Biology subjects

Bandow, J. E.

Publications and source records attributed to Bandow, J. E..

9 recordsLinked to original sources

bioRxiv2025

Plasma-driven biocatalysis using the cytochrome P450 enzyme CYP152BSβ

Plasma-driven biocatalysis utilizes in situ H2O2 production by atmospheric pressure plasmas to drive H2O2-dependent enzymatic reactions. Having previously established plasma-driven biocatalysis using recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) to produce (R)-1-phenylethanol from ethylbenzene (ETBE), we here employed CYP152 from Bacillus subtilis (CYP152BS{beta}). CYP152BS{beta} naturally hydroxylates medium and long-chain carboxylic acids, and, with short-chain carboxylic acids as decoy molecules, also converts non-natural substrates such as ETBE. To produce active CYP152BS{beta} overexpression and heme loading were optimized. The conversion of the non-natural substrates guaiacol and ABTS with heptanoic acid as decoy molecule and H2O2 from stock solution yielded 18.28 and 21.13 nmol product min-1 [Formula], respectively. These reactions also served to assess compatibility of CYP152BS{beta} with plasma-driven biocatalysis regarding temperature and H2O2 operating windows. To establish CYP152BS{beta}-based plasma-driven biocatalysis, immobilized enzyme in a rotating bed reactor (5 ml reaction volume) was then supplied with H2O2 from a capillary plasma jet operated with 1280 ppm H2O in helium. After a 120 min run time a turnover number (TON) of 18.82 mol(R)-1-PhOl [Formula] was reached. We conclude that plasma-driven biocatalysis can be extended to other H2O2-dependent enzymes. Future efforts will be directed at increasing the TON and product range.

biochemistry↗

bioRxiv2025

Iron-sulfur cluster proteins present the weak spot in plasma-treated Escherichia coli

Non-thermal atmospheric pressure plasmas have an antiseptic activity beneficial in different medical applications. In a genome-wide screening, hydrogen peroxide and superoxide were identified as key species contributing to the antibacterial effects of plasma while [FeS] cluster proteins emerged as potential cellular targets. We investigated the impact of plasma treatment on [FeS] cluster homeostasis in Escherichia coli treated for 1 min with the effluent of a microscale atmospheric pressure plasma jet ({micro}APPJ). Mutants defective in [FeS] cluster synthesis and maintenance lacking the SufBC2D scaffold protein complex or desulfurase IscS were hypersensitive to plasma treatment. Monitoring the activity of [FeS] cluster proteins of the tricarboxylic acid cycle (aconitase, fumarase, succinate dehydrogenase) and malate dehydrogenase (no [FeS] clusters), we identified cysteine, iron, superoxide dismutase, and catalase as determinants of plasma sensitivity. Survival rates, enzyme activity, and restoration of enzyme activity after plasma treatment were superior in mutants with elevated cysteine levels and in the wildtype under iron replete conditions. Mutants with elevated hydrogen peroxide and superoxide detoxification capacity over-expressing sodA and katE showed full protection from plasma-induced enzyme inactivation and survival rates increased from 34% (controls) to 87%. Our study indicates that metabolic and genetic adaptation of bacteria may result in plasma tolerance and resistance, respectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/631878v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@119626eorg.highwire.dtl.DTLVardef@18d2973org.highwire.dtl.DTLVardef@9c5568org.highwire.dtl.DTLVardef@1ab4e58_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG HighlightsO_LIEnzymes with [FeS] clusters are rapidly inactivated by plasma C_LIO_LIClusters damaged by plasma are repaired in vivo using iron and cysteine C_LIO_LIOver-expression of sodA and katE completely prevents disruption of [FeS] clusters by plasma C_LIO_LIPlasma resistance is increased threefold by SodA and KatE over-production C_LIO_LIPre-adaptation of E. coli to O2- increases plasma tolerance C_LI

microbiology↗

bioRxiv2025

The atmospheric pressure capillary plasma jet is well-suited to supply H2O2 for plasma-driven biocatalysis

Plasma-generated H2O2 can be used to fuel biocatalytic reactions that require H2O2 as co-substrate such as the conversion of ethylbenzene to (R)-1-phenylethanol ((R)-1-PhOl) catalyzed by unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). Immobilization was recently shown to protect biocatalysts from inactivation by highly reactive plasma-produced species, however, H2O2 supply by the employed plasma sources ({micro}APPJ and DBD) was limiting for rAaeUPO performance. In this study we evaluated a recently introduced capillary plasma jet for suitability to supply H2O2 in situ. H2O2 production was modulated by varying the water concentration in the feed gas, providing a greater operating window for applications in plasma-driven biocatalysis. In a static system after 80 min of biocatalysis, a turnover number of 44,199 mol(R)-1-PhOl mol-1rAaeUPO was achieved without significant enzyme inactivation. By exchanging the reaction solution every 5 min, a total product yield of 122 {micro}mol (R)-1-PhOl was achieved in 700 min run time, resulting in a total turnover number of 174,209 mol(R)-1-PhOl mol-1rAaeUPO. We conclude that the capillary plasma jet, due to its flexibility regarding feed gas, admixtures, and power input, is well-suited for in situ H2O2 generation for plasma-driven biocatalysis tailoring to enzymes with high H2O2 turnover.

biochemistry↗

bioRxiv2024

Trans-Translation inhibitors and copper ions synergize for enhanced antibiotic activity

Trans-Translation is the most effective ribosome rescue mechanism and a compelling target for new antimicrobial agents. A recent proteomic study revealed similarities between the responses of Bacillus subtilis to the inhibitors small-molecule inhibitors oxadiazole KKL-40 and tetrazole KKL-55 and divalent cation ionophores, indicating the disturbance of metal homeostasis as potential secondary mechanism of action. Here, we report increased copper levels in KKL-40 and KKL-55-treated B. subtilis. Both inhibitors form copper complexes that enter large unilamellar vesicles. Copper supplementation enhanced the antibacterial activity against B. subtilis by simultaneously increasing inhibitor and copper uptake. The co-treatment of B. subtilis with trans-translation inhibitors and copper concentrations normally benign for trans-translation-competent cells, caused an immediate stalling of growth and translation, as observed at higher KKL-40 and KKL-55 concentrations without copper supplementation. Proteome analysis showed that during translation stalling cells were unable to mount an effective copper toxicity response. Taken together, the synergetic uptake of KKL-40 and KKL-55 with copper leads to a quick-onset translation stalling, preventing B. subtilis from counteracting the toxic effects of rapid copper influx. Significance statementThe challenge of antimicrobial resistance is growing, necessitating an exploration of novel antibiotic targets. Among these, trans-translation has attracted considerable attention due to its ubiquitous presence in bacteria as well as its role in virulence and pathogenesis. Several inhibitors of trans-translation have been identified in a target-based screening using a whole-cell assay. However, recent proteomic profiling studies suggested that the tested trans-translation inhibitors might have an additional mode of action. In this work, we shed light on their previously undiscovered copper ionophore activity and explore the consequences of co-treating B. subtilis with KKL-40 or KKL-55 and CuCl2. This co-treatment results in a rapid antibiotic influx, and, consequently to the stalling of ribosomes, translation, and bacterial growth. Simultaneously, massive amounts of copper accumulate in the cells, the toxic effects of which require a copper stress response to mitigate. However, such a response is averted by the stalled translation. Dual mechanism antibacterial agents are attractive because they are typically associated with slow emergence of resistance. A deep understanding of the complex interplay of KKL-40 and KKL-55 with metal ions will help to fully exploit trans-translation as an antibacterial target and to develop KKL-40 and KKL-55-based antibiotics into novel treatments for bacterial infections.

microbiology↗

bioRxiv2024

HOCl Forms Lipid N-Chloramines in Cell Membranes of Bacteria and Immune Cells

Neutrophils orchestrate a coordinated attack on bacteria, combining phagocytosis with a potent cocktail of oxidants, including the highly toxic hypochlorous acid (HOCl), renowned for its deleterious effects on proteins. Here, we examined the occurrence of lipid N-chloramines in vivo, their biological activity and neutralization. Using a chemical probe for N-chloramines, we demonstrate their formation in the membranes of bacteria and monocytic cells exposed to physiologically relevant concentrations of HOCl. N-chlorinated model membranes composed of phosphatidylethanolamine, the major membrane lipid in Escherichia coli and an important component of eukaryotic membranes, exhibited oxidative activity towards the redox-sensitive protein roGFP2, suggesting a role for lipid N-chloramines in protein oxidation. Conversely, the cellular antioxidant glutathione neutralized lipid N-chloramines by removing the chlorine moiety. We propose that lipid N-chloramines, like protein N-chloramines, are involved in inflammation and accelerate the host immune response.

microbiology↗

bioRxiv2024

Auranofin induces disulfide bond-mimicking S-Au-S bonds in protein thiol pairs

Auranofin is an inhibitor of human thioredoxin reductase, clinically used in the treatment of rheumatoid arthritis. More recently, it has been shown to possess strong antibacterial activity. Despite the structural dissimilarity and the independent evolutionary origins of human thioredoxin reductase and its bacterial counterpart (TrxB), inhibition of bacterial thioredoxin reductase is often suggested to be a major factor in auranofins antibacterial mode of action. To test this hypothesis, we attempted to determine the mechanism of inhibition of auranofin for bacterial TrxB in the presence of thioredoxin, TrxBs natural substrate. However, the data obtained in these experiments was not consistent with a specific and exclusive interaction between TrxB and auranofin. Instead, it suggested that auranofin directly interacts with the cysteine thiols in thioredoxin, TrxBs substrate. Using the fluorescent redox protein roGFP2, we showed that auranofin does indeed directly interact with cysteine pairs in proteins, forming a thiol modification that is similar to, but clearly distinct from a disulfide bond. The Au:S stoichiometries of auranofin-treated roGFP2 and thioredoxin strongly suggest the presence of an S-Au-S bridge between two cysteines in those proteins. These S-Au-S bonds form independent of thioredoxin reductase at a rate that indicates their pertinence in auranofins antibacterial mode of action.

biochemistry↗

bioRxiv2023

Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

The antirheumatic gold(I) compound auranofin has been suggested to exhibit bactericidal activity by disrupting the thiol homeostasis through direct inhibition of bacterial thioredoxin reductase (TrxB). While highly effective at killing Gram-positive bacteria, it lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis. A change in the proteome of E. coli exposed to auranofin suggested that the effect of this gold compound is a combination of inactivation of thiol-containing enzymes, upregulation of proteins involved in basal metabolism, and the consequent induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking the proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. colis high tolerance to auranofin. To elucidate factors that could make Gram-negative bacteria less susceptible to auranofin, we tested E. coli cells lacking the efflux pump component TolC. These cells were more sensitive to auranofin treatment than the wild type, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested if E. colis thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis, which was not the case. E. coli cells lacking components of the thioredoxin-system were also only marginally more susceptible to auranofin. However, E. coli strains lacking the low molecular weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols in situ. In line with their susceptibility, the kinetics of probe oxidation and the degree of oxidation promoted by auranofin is significantly higher in Gram-positive bacteria when compared to Gram-negative bacteria. Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.

biochemistry↗

bioRxiv2023

Same same but different; The global response of Escherichia coli to five different LpxC inhibitors

A promising but yet clinically unexploited antibiotic target in difficult-to-treat Gram-negative bacteria is LpxC, the key enzyme in the biosynthesis of lipopolysaccharides (LPS), which are the major constituents of the outer membrane. To gain insights into the mode of action of five different LpxC inhibitors, we conducted a comparative phenotypic and proteomic analysis. All five compounds bound to purified LpxC from Escherichia coli. Treatment of E. coli with these compounds changed the cell shape and stabilized LpxC suggesting that the FtsH-mediated turnover is impaired. LpxC inhibition sensitized E. coli to the cell wall antibiotic vancomycin, which typically does not cross the outer membrane. Four of the five compounds led to an accumulation of lyso-PE, a cleavage product of phosphatidylethanolamine (PE), generated by the phospholipase PldA. The combined results suggested an imbalance in phospholipid (PL) and LPS biosynthesis, which was corroborated by the global proteome response to treatment with the LpxC inhibitors. Apart from LpxC itself, FabA and FabB responsible for the biosynthesis of unsaturated fatty acids, were consistently upregulated. Our work also shows that antibiotics targeting the same enzyme do not necessarily elicit identical cellular responses. Compound-specific marker proteins belonged to different functional categories, like stress responses, nucleotide or amino acid metabolism and quorum sensing. These findings provide new insights into common and distinct cellular defense mechanisms against LpxC inhibition. Moreover, they support a delicate balance between LPS and PL biosynthesis with great potential as point of attack for antimicrobial intervention. ImportanceThe alarming spread of antimicrobial resistance among Gram-negative bacteria calls for novel intervention strategies. Inhibitors of LpxC, the first committed enzyme of lipopolysaccharide biosynthesis have been recognized as promising broad-spectrum antibiotics against Gram-negative pathogens. Despite the development of dozens of chemically diverse LpxC inhibitor molecules, it is essentially unknown how bacteria counteract LpxC inhibition. Our study provides comprehensive insights into the bacterial defense strategies against five different LpxC inhibitors. We show that the cellular response of Escherichia coli is compound-specific but shares a common pattern. Inhibition of LpxC is toxic, disrupts membrane integrity, and elicits a stress response, including upregulation of fatty acid biosynthesis proteins. Pre-treatment of E. coli with low doses of LpxC inhibitors increased the sensitivity to the cell wall antibiotic vancomycin suggesting new directions in combination therapies.

microbiology↗

bioRxiv2021

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated proteins

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidAs chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidAs chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N- chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCls chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

biochemistry↗