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An evolutionary conserved detoxification system for membrane lipid–derived peroxyl radicals in Gram-negative bacteria

['Marwa Naguib', 'Wellcome-Wolfson Institute For Experimental Medicine', 'Queen S University Belfast', 'Belfast', 'United Kingdom', 'Department Of Microbiology', 'Immunology', 'Faculty Of Pharmacy', 'Damanhour University', 'Damanhour']

Date: 2022-05

How double-membraned Gram-negative bacteria overcome lipid peroxidation is virtually unknown. Bactericidal antibiotics and superoxide ion stress stimulate the transcription of the Burkholderia cenocepacia bcnA gene that encodes a secreted lipocalin. bcnA gene orthologs are conserved in bacteria and generally linked to a conserved upstream gene encoding a cytochrome b 561 membrane protein (herein named lcoA, lipocalin-associated cytochrome oxidase gene). Mutants in bcnA, lcoA, and in a gene encoding a conserved cytoplasmic aldehyde reductase (peroxidative stress-associated aldehyde reductase gene, psrA) display enhanced membrane lipid peroxidation. Compared to wild type, the levels of the peroxidation biomarker malondialdehyde (MDA) increase in the mutants upon exposure to sublethal concentrations of the bactericidal antibiotics polymyxin B and norfloxacin. Microscopy with lipid peroxidation–sensitive fluorescent probes shows that lipid peroxyl radicals accumulate at the bacterial cell poles and septum and peroxidation is associated with a redistribution of anionic phospholipids and reduced antimicrobial resistance in the mutants. We conclude that BcnA, LcoA, and PsrA are components of an evolutionary conserved, hitherto unrecognized peroxidation detoxification system that protects the bacterial cell envelope from lipid peroxyl radicals.

Funding: This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (BBSRC grant BB/S006281/1) to MAV, and by grants from the Natural Sciences and Engineering Research Council (NSERC RGPIN-2019-05935) and the Canada Foundation for Innovation (Project Numbers 10401 and 36368) to G.C. MN was supported from a Scholarship Award by the Newton-Moshrafa PhD Programme, a partnership of the British Council and Egypt; N.F. was supported by an International PhD Fellowship from the Northern Ireland Department of Economy; J.M. was supported by an NSERC Post-Graduate Scholarship (CGSD3-519527-2018). OME is supported by a Canada Research Chair in Chemogenomics and Antimicrobial Research (Project Number 950-232965). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The B. cenocepacia bcnA and bcnB genes are in a 3-gene operon; they are located downstream from a gene encoding a predicted membrane cytochrome b 561 protein [ 27 ], which we herein named lipocalin-associated cytochrome oxidase gene (lcoA; S1 Fig ). We previously showed that lcoA (formerly bcoA), bcnA, and bcnB are cotranscribed [ 29 ]; lcoA and bcnA are also up-regulated in response to sublethal concentrations of bactericidal antibiotics and the pro-oxidant herbicide paraquat [ 25 , 27 ]. Similarly, the transcription of the P. aeruginosa PA0423 bcnA ortholog also increases in response to hydrogen peroxide and paraquat [ 38 ]. The physical and regulatory link between bcnA and lcoA suggests that their protein products could provide an antioxidant function. Due to the periplasmic and membrane locations of BcnA and LcoA, respectively, we hypothesized that these proteins are involved in protecting outer membrane phospholipids from peroxyl radicals, particularly under oxidative stress mediated by sublethal concentrations of antibiotics and other pro-oxidant molecules, such as paraquat or toxic metals. In this study, we demonstrate that both BcnA and LcoA, together with the conserved cytoplasmic aldehyde reductase PsrA, are components of a hitherto unrecognized system to protect Gram-negative bacteria from membrane lipid–derived peroxidation damage.

In addition to BcnA and BcnB (PDB IDs 5IXH and 5IXG , respectively) [ 27 ], several YceI bacterial lipocalins have been crystallized from Escherichia coli (PDB ID 1Y0G ), Pseudomonas syringae (PDB ID 3Q34 ), Campylobacter jejuni (PDB ID 2FGS ), Helicobacter pylori (PDB ID 3HPE ) [ 30 ], Pseudomonas aeruginosa (PDB ID 7BWL ) [ 31 ], Thermus thermophilus (PDB ID 1WUB ) [ 32 ], Saccharophagus degradans (PDB ID 2X32 ) [ 33 ], and Treponema pallidum (PDB ID 5JK2 ) [ 34 ]. These molecules reveal remarkable structural conservation despite notable differences in their amino acid sequences. Further, an isoprenoid molecule, typically octaprenylphosphate, octaprenylphenol, or ubiquinone-8 (Ubi-8, coenzyme Q), has been detected in the lipocalin β-barrel tunnel of most of the structures. From these, Ubi-8 plays a role in electron transfer reactions and as an antioxidant [ 35 , 36 ]. The observation that the Neisseria meningitidis YceI lipocalin has Ubi-8 bound when purified from periplasmic extracts [ 37 ] argues that Ubi-8 could be a natural substrate for YceI lipocalins.

In addition to conventional mechanisms of antibiotic resistance, we previously discovered that the opportunistic human pathogen Burkholderia cenocepacia can resist bactericidal antibiotics by mechanisms operating extracellularly [ 25 – 27 ]. Key molecules involved in these mechanisms are the polyamine putrescine [ 26 ] and the BcnA lipocalin protein [ 27 ]. BcnA, which belongs to the widely conserved YceI family of bacterial lipocalins, protects bacteria from bactericidal antibiotics by molecular scavenging [ 27 ]. B. cenocepacia also produces BcnB, another lipocalin that lacks antibiotic binding activity [ 27 ]. BcnA and BcnB proteins share a characteristic lipocalin β-barrel shape [ 27 , 28 ] composed of 8 antiparallel β-strands and relatively unstructured (flexible) loops at the open end, which form a “cup” domain [ 27 ]. In solution, BcnB forms a dimer, while BcnA is a monomeric protein. Both proteins are predicted to be in the periplasm, but only BcnA is secreted extracellularly in B. cenocepacia by an unknown mechanism [ 27 ]. Molecular dynamics and in silico docking experiments revealed that antibiotics interact with the rim of BcnA’s cup domain [ 27 ]; their binding interactions are weaker compared to that of hydrophobic ligands that can access the interior of the tunnel such as Nile red, polymyxin B, and α-tocopherol [ 27 , 29 ]. The structural and molecular binding characteristics of BcnA argue against the notion that antibiotic binding is the primary function of this lipocalin.

Gram-positive bacteria contain a single cell membrane surrounded by a thick cell wall peptidoglycan layer, while the Gram-negative bacterial cell envelope consists of 2 membranes, inner and outer membrane, separated by a periplasmic region containing cell wall peptidoglycan. The inner membrane is a phospholipid bilayer; the outer membrane is an asymmetrical bilayer consisting of phospholipids at the inner leaflet and lipopolysaccharide (LPS) at the outer leaflet [ 8 ]. The most abundant phospholipids in Gram-negative bacteria are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL), all carrying monounsaturated acyl chains [ 9 – 11 ]; the lipid A moiety of the LPS generally contains saturated acyl chains [ 8 ]. Upon oxidative stress, bacteria produce antioxidant enzymes (e.g., superoxide dismutases and catalases/peroxidases) that attenuate ROS-generated oxidative damage [ 12 , 13 ]. The response to ROS has been well studied [ 14 ], but how bacterial cells, especially the double-membraned Gram-negatives, overcome membrane lipid peroxidation stress is unclear. This partly relates to the prevailing notion that membrane lipids containing polyunsaturated fatty acids with labile bis-allylic hydrogen atoms, which are highly susceptible to lipid peroxidation [ 15 ], are generally absent from bacteria, whose membranes are rich in poorly oxidizable saturated or monounsaturated lipid molecules [ 10 , 16 , 17 ]. However, lipid peroxidation occurs in bacteria under many conditions, such as exposure to tert-butyl hydroperoxide (TBH) [ 18 ], hydrogen peroxide [ 19 ], potassium tellurite [ 2 , 20 ] and titanium oxide [ 21 ], defects in the synthesis of polyamines [ 22 ], and sublethal concentrations of antibiotics [ 3 , 23 , 24 ]. Because of the destructive nature of the lipid peroxidation reaction, bacterial cells must deal with both lipid radical formation and membrane repair. While restoring oxidized lipids in the inner membrane (or the cell membrane in Gram-positives) would be expected through regulatory responses influencing phospholipid synthesis, a mechanism to protect Gram-negative bacterial cells against lipid peroxyl radicals and toxic reactive aldehydes arising from the peroxidation of outer membrane phospholipids is presently unknown.

Aerobic bacteria use molecular oxygen for respiration and oxidation of nutrients with the concomitant production of toxic reactive by-products, which include reactive oxygen species (ROS), such as superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals [ 1 ], as well as nitrogen and electrophilic species. Oxidative stress may also arise from bacterial exposure to physical agents (e.g., ultraviolet radiation), metals, and chemicals (e.g., potassium tellurite, paraquat, and bactericidal antibiotics) [ 1 – 3 ]. Free radicals damage cell membranes via the formation of lipid peroxides. Lipid peroxidation is a nonenzymatic autocatalytic radical chain reaction resulting in the destruction of the phospholipid acyl chains and production of toxic reactive aldehydes such as acrolein, 4-hydroxynonenal, and malondialdehyde (MDA), and eventually alters membrane fluidity and barrier function [ 4 – 7 ]. Like oxygen radicals, lipid aldehydes can react with DNA and proteins, but they are more stable and can cause sustained damage [ 1 ].

Results

bcnA and lcoA gene orthologs are genetically linked in many different Gram-negative bacterial species To support the notion that BcnA and LcoA proteins could be functionally linked, we examined the synteny of their respective gene orthologs in the β-proteobacteria using MultiGeneBlast [39]. The results revealed that lcoA-bcnA-bcnB gene orthologs are present in the same organization in most species of the β-proteobacteria, including all members of the Burkholderia genus (S1 Fig). Exceptions were found in Neisseria, Bordetella, and Achromobacter species, where bcnA was identified as a monocistronic gene unlinked to lcoA or bcnB (S1 Fig). The lcoA-bcnA genes are also conserved as a predicted 2-gene operon in some of the γ-proteobacteria (e.g., Escherichia, Enterobacter, Proteus, and Pseudomonas, while bcnA gene orthologs are monocistronic in others (e.g., Klebsiella and Acinetobacter) (S1 Fig). A conserved gene organization within bacterial operons is generally related to function [40]. In most of the α-proteobacteria (e.g., Caulobacter, Agrobacterium, Sphingomonas, Rhizobium, and Parvularcula), lcoA and bcnA genes are fused, suggesting that they encode a predicted chimeric protein with a cytochrome b 561 amino-terminal domain and a BcnA lipocalin carboxyl-terminal domain. In these cases, the predicted LcoA-BcnA chimeras have a linker domain that replaces the amino acids corresponding to the signal peptide of the lipocalin, suggesting that BcnA is tethered to the membrane-embedded cytochrome b 561 . The conserved gene neighborhood and examples of lcoA-bcnA fused genes imply a functional link between the LcoA cytochrome b 561 and the BcnA lipocalin.

BcnA, LcoA, and PsrA show oxygen-dependent increased accumulation of lipid peroxidation by-products upon antibiotic-mediated stress The involvement of BcnA, LcoA, and PsrA in reducing the level of membrane lipid peroxidation suggested that mutants in these genes accumulate lipid peroxidation by-products. At sublethal doses, bactericidal antibiotics (e.g., β-lactams, quinolones, and colistin/polymyxin B) induce the formation of hydroxyl radicals that oxidize lipid membranes leading to the accumulation of reactive toxic aldehydes such as MDA [3]. The levels of MDA therefore serve as a proxy to assess the magnitude of lipid peroxidation and toxic aldehydes production. We pretreated bacteria with 2 different bactericidal antibiotics, norfloxacin and polymyxin B, at concentrations equivalent to half their respective MICs. The results using norfloxacin (S3A Fig) show that in the absence of the BcnA protein, MDA levels increased by 3-fold compared to wild type under exposure to sublethal concentration of norfloxacin (S3A Fig, p < 0.0001). MDA levels returned to wild-type concentrations upon genetic complementation (p = 0.99). Similarly, a statistically significant increase in the levels of MDA was seen in ΔlcoA mutant versus the wild-type strain (p = 0.02), which returned to wild-type levels upon complementation with LcoA. In the case of ΔpsrA, there was also a significant 2-fold increase in the level of modified protein in comparison to wild type (p < 0.001). As with the DPPP experiments, there was no difference in MDA levels between wild type and ΔbcnB. Similar results were obtained in bacteria incubated with polymyxin B (S4 Fig). From these experiments, we concluded that bacteria lacking BcnA, LcoA, and PsrA accumulate the lipid peroxidation by-product MDA upon antibiotic-induced stress. We reasoned that if BcnA, LcoA, and PsrA are required during peroxidation stress, these proteins would be dispensable under reduced concentrations of molecular oxygen. Bacteria were challenged with 0.25 MIC of norfloxacin, and surviving cells were enumerated after growth under microaerophilic conditions that allow survival of B. cenocepacia, for over 48 hours [49]. Control experiments showed that B. cenocepacia, which is an obligate aerobic bacterium, did not grow under these microaerophilic conditions with concentrations of norfloxacin above 0.25 MIC. No significant differences were found in the survival of wild-type and mutant strains in the absence or in the presence of 0.25 MIC norfloxacin (S3B Fig). Despite that in the presence of antibiotic the number of surviving bacteria was lower than in the untreated control (probably due the added stress of the antibiotic treatment under microaerophilia), no differences were observed among the strains (S3B Fig), indicating that both wild type and mutants exhibit similar antibiotic susceptibility under microaerophilic conditions. In contrast, the control experiment under aerobiosis revealed differences in norfloxacin susceptibility, consistent with the loss of BcnA, LcoA, and PsrA function (S3C Fig). Indeed, all mutants lacking the bcnA gene showed significant reduction in the number of surviving bacteria relative to wild type (p < 0.0001). Similar results were obtained with the single ΔlcoA and ΔpsrA mutants (p < 0.0001 and p < 0.001, respectively). Bacterial growth was restored to wild type or near wild-type levels in mutants genetically complemented with either BcnA or LcoA encoding plasmids. As before, the ΔbcnB mutant treated with norfloxacin displayed no difference in susceptibility when compared to wild type (p = 0.79). From these results, we conclude that the function of BcnA, LcoA, and PsrA proteins are not required under reduced oxygen levels, consistent with their role in reducing the accumulation of lipid peroxidation products under oxidative stress.

BcnA, LcoA, and PsrA promotes reduced levels of cell envelope peroxidation especially at the cell poles and the septum We directly assessed the level of membrane peroxidation by fluorescence microscopy using 2,2,6-trimethyl-4-(4-nitrobenzo [1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen), a fluorogenic probe that becomes emissive [50] upon scavenging lipid peroxyl radicals in living cells due the presence of an α-substituted nitroxide [51,52]. In these experiments, we compared the ΔbcnA mutant and the wild-type strain K56-2, both without and with antibiotic stress caused by exposure to sub-MIC of norfloxacin (4 μg ml−1 and 48 μg ml−1, respectively [29]) over 4 hours before adding the fluorescent probe. Fluorescence images of untreated cells showed that K56-2 bacteria fluoresced with lower intensity than ΔbcnA (Fig 3A). In contrast, the ΔbcnA cells treated with norfloxacin show higher levels of fluorescence around cells, with a punctate distribution at the poles and in some cells at the midbody, corresponding to cell division sites. K56-2 cells treated with norfloxacin also displayed discrete fluorescence patches at the poles, with some cells lacking detectable peroxidation when comparing fluorescence and phase contrast images. K56-2 bacterial cells treated with norfloxacin gave a fluorescence intensity comparable to that of the ΔbcnA control without norfloxacin, further supporting the idea that at steady state, bacteria lacking BcnA produce higher levels of peroxidation. Quantification of fluorescence (Fig 3B) indicated significant differences between norfloxacin-treated and norfloxacin-untreated bacteria, as well as significant differences between norfloxacin-treated K56-2 and ΔbcnA, supporting the notion that BcnA has a role in reducing lipid peroxyl radical formation. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Detection of lipid peroxidation in ΔbcnA by fluorescence microscopy using NBD-Pen. (A) Fluorescence (left) and phase contrast (right) images of ΔbcnA and K56-2 strains untreated or treated with 75% MIC for norfloxacin (Nfx) and incubated with NBD-Pen. The insets are zoomed sections of the images to provide more detail of the location of the fluorescent at the cell poles and mid cell (arrowheads). Scale bars, 10 μm. (B) Quantitative analysis of fluorescence intensity based on fluorescence images in panel A. SD shown as error bars, and mean values shown as horizontal black lines. Statistical significance was determined by 1-way ANOVA with Tukey multiple comparisons test post hoc analysis. ****, p < 0.0001; ns, nonsignificant. Data underlying the graphs in this figure can be found in S2 Data. MIC, minimum inhibitory concentration; NBD-Pen, 2,2,6-trimethyl-4-(4-nitrobenzo [1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl; SD, standard deviation. https://doi.org/10.1371/journal.pbio.3001610.g003 We also examined the level of membrane peroxidation by quantitative fluorescence in the ΔlcoA and ΔpsrA mutants. Unlike ΔbcnA, ΔlcoA produced higher levels of membrane lipid peroxidation in the absence of antibiotic compared to the wild-type K56-2 treated with norfloxacin (S5A Fig). We speculate that the ΔlcoA mutant has a higher level of basal lipid peroxidation arising from an increased intracellular level of ROS [53]. The ΔpsrA mutant, in contrast, behave like ΔbcnA by showing peroxidation levels in the untreated condition that were like those of antibiotic treated K56-2. Quantification of fluorescence (S5B Fig) demonstrated significant differences between treated and untreated bacteria, as well as a significant difference between the wild-type strain K56-2 and the mutants, ΔlcoA, and ΔpsrA. Similar results were obtained using polymyxin B for all the mutants (S6 and S7 Figs). To confirm that lipid peroxyl radicals at the cell poles are more abundant under antibiotic stress, we quantified the fluorescence intensity across the long axis of the cells. The results show that, in contrast to cells under no antibiotic stress, where the fluorescence is homogenous along the bacterial cell length, peaks of higher intensity are present at the poles when the bacteria are exposed to antibiotics (S8 Fig). Given that nitroxides and their fluorogenic analogues are radical trapping antioxidant scavengers both in solution and in micelles [54,55], the emission intensity enhancement with nitroxide–fluorophore adducts may partly arise because of radical trapping antioxidant scavenging in solution, rather than scavenging lipid peroxyl radicals [56]. Therefore, we also investigated membrane lipid peroxidation with the lipophilic fluorogenic antioxidant probe 8-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-methyl)-1,5-di(3-chloropropyl)-pyrromethene fluoroborate (H 4 BPMHC) [56–58], which consists of α-tocopherol chromanol moiety (PMHC, a chromanol-based free radical scavenger) and a BODIPY lipophilic fluorophore that renders the lipophilic character of the fluorogenic probe and facilitates its incorporation into membranes. The α-tocopherol chromanol moiety in H 4 BPMHC quenches the BODIPY fluorescence via photo-induced electron transfer until this moiety is oxidized by lipid peroxyl radicals, restoring fluorescence, and allowing the generation of these lipid peroxyl radicals to be visualized [56–58]. In this experiment, the fluorescence was seen in the cell envelope of the mutant bcnA at its normal state, correlating with peroxidation of membrane lipids (S9A Fig). The distribution of fluorescence adopted a punctate pattern with strong accumulation of fluorescent spots at bacterial cell poles and septa. Image quantification demonstrated that ΔbcnA had 3-fold higher fluorescence intensity than K56-2 (p = 0.0001) and the complemented mutant ΔbcnA(pBcnA) (S9B Fig). We therefore conclude that the localization of the peroxidation in the membrane was independent of the chemistries of the fluorogenic probes. The combined results from all these experiments demonstrate that the ΔbcnA, ΔlcoA, and ΔpsrA mutant cells display high level of peroxidation at the cell envelope with a discrete spatial distribution, being in general more pronounced at the poles and septal regions.

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