Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane

Manioğlu, Selen; Modaresi, Seyed Majed; Ritzmann, Noah; Thoma, Johannes; Overall, Sarah A.; Harms, Alexander; Upert, Grégory; Luther, Anatol; Barnes, Alexander B.; Obrecht, Daniel; Müller, Daniel J.; Hiller, Sebastian

doi:10.1038/s41467-022-33838-0

Cited by 39 publications

(42 citation statements)

References 60 publications

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“…More care would need to be taken for labeling Gram-negative bacteria to ensure that the used fluorescent marker labels only the membrane of interest, either the inner or the outer membrane. Third, additional care must be taken when performing FCS experiments in Gram-negative bacteria, as the topography of the outer membrane was previously shown to be affected by various compounds (26, 64) which itself affects measurement of diffusion speeds with FCS (65). Our work confirmed the results of previous experiments on the effect of a cold shock on membrane fluidity.…”

Section: Discussionmentioning

confidence: 99%

Quantification of membrane fluidity in bacteria using TIR-FCS

Barbotin,

Billaudeau,

Sezgin

et al. 2023

Preprint

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Cell membrane fluidity is an important phenotypic feature that regulates the diffusion, function and folding of transmembrane and membrane-associated proteins. It is particularly interesting to study it in bacteria as variations in membrane fluidity are known to affect fundamental cellular processes such as respiration, transport and antibiotic resistance. As such key parameter, membrane fluidity is regulated to adapt to environmental variations and stresses like temperature fluctuations or osmotic shocks. Membrane fluidity has been however scarcely studied quantitatively in bacterial cells, mostly because of the lack of available tools. Here, we developed an assay based on total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. We used this assay to quantify the fluidity of the cytoplasmic membrane of the Gram-positive model bacterium Bacillus subtilis in response to a cold shock, caused by a shift from 37°C to 20°C. In our experimental conditions, steady-state fluidity was recovered within 30 mins, and the steady-state fluidity at 20°C was about half of that at 37°C. Our minimally invasive assay opens up exciting perspectives and could be used to study a wide range of phenomena affecting the bacterial membrane, from disruption by antibiotics, antimicrobial peptides, or osmotic shocks.

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Section: Discussionmentioning

confidence: 99%

Quantification of membrane fluidity in bacteria using TIR-FCS

Barbotin,

Billaudeau,

Sezgin

et al. 2023

Preprint

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“…Nevertheless, improved dosing regimens and the rise of Gram-negative MDR strains led to a renaissance of their clinical use when everything else was failing 4 . Sadly, PM resistance has also emerged, mostly due to outer membrane lower permeability 7 , but also through nonspecific mechanisms (e.g.…”

Section: Introductionmentioning

confidence: 99%

Machine Learning Modeling and Insights into the Structural Foundations of Polymyxin-like Antimicrobials

Machado

Inácio

Jorge

et al. 2023

Preprint

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Antimicrobial resistance (AMR) is a silent pandemic that represents an urgent threat to human health. Unfortunately, the antibiotic development pipeline is slow even though AMR has been escalating uncontrollably fast, namely amongst Gram-negative pathogens. Although out of use until recently due to their toxic side effects, polymyxins have been revived as a last-line therapeutic option since all other antibiotics are currently failing. In an attempt to ameliorate their toxicity and improve antimicrobial activity, many studies have been generating polymyxin analogues through different strategies, mostly empirical. As such, there is still a lack of faster and more reliable approaches to make analog design efficient in order to tackle AMR in a timely fashion. The solution to accelerate the discovery of new drugs probably lies in the use of in silico approaches, such as machine learning, due to their faster pace and time and cost efficiency. In this work, machine learning was applied to Quantitative Structure-Activity Relationship (QSAR) modeling with the objective of providing a working semi-quantitative model capable of predicting the activity of polymyxin-like molecules for a given species. For this, we applied four different learning algorithms and ten different families of molecular descriptors to our dataset of 408 molecule/microorganism pairs retrieved from PubChem. The AdaBoost model devised using the CKP set of descriptors was the best performer, with good accuracies and very low false negative and positive predictions. Preliminary exploration of the model's response to systematic changes in the structure of polymyxin B reveals a trend towards increased antimicrobial activity when exchanging some of its constituent amino acids for more lipophilic ones. Experimental studies are already underway based on this model's application and we believe it will become a crucial tool for drug development.

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“…In many organisms, like Escherichia coli, the lipid A portion of LPS is negatively charged due to the presence of phosphate groups on the glucosamine disaccharide (3). These phosphates are the binding sites for cationic antimicrobial peptides (CAMPs) like polymyxins (4,5). While LPS is generally considered to be essential, LPS-null mutants of several Gram-negative organisms have been isolated including Acinetobacter baumannii (6), Moraxella catarrhalis (7), Neisseria meningitidis (8), and Caulobacter crescentus (9).…”

Section: Introductionmentioning

confidence: 99%

Characterization of an evolutionarily distinct bacterial ceramide kinase fromCaulobacter crescentus

Dhakephalkar

Stukey

Guan

et al. 2023

Preprint

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A common feature among nearly all Gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides structural integrity to the bacterial membrane which aids bacteria in maintaining their shape and acts as a barrier from environmental stress and harmful substances such as detergents and antibiotics. Recent work has demonstrated that Caulobacter crescentus can survive without LPS due to the presence of the anionic sphingolipid ceramide-phosphoglycerate. Based on genetic evidence, we predicted that protein CpgB functions as a ceramide kinase and performs the first step in generating the phosphoglycerate head group. Here, we characterized the kinase activity of recombinantly expressed CpgB and demonstrated that it can phosphorylate ceramide to form ceramide 1-phosphate. The pH optimum for CpgB was 7.5, and the enzyme required Mg2+ as a cofactor. Mn2+, but not other divalent cations, could substitute for Mg2+. Under these conditions, the enzyme exhibited typical Michaelis-Menten kinetics with respect to NBD-C6-ceramide (Km,app=19.2 ± 5.5 uM; Vmax,app=2586.29 ± 231.99 pmol/min/mg enzyme) and ATP (Km,app=0.29 ± 0.07 mM; Vmax,app=10067.57 ± 996.85 pmol/min/mg enzyme). Phylogenetic analysis of CpgB revealed that CpgB belongs to a new class of ceramide kinases which is distinct from its eukaryotic counterpart; furthermore, the pharmacological inhibitor of human ceramide kinase (NVP-231) had no effect on CpgB. The characterization of a new bacterial ceramide kinase opens avenues for understanding the structure and function of the various microbial phosphorylated sphingolipids.

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Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane

Cited by 39 publications

References 60 publications

Quantification of membrane fluidity in bacteria using TIR-FCS

Quantification of membrane fluidity in bacteria using TIR-FCS

Machine Learning Modeling and Insights into the Structural Foundations of Polymyxin-like Antimicrobials

Characterization of an evolutionarily distinct bacterial ceramide kinase fromCaulobacter crescentus

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