Martin Jakubec’s research while affiliated with University of Bergen and other places

The polar bear ( Ursus maritimus ) is the only Arctic land mammal that dives into water to hunt. Despite thermal insulation provided by blubber and fur layers and low Arctic temperatures, their fur is typically observed to be free of ice. This study investigates the anti-icing properties of polar bear fur. Here, we show that polar bear fur exhibits low ice adhesion strengths comparable to fluorocarbon-coated fibers, with the low ice adhesion a consequence of the fur sebum (hair grease). Lipid analyses reveal the presence of cholesterol, diacylglycerols, anteisomethyl-branched fatty acids, and the unexpected absence of squalene. Quantum chemical calculations predict low ice adsorption energies for identified lipids and high adsorption for squalene, suggesting that sebum composition is responsible for the observed anti-icing properties. Our work enhances understanding of polar bears and their interactions with their environment and builds on Inuit knowledge of natural anti-icing materials.

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point need to either cross or just interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions with novel molecules -both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing relevant synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Bacterial extracellular vesicles (bEVs) are naturally secreted by Gram-negative and Gram-positive bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. bEVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work, we have isolated and characterized bEVs from one Escherichia coli mutant and three clinical strains of the ESKAPE pathogens Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The bEVs were shown to be representative models for the bacterial membrane in terms of lipid composition with speciesstrain specific variations. The bEVs were further used to probe the interactions between bEV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that bEVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect induced by antibiotics, or the response to host-pathogen interactions.

One strategy to combat antimicrobial resistance is the discovery of new classes of antibiotics. Most antibiotics will at some point interact with the bacterial membrane to either interfere with its integrity or to cross it. Reliable and efficient tools for determining the dissociation constant for membrane binding ( K D ) and the partitioning coefficient between the aqueous- and membrane phases ( K P ) are therefore important tools for discovering and optimizing antimicrobial hits. Here we demonstrate that microscale thermophoresis (MST) can be used for label-free measurement of K D by utilising the intrinsic fluorescence of tryptophan and thereby removing the need for chromophore labelling. As proof of principle, we have used the method to measure the binding of a set of small cyclic AMPs to large unilamellar vesicles (LUVs) and two types of lipid nanodiscs assembled by styrene maleic acid (SMA) and quaternary ammonium SMA (SMA-QA). The measured K D values correlate well with the corresponding measurements using surface plasmon resonance (SPR), also broadly reflecting the tested AMPs’ minimal inhibition concentration (MIC) towards S. aureus and E. coli. We conclude that MST is a promising method for fast and cost-efficient detection of peptide-lipid interactions or mapping of sample conditions in preparation for more advanced studies that rely on expensive sample preparation, labelling and/or instrument time.

Antimicrobial peptides (AMPs) are generally membrane-active compounds that physically disrupt bacterial membranes. Despite extensive research, the precise mode of action of AMPs is still a topic of great debate. This work demonstrates that the initial interaction between the Gram-negative E. coli and AMPs is driven by lipopolysaccharides (LPS) that act as kinetic barriers for the binding of AMPs to the bacterial membrane. A combination of SPR and NMR experiments provide evidence suggesting that cationic AMPs first bind to the negatively charged LPS before reaching a binding place in the lipid bilayer. In the event that the initial LPS-binding is too strong (corresponding to a low dissociation rate), the cationic AMPs cannot effectively get from the LPS to the membrane, and their antimicrobial potency will thus be diminished. On the other hand, the AMPs must also be able to effectively interact with the membrane to exert its activity. The ability of the studied cyclic hexapeptides to bind LPS and to translocate into a lipid membrane is related to the nature of the cationic charge (arginine vs. lysine) and to the distribution of hydrophobicity along the molecule (alternating vs. clumped tryptophan).

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point needs to either cross or interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions to novel antimicrobials – both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing realistic synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Outer membrane vesicles (OMVs) are naturally secreted by Gram-negative bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. OMVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work we have isolated and characterized OMVs from E. coli mutant strains and clinical isolates of the ESKAPE members Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa . The OMVs were shown to be representative models for the bacterial membrane in terms of lipid composition with strain specific variations. The OMVs were further used to probe the interactions between OMV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that OMVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect of induction by antibiotics, or the response to host-pathogen interactions.

Antimicrobial peptides (AMPs) are generally membrane-active compounds that physically disrupt bacterial membranes. Despite extensive research, the precise mode of action of AMPs is still a topic of great debate. This work demonstrates that the initial interaction between the Gram-negative E. coli and AMPs is driven by lipopolysaccharides (LPS) that act as kinetic barriers for the binding of AMPs to the bacterial membrane. A combination of SPR and NMR experiments provide evidence suggesting that cationic AMPs first bind to the negatively charged LPS before reaching a binding place in the lipid bilayer. In the event that the initial LPS-binding is too strong (corresponding to a low dissociation rate), the cationic AMPs cannot effectively get from the LPS to the membrane and their antimicrobial potency will thus be diminished. On the other hand, the AMPs must also be able to effectively interact with the membrane to exert its activity. The ability of the studied cyclic hexapeptides to bind LPS and to translocate into a lipid membrane is related to the nature of the cationic charge (arginine vs lysine) and to the distribution of hydrophobicity along the molecule (alternating vs clumped tryptophan).

One strategy to combat antimicrobial resistance is the discovery of new classes of antibiotics. Most antibiotics will at some point interact with the bacterial membrane to either interfere with its integrity or to cross it. Reliable and efficient tools for determining the dissociation constant for membrane binding (KD) and the partitioning coefficient between the aqueous- and membrane phases (KP) are therefore important tools for discovering and optimizing antimicrobial hits. Here we demonstrate that microscale thermophoresis (MST) can be used for label-free measurement of KD by utilising the intrinsic fluorescence of tryptophan and thereby removing the need for chromophore labelling. As proof of principle, we have used the method to measure the binding of a set of small cyclic AMPs to large unilamellar vesicles (LUVs) and two types of lipid nanodiscs assembled by styrene maleic acid (SMA) and quaternary ammonium SMA (SMA-QA). The measured KD values correlate well with the corresponding measurements using surface plasmon resonance (SPR), also broadly reflecting the tested AMPs’ minimal inhibition concentration (MIC) towards S. aureus and E. coli. We conclude that MST is a promising method for fast and cost-efficient detection of peptide-lipid interactions.

Lipids have been implicated in Parkinson's Disease (PD). We therefore studied the lipid profile of the neuroblastoma SH-SY5Y cell line, which is used extensively in PD research and compared it to that of the A431 epithelial cancer cell line. We have isolated whole cell extracts (WC) and plasma membrane (PM) fractions of both cell lines. The isolates were analyzed with 31P NMR. We observed a significant higher abundance of phosphatidylcholine (PC) for SH-SY5Y cells for both WC (55 ± 4.1%) and PM (63.3 ± 3.1%) compared to WC (40.5 ± 2.2%) and PM (43.4 ± 1.3%) of A431. Moreover, a higher abundance of phosphatidylethanolamine was detected for the WC of A431 compared to the SH-SY5Y. Using LC-MS/MS, we also determined the relative abundance of fatty acid (FA) moieties for each phospholipid class, finding that SH-SY5Y had high polyunsaturated FA levels, including arachidonic acid compared to A431 cells. When comparing our results to reported compositions of brain and neural tissues, we note the much higher PC levels, as well as very low levels of docosahexaenoic acid. However, relative levels of arachidonic acid and other polyunsaturated fatty acids were elevated, in line with what is desirable for a neural model system.

Antimicrobial peptides (AMPs) are a promising source of inspiration for new antibiotics discovery, in part because they believed to not trigger rapid resistance development since AMPs have co-existed with bacteria throughout evolution and do not target a single enzyme encoded by a single gene. AMPs is a diverse class of molecules that have diverse and often unspecific modes of action interfering with the membrane potential or the bacterial cell wall integrity, but also intracellular targets have been reported. Regardless of the mode of action(s), the AMPs will first have to interact with-, or penetrate through-, the bacterial cell well, and early characterization of AMP activity relies on the determination of the KD (dissociation constant for binding affinity) and the KP (partitioning constant) of AMP:lipid interactions. Here we demonstrate that microscale thermophoresis (MST) can be used for reliable unlabelled measurement of KD and KP utilising the intrinsic tryptophan fluorescence, thus removing the need for chromophore labelling. The MST results of binding to small unilamellar vesicles (SUVs) and stryrene maleic acid (SMA) based nanodiscs are compared to the corresponding surface plasmon resonance (SPR) measurements. SMA-QA nanodiscs are shown to be best suited for accurate measurements, while vesicles are a viable alternative. Unmodified SMA-nanodiscs proved unsuitable due to interactions between the cationic AMPs and the anionic polymer belt. Significant reduction of KD was observed when 5% anionic lipids were included in the lipid composition of the membrane models. This highlights the preference of the tested AMPs for anionic bacterial membranes, and the measured KD and KP values correlate well with their activity towards S. aureus and E. coli. We conclude that MST is a promising method for fast and efficient detection of peptide-lipid interactions, and the relative strength of the interactions can be reliably ranked within a library of screened compounds.

Antimicrobial peptides (AMPs) are a promising source of inspiration for new antibiotic discovery, in part because they believed to not trigger rapid resistance. AMPs can target many features of the cell surface, and effective resistance development may require multiple mutations in parallel that have large fitness costs. Although they have diverse modes of action, AMPs will often begin by binding to the membrane; cell-penetrating peptides will subsequently need to cross it. Characterization of AMP activity can thus be inferred by the determination of KD (dissociation constant for binding affinity) and KP (partitioning constant). Here we demonstrate that microscale thermophoresis (MST) can be used for reliable label-free measurement of KD and KP utilising the intrinsic tryptophan fluorescence - removing the need for chromophore labelling. The MST results of binding to small unilamellar vesicles (SUVs) and styrene maleic acid (SMA) based nanodiscs are compared to the corresponding surface plasmon resonance (SPR) measurements. SMA-QA nanodiscs are shown to be best suited for accurate measurements, while vesicles are a viable alternative. Unmodified SMA-nanodiscs proved unsuitable due to interactions between the cationic AMPs and the anionic polymer belt. Significant reduction of KD was observed when 5% anionic lipids were included in the lipid composition of the membrane models. This highlights the preference of the tested AMPs for anionic bacterial membranes, and the measured KD and KP values correlate well with their activity towards S. aureus and E. coli. We conclude that MST is a promising method for fast and efficient detection of peptide-lipid interactions, and the relative strength of the interactions can be reliably ranked within a library of screened compounds.