Polymyxins are clinically used antibiotics, discovered in mid-20th century. Once abandoned due to excessive nephrotoxicity, they are now used increasingly to address infections caused by multi-drug... Show morePolymyxins are clinically used antibiotics, discovered in mid-20th century. Once abandoned due to excessive nephrotoxicity, they are now used increasingly to address infections caused by multi-drug resistant Gram-negative bacteria.In this thesis, we describe the development and synthesis of analogues of polymyxin, aimed at reducing its associated nephrotoxicity. Analogues were made by semisynthesis, with modifications introduced mostly in the exocyclic portion of the molecule. Especially the introduction of a disulfide bond within the linked lipid helped in reducing the toxicity of the molecules, as evidenced by testing on proximal tubule epithelial cells. For most potent analogues, the antimicrobial activity was completely retained.In addition, this thesis describes studies on the mechanism of action of polymyxin, mostly based on the full stereoisomer of polymyxin B4. This analogue lacks antimicrobial activity, indicating its original stereochemistry to be of utmost importance for its use as an antibiotic.Hybrids based on polymyxin B derivatives are described, addressing non-conventional targets. A hybrid with vancomycin (typically active on Gram-positive bacteria only) shows activity on Gram-negative bacteria. A polymyxin-based hybrid coupled to a peptide with a beta-hairpin motif addresses Gram-negative bacteria, presumably by binding to outer membrane protein BamA. Show less
Lipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis... Show moreLipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis or bacterial virulence, depending on the bacterial lifestyle. Plant microbiota shows great complexity, promotes plant health and growth and assures protection from pathogens. How plants perceive LPS from plant-associated bacteria and discriminate between beneficial and pathogenic microbes is an open and urgent question. Here, we report on the structure, conformation, membrane properties and immune recognition of LPS isolated from the Arabidopsis thaliana root microbiota member Herbaspirillum sp. Root189. The LPS consists of an O-methylated and variously acetylated Drhamnose containing polysaccharide with a rather hydrophobic surface. Plant immunology studies in A. thaliana demonstrate that the native acetylated O-antigen shields the LPS from immune recognition whereas the O- deacylated one does not. These findings highlight the role of Herbaspirillum LPS within plant-microbial crosstalk, and how O-antigen modifications influence membrane properties and modulate LPS host recognition. Show less
Hegedus, J.H. von; Kahnt, A.S.; Ebert, R.; Heijink, M.; Toes, R.E.M.; Giera, M.; Ioan-Facsinay, A. 2020
Inflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic... Show moreInflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic inflammation. Several cells and soluble mediators, including lipid mediators, regulate the course of inflammation and its resolution. It is, however, unclear which signals and cells are involved in initiating the resolution process. Macrophages are tissue resident cells and key players in regulating tissue inflammation through secretion of soluble mediators, including lipids. We hypothesize that persistent inflammatory stimuli can initiate resolution pathways in macrophages.In this study, we detected 21 lipids in LPS-stimulated human monocyte-derived macrophages by liquid chromatography coupled to tandem mass spectrometry. Cyclooxygenase-derived Prostaglandins were observed in the first six hours of stimulation. Interestingly, a switch towards 15-lipoxygenase products, such as the proresolving lipid precursors 15-HEPE and 17-HDHA was observed after 24 h. The RNA and protein expression of cyclooxygenase and 15-lipoxygenase were in line with this trend. Treatment with 17-HDHA increased IL-10 production of monocyte-derived macrophages and decreased LTB4 production by neutrophils, indicating the anti-inflammatory property of this lipid.These data reveal that monocyte-derived macrophages contribute to the resolution of inflammation in time by the production of pro-resolving lipids after an initial inflammatory stimulus. Show less
The incidence of bacterial infections and sepsis, as well as the mortality risk from sepsis, is sex specific. These clinical findings have been attributed to sex differences in immune... Show moreThe incidence of bacterial infections and sepsis, as well as the mortality risk from sepsis, is sex specific. These clinical findings have been attributed to sex differences in immune responsiveness. The aim of the present study was to investigate sex differences in monocyte-derived cytokine production response upon stimulation with the gram-negative stimulus lipopolysaccharide (LPS) using cytokine data from 15 study populations. Individual data on ex vivo cytokine production response upon stimulation with LPS in whole blood were available for 4,020 subjects originating from these 15 study populations, either from the general population or from patient populations with specific diseases. Men had a stronger cytokine production response than women to LPS for tumour necrosis factor-alpha, interleukin (IL)-6, IL-12, IL-1 beta, IL-1RA, and IL-10, but not for interferon-gamma. The granulocyte-macrophage colony-stimulating factor production response was lower in men than in women. These sex differences were independent of chronological age. As men had higher monocyte concentrations, we normalized the cytokine production responses for monocyte concentration. After normalization, the sex differences in cytokine production response to LPS disappeared, except for IL-10, for which the production response was lower in men than in women. A sex-based approach to interpreting immune responsiveness is crucial. Show less
The research described in this thesis focussed on the role of apolipoproteins in lipid metabolism, inflammation and bacterial sepsis, with specific emphasis on apoCI. From studies in human APOC1_... Show moreThe research described in this thesis focussed on the role of apolipoproteins in lipid metabolism, inflammation and bacterial sepsis, with specific emphasis on apoCI. From studies in human APOC1_-transgenic and apoc1-/- mice, we were able to identify apoCI as a potent inhibitor of triglyceride hydrolysis by inhibiting lipoprotein lipase. Since APOC1 mice have thus increased VLDL levels, and VLDL protects against bacterial infection, we studied whether apoCI could play a role in inflammation and infection. We found that apoCI was able to bind lipopolysaccharide (LPS), the main toxic component of Gram-negative bacteria. Interestingly, although other apolipoproteins which have been studied have anti-inflammatory properties, we found that apoCI is a pro-inflammatory protein. By enhancing the biological response towards LPS and Gram-negative bacteria, apoCI dose-dependently improved the anti-bacterial attack, and protected against intrapulmonal Klebsiella pneumoniae-induced sepsis. Consistent with these experimental findings we also found that subjects with high plasma apoCI levels were less prone to infection-related mortality during follow-up, independent of plasma lipid levels. Likewise, survivors of severe sepsis showed higher plasma apoCI levels as compared to non-survivors, again independent of lipid levels. Taken together, our findings indicate that apoCI is an important determinant of the inflammatory response in mice and humans. Show less
The host response to bacterial invasion requires first the recognition of the presence of pathogens. The presence of a pathogen is detected by binding of pathogen-preserved molecules called ... Show moreThe host response to bacterial invasion requires first the recognition of the presence of pathogens. The presence of a pathogen is detected by binding of pathogen-preserved molecules called 'pathogen-associated molecular patterns' or PAMPs on cell surface receptors known as 'pattern recognition receptors' or PRRs. Lipopolysaccharide (LPS; endotoxin), one of the major outer membrane components of the Gram-negative bacterial cell wall, is an important PAMP. In the last decade the mechanisms by which the host responds to the presence of microbial pathogens on its mucosal surfaces or in the tissues, have been elucidated, most up to their molecular level. With the sequencing of the genome and recognition of the genetic variation within the human population, it became clear this variation is responsible for the inter-individual differences in cytokine release after challenge with microbial products. Such genetically determined variations in cytokine release could be relevant to explain the variability in susceptibility too, and outcome from, sepsis. The thesis focuses on genes that encode for proteins involved in the pathogenesis of sepsis including those involved in the initial recognition of bacterial products such as Toll-like receptors, and cytokines known to mediate the host inflammatory response, such as tumor necrosis factor (TNF)-α, interleukin(IL)-6 and IL-10. Show less
