Some Gram-negative bacteria produce a protective protein pair that shields their outer membrane from antibiotic-induced chemical damage, and researchers want to understand exactly how that shield works. This matters because Gram-negative bacteria already resist most available antibiotics, and a phenomenon called heteroresistance makes treatment even harder: within a single bacterial population, some cells survive antibiotic doses that kill others. The team has discovered that when antibiotics attack, they trigger lipid peroxidation—a destructive process that degrades the bacterial membrane’s fatty molecules. In response, bacteria ramp up production of a lipocalin protein (BcnA) and a membrane cytochrome (LcoA), which together form a recycling system that neutralises the peroxidised lipids. The researchers have also identified that this damage concentrates at the bacterial poles and division sites, areas rich in anionic phospholipids. This is fundamental science. The project aims to answer two basic questions: how the BcnA/LcoA system works at a molecular level, and which specific lipids are being damaged. If successful, it will reveal a previously unknown bacterial defence mechanism. That knowledge could eventually guide the design of molecules that disable this protective system, making existing antibiotics more effective against stubborn Gram-negative infections—but any practical application remains years away.
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The knowledge gap–Treating infections by Gram-negative bacteria is becoming increasingly difficult since these bacteria often show intrinsic high-level resistance to virtually all clinically approved antibiotics. Compounding this problem, many bacteria display heteroresistance, a phenomenon where subpopulations of seemingly isogenic bacteria exhibit a range of susceptibilities to a particular antibiotic, resulting in ineffective microbial killing. Bacterial exposure to sublethal antibiotic concentrations elicit adaptive bacterial stress responses, enhancing antibiotic resistance and tolerance. Our research has revealed that these adaptive responses occur against bactericidal antibiotics, irrespective of their chemical nature. We have discovered that under conditions of bactericidal stress bacteria hyperproduce a lipocalin and its associated cytochrome (BcnA/LcoA), and polyamines, all of which contribute to increased antibiotic resistance and heteroresistance. A major stressor resulting from antibiotic action, is membrane-lipid peroxidation, which destroys lipids in the bacterial membrane eliciting adaptive bacterial responses that result in even higher antibiotic resistance. Therefore, elucidating the fundamental biological question of how the double-membraned Gram-negatives protect their outer membranes from lipid peroxidation may reveal novel ways to overcome resistance and heteroresistance. Our discovery–Our research supported by BBSRC BB/S006281/1, recently published in PlosBiology, described a hitherto unknown evolutionary conserved peroxidation detoxification system that protects the bacterial cell envelope from lipid peroxidation and plays a key role in intrinsic resistance against multiple classes of antibiotics. This system comprises three proteins which are conserved in all Gram-negative bacteria: the BcnA lipocalin (putative scavenger of lipid peroxyl radicals), the LcoA membrane cytochrome-b561, and PsrA, a cytoplasmic aldehyde reductase. New preliminary data show that BcnA binds quinone (Ubi8), and structural modelling predicts that BcnA and LcoA interact forming a complex. This information underpins our working model indicating that BcnA/LcoA form a novel cyclic redox system in which the BcnA-bound Ubi8 reduces oxidised lipid species by becoming itself oxidised, while LcoA interacts with BcnA to reduce Ubi8, recycling the redox scavenger form of BcnA. The challenge of this proposal is to elucidate the detailed mechanism of BcnA/LcoA function and the nature of membrane lipids targeted by peroxidation. Aims–The nature of the peroxidised lipids in the Gram-negative bacterial outer membrane remains a longstanding unanswered question. However, using specialised fluorescent probes to image both peroxidation and the distribution of certain classes of lipids called anionic phospholipids, we showed that peroxidation and lipid accumulation predominates at the bacterial poles and the septa (sites of cell division), both areas of membrane curvature known to be rich in anionic phospholipids. These data suggest a bacterial response to increase the production of anionic lipids, likely to replenish oxidised membrane lipids at these sites. Employing state-of-the-art technologies with robust research methodologies underpinned by published and new preliminary data, together with the expertise of key collaborators, this innovative research program will investigate two fundamental biological questions: (1)How do the LcoA cytochrome and BcnA can combat lipid peroxidation? (2) What are the lipids targeted by peroxidation in Gram-negative bacteria? Impact–Elucidating these questions will help us understand the role of peroxidation in the context of antibiotic resistance, aligning with the BBSRC priorities of "understanding the rules of life" and "research to combat antimicrobial resistance". This new biology may ultimately bring societal benefit to the economy and health by leveraging the discovery of novel molecules to maximise the action of antibiotics through interference with bacterial protection against peroxidative stress and/or acceleration of bacterial lipid oxidative damage.
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