients and antibiotics.…”