As described in the “in brief” section, our laboratory has two main objectives. The first is to investigate how cellular membranes intervene in bacterial and cancer cell processes that could constitute targets for antibacterial or anticancer treatment. The second is the discovery and optimization of bacterial treatments especially with a regard on antibiotic resistances. To attain these objectives, we use a multidisciplinary approach and a variety of techniques that range from membrane biophysical assays to complex pharmacokinetic/pharmacodynamic models. If you are interested in the different projects or if you want eventually to apply for an undergraduate/PhD or postdoc position related to one of the projects, please click on the corresponding subjects below to get more information. You can contact us by email under francoise.vanbambeke@uclouvain.be and joseph.lorent@uclouvain.be.
Discovery and description of membrane related processes to develop new antibiotic and anticancer agents
Targeting cell membranes
The cell membrane displays a fascinating structural and compositional complexity including hundreds to thousands of lipids and proteins that are heterogeneously distributed upon the cell surface. Lipids and proteins can arrange into lateral functional domains that can constitute either signalling platforms or transport hubs. Further, lipids are asymmetrically distributed upon both membrane monolayers which confer special biophysical and mechanical properties to the cell membrane. This structural complexity translates into the numerous functions that are fulfilled by cellular membranes.
Membranes are interesting pharmacological targets because they have tissue and species- specific compositions and properties. They act as diffusion barriers for drugs such as antibiotics or anticancer agents and influence availability to drug targets in bacterial or cancer resistance. Further, the lipid portion of membranes constitutes interesting pharmacological targets for antibiotic or anticancer therapy. Membranes are also the siege of membrane receptors and transporters that constitute the most important targets encountered in pharmacology. They are further involved in processes such as cell migration during cancer invasion, cell division, or synthesis of the cell wall in bacterial cells. Our main goal is to investigate how membranes intervene in cellular processes that are vital or confer resistance to pathogenic bacteria and cancer cells with the long term aim to develop treatments that target these processes.
Different aspects of cell membranes that are studied in our projects
Approaches
In all the following projects investigating membrane properties and drug-membrane interactions, we will globally use a combination of techniques related to cell culture (mammalian and microbial cell culture), microbiology such as MIC determination, advanced microscopy techniques such as two-photon, fluorescence lifetime imaging (FLIM) and spectral microscopy, advanced image analysis (image J, Matlab), lipidomics approaches (LC-DAD/MS, TLC), membrane models to pinpoint specific interactions of drugs with lipids and to anticipate certain biophysical membrane properties such as domain formation and membrane asymmetry (LUV, SUV, SPB, GUV), bioinformatics approaches to analyze and predict protein properties and lipid membrane properties, molecular biology to construct artificial protein constructs (fluorescence/protein isolation/membrane properties) and to create knock-out cells, biochemistry (protein isolation, western blot, biochemical assays) and fluorescence spectroscopy (anisotropy, spectral shifts, FRET,…).
Glioblastoma multiforme (GBM), which develops from glioma, is the most aggressive and most frequent brain tumour in adults and is associated with a very poor survival of 12-18 months. Because of the devastating outcome of the disease, new therapeutic approaches are urgently needed. In tumours, mechanical properties of cell membranes are generally altered, possibly influencing mechanosensation and leading to exaggerated proliferation and increased invasiveness of tumour cells. In addition, GBM growth induces increased intracranial pressure to which tumours are later submitted. Unfortunately, it is barely known how mechanosensation in GBM is altered to ensure continuous growth and invasion (Figure). The present project will therefore address if and how transmembrane mechanosensitive (MS) channels are deregulated in GBM and how membrane lipid composition and biophysical properties are involved in the process.
Mechanoreceptor Piezo1 opening and signalling is guided by membrane tension upon glioblastoma invasion/migration of the brain
One interesting aspect of the outer membrane in Gram negative bacteria (Figure A) is the asymmetric composition of lipids in the outer versus inner monolayer. While the outer monolayer is almost completely composed of lipopolysaccharides (LPS), contains the inner monolayer mainly phospholipids. This lipid asymmetry is established by a pathway of several proteins which first transport LPS to the outer membrane and then flop LPS to the outer monolayer of the outer membrane. The asymmetric lipid composition provides certain biophysical properties to the outer membrane such as a high lipid packing of the outer monolayer which seems to be associated with a low permeability towards antibiotics. In Gram negative bacteria such as Pseudomonas aeruginosa, which invade hospitals and cause multi-resistant infections, the outer membrane forms an almost impenetrable layer for antibiotics (Figure A). One goal of our laboratory is to counteract this low permeability by using agents that specifically alter outer membrane properties. This can be achieved either by inhibiting the associated pathways or by targeting the outer monolayer directly. We therefore focus on the discovery and characterization of pathways that lead to membrane asymmetry and on the biophysical properties that are associated with this asymmetry. We specifically search for agents that can selectively modulate these properties and thus might reverse antibiotic resistance.
Outer membrane asymmetry in Gram negative bacteria and associated biophysical properties that constitute possible antibiotic targets or resistance.
Staphylococci constitute a large branch of infective bacteria that developed various mechanisms to escape antimicrobial therapy (see also project three). These bacteria cause enormous burdens on healthcare systems all over the planet right now since some of them have become untreatable by current antibiotics. One way of escaping antimicrobial therapy is by modulating membrane asymmetric composition and properties. The outer monolayer of cytoplasmic membranes in Staphylococci have usually a negative surface charge because of the high amount of negatively charged lipids such as phosphatidylglycerol (Figure). This makes these bacteria susceptible towards cationic antimicrobial peptides (CAMPs) such as daptomycin. Interestingly, certain Staphylococci can produce Lysyl-phospholipids that bear positive charges and thereby increase the membrane surface charge. To transport these positively charged lipids to the outer monolayer of the membrane (flopping), bacteria use a floppase (MprF1), which reduces specifically the outer monolayer surface charge and renders bacteria impervious towards multiple CAMPs. In a current project we want to characterize the extend by which this floppase can change the surface charge of both monolayers and investigate how this mechanism alters antibiotic susceptibility, with the aim to develop adjuvant treamtents that reinitialize susceptibility to cationic antibiotics.
Membrane biophysical properties of S. aureus that constitute possible targets for antibacterial activity or antibiotic resistance.
Methicillin resistant Staphylococcus aureus (MRSA) are bacteria that cause immense havoc because they acquire easily other resistances towards last resort antibiotics. In MRSA, formation of lateral lipid microdomains in the membrane induces the recruitment and later aggregation of penicillin binding protein 2a (PBP2a), which renders them resistant towards meticillin (Figure). Formation of membrane microdomains seems to depend on specific carotenoids and proteins (flotillins) in S. aureus. But so far it is not completely clear how these microdomains are formed, how they recruit certain proteins and how they render bacteria resistant. We therefore launched a project that aims at characterizing these domains in clinically relevant MRSA strains and the mechanism(s) by which they are able to recruit proteins like PBP2a involved in β-lactam resistance with the aim to disrupt the formation of these and reverse antibiotic resistance. In a long run, we would like to determine also microdomain properties and function in other bacteria, since there presence seem to be somewhat universal.
Membrane biophysical properties of S. aureus that constitute possible targets for antibacterial activity or antibiotic resistance.
During the innate immune response, macrophages phagocyte invading microorganisms and induce a process called oxidative burst in phagolysosomes. During this process, reactive oxygen and nitrogen species (RONS) are enzymatically produced in phagolysosomes. These RONS have the potential to kill certain bacteria efficiently. Unfortunately, the mechanisms by which bacteria are specifically killed by RONS in phagolysosomes without inducing major injuries to the host cells are not known. We recently discovered that lipid oxidation from short living oxygen species does not specifically depend on lipid unsaturation (Figure). This is of particular interest since bacteria do not possess large amount of polyunsaturated lipids conversely to human cell membranes. We also realized that only short-living RONS are efficiently able to kill bacteria probably through a cell envelope dependent mechanism. We therefore want to investigate how membranes of bacteria and lysosomes are affected during oxidative burst in terms of membrane biophysical properties, molecular damage and physiological consequences for the macrophages and bacteria. This should give us clues on how our immune system is able to selectively kill bacteria and how we light use this as an advantage to develop therapies based on the generation of RONS.
Molecular membrane damage (left) and structural damage (right) in a human cell membrane induced by reactive oxygen and nitrogen species (RONS)
Microbial persistence mechanisms
Bacterial persistent or recurrent infections are associated with two specific lifestyles, namely intracellular survival and biofilms. We are studying antibiotic activity against these two forms of infections, with the aim to understand why bacteria do not respond to antibiotics in the absence of resistance mechanism and to develop innovative adjuvant therapeutic strategies that can restore antibiotic activity against these specific forms of infection.
Antibiotics are unable to fully eradicate intracellular Staphylococcus aureus, even when used at high concentrations. Using single cell analyses, we showed that the subpopulation of surviving bacteria showcases the hallmarks of so-called persisters, i.e. non-dividing bacteria with reduced metabolic activity, which revert to a normal phenotype once the antibiotic pressure has been relieved. We also showed that the level of dormancy (evaluated by the lag-time needed to recover their capacity to multiply) is depending on the level of oxidative stress imposed by the host cells. We are now expanding these experiments to other intracellular bacteria (notably Escherichia coli and Pseudomonas aeruginosa, studying their response to antibiotics from different classes, their intracellular fate and their response to antibiotic and cellular stresses. Lastly, we evaluate the activity of original molecules (amphiphilic molecules disturbing the membranes, bacteriocins) that synergize with antibiotics against these persisters and try to establish their mechanism of action.
Left: A biphasic kill curve is observed for intracellular S. aureus exposed to different antibiotics at 50x MIC, indicating that a subpopulation does not respond to antibiotics
Middle: flow cytometry profiles of intracellular S. aureus expressing an inducible green-fluorescence protein (GFP) in J774 (permissive) macrophages after 24 h incubation with oxacillin at 50x MIC (blue) or 24 h after removal of the drug (red). The fluorescence signal is elevated in the presence of antibiotic, indicating that GFP was not diluted (no bacterial division), but low after drug removal, indicating that dilution had occurred along with bacterial divisions.
Right: flow cytometry profiles of intracellular S. aureus expressing an inducible GFP in human (non permissive) macrophages after 24 h incubation with oxacillin at 50x MIC (red) or 24 h after collection and reinoculation in J774 permissive cells (blue). The fluorescence signal is elevated in human non permissive cells, indicating the absence of bacterial division), but low after transfer in permissive cells, indicating that dilution had occurred along with bacterial divisions.
Adapted from Peyrusson et al, doi: 10.1007/978-1-0716-1621-5_16; 10.1038/s41467-020-15966-7; 10.1128/spectrum.02313-21
Experimental approaches include models of intracellular infections in vitro, construction of reporter strains expressing fluorescent proteins, fluorescence microscopy, transcriptomic, proteomic, and genomic analyses.
Biofilms are communities of bacteria embedded in a self-produced matrix of polysaccharides, proteins and extracellular DNA. This matrix protects them physically from immune defenses and antibiotics but also contribute to increase their tolerance to antibiotics by impairing the access of nutriments and oxygen, pushing them to reduce their metabolic activity. We are mainly interested in biofilms responsible for chronic lung infections in patients with cystic fibrosis (mixed biofilms of Staphylococcus aureus and Pseudomonas aeruginosa) and for prosthetic joint infections (Staphylococci). We have set up in vitro models in media mimicking the in-vivo environment (artificial sputum medium to mimic the mucus of patients with cystic fibrosis, biofilms growing on titanium support to mimic prosthesis material) as well as in vivo models of implanted material (titanium beads, 3D-printed knee prosthesis in collaboration with the MNSK pole at IREC) to evaluate the efficacy of innovative strategies capable to disrupt the biofilms to help antibiotic penetration. We demonstrated previously that the antifungal caspofungin inhibits the synthesis of exopolysaccharides by S. aureus, improving thereby the activity of fluoroquinolones against S. aureus biofilms. We are now studying combinations of antibiotics with enzymes acting on different types of matrix constituents or with bacteriophages (in collaboration with the Queen Astrid Military hospital).
Comparison of the residual biomass of three-species biofilms constructed with reference (ATCC) or clinical isolates of S.aureus:E.coli:C.albicans (5706:6081:2522 and 8066:5701:7729) grown on Titanium coupons after sequential incubation with hydrolytic enzymes during 1 h (subtilisin A 0.5 U/mL [orange] or a cocktail of cellulase 7 U/mL/denarase 250 U/mL/dispersin B 1.25 U/mL/lyticase 12.6 U/mL [Ce/De/Di/Ly, purple]), and antimicrobials during 24 h (moxifloxacin 4 mg/L/caspofungin 13.8 mg/L [MXF/CAS; dark colors] or meropenem 40 mg/L/caspofungin 13.8 mg/L [MEM/CAS; light colors]).
Combining enzymes and antibiotics is more effective to reduce biofilm biomass for clincial isolates in this complex three-species model.
Reproduced from Ruiz-Sorribas et al, 2022; doi: 10.1128/spectrum.02589-21
Experimental approaches include models of biofilms in vitro and in vivo (with MNSK/IREC), confocal microscopy, biochemical and metabolomic analyses of biofilms constituents.
Peyrusson F, Varet H, Nguyen TK, Legendre R, Sismeiro O, Coppée JY, Wolz C, Tenson T, Van Bambeke F. Intracellular Staphylococcus aureus persisters upon antibiotic exposure. Nature Communications (2020) 11:2200
Ruiz-Sorribas A, Poilvache H, Kamarudin NHN, Braem A, Van Bambeke F. Hydrolytic enzymes as potentiators of antimicrobials against an inter-kingdom biofilm model. Microbiology Spectrum 2022; 10(1):e0258921
Wang G, Brunel JM, Preusse M, Mozaheb N, Willger SD, Larrouy-Maumus G, Baatsen P, Häussler S, Bolla JM, Van Bambeke F. The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes Pseudomonas aeruginosa to antibiotics and reduces bacterial virulence. Communications Biology 2022; 5(1):871.
Pharmacokinetics and pharmacodynamics of antibiotics
To act against intracellular bacteria, antibiotics need to accumulate inside the infected subcellular compartment and express their activity in the local environmental conditions. We study the cellular pharmacokinetics of antibiotics representative of the main classes used in the clinics or in preclinical development in order to characterize their accumulation, subcellular distribution, and efflux in eukaryotic cells. We also examine how the local conditions (pH, oxidative stress) could affect their expression of activity (pharmacodynamics).
We study the mechanism of entry of antibiotics inside eukaryotic cells (diffusion, endocytosis, active transport), their level of accumulation, their subcellular distribution (cytosol, lysosomes, other organelles) and their efflux, including their active transport. Over the last years, we have focused our interest on new antibiotic classes, like lipoglycopeptides, ketolides, new fluoroquinolones and new oxazolidinones. Currently, in collaboration with PMGK, we have started to examine whether genetic polymorphisms in efflux transporters (ABCB1, ABCC2 or 4, ABCG2) can affect the efflux of fluoroquinolone antibiotics.
Experimental approaches include cellular and molecular biology, cell fractionation, biochemistry, analytical chemistry.
In parallel, we study the activity of antibiotics against intracellular bacteria sojourning in different subcellular compartments, mainly Listeria monocytogenes (cytosol), Staphylococcus aureus (phagolysosomes), Pseudomonas aeruginosa, or Escherichia coli using in vitro models of infected phagocytic and non-phagocytic cells. We have also extended these models to many other bacterial species of medical interest. We developed an in vitro pharmacodynamic approach to compare the efficacy and the potency of the drugs. In brief, we showed that antibiotics are in general less effective but equipotent against intracellular than against extracellular bacteria, irrespective of their accumulation level. The data generated with these models have been incorporated in the dossier having led to the registration of the last antibiotics brought on the market. While still continuing to characterize on a pharmacological basis this intracellular profile of activity, we also try to decipher the reasons of antibiotic failure to eradicate these infections, as described in the page dedicated to persistent forms of infections. We also aim at defining synergistic drug combinations against intracellular bacteria and at elucidating the molecular mechanisms of these synergies.
Experimental approaches include cellular biology and pharmacology.
Peyrusson F, Van Wessem A, Dieppois G, Van Bambeke F, Tulkens PM. Cellular pharmacokinetics and intracellular activity of the bacterial fatty acid synthesis inhibitor afabicin desphosphono against different resistance phenotypes of Staphylococcus aureus in models of cultured phagocytic cells. International Journal of Antimicrobial Agents (2020) 55:105848
Chalhoub H, Harding S, Tulkens PM, Van Bambeke F. Influence of pH on the activity of finafloxacin against extracellular and intracellular Burkholderia thailandensis, Yersinia pseudotuberculosis, and Francisella philomiragia and on its cellular pharmacokinetics in THP-1 monocytes. Clinical Microbiology and Infection (2020) 26: 1254.e1-1254.e8
Cellular toxicity of antibiotics
While cellular accumulation of antibiotics is useful when dealing with intracellular infections, it can also cause cellular toxicity. Over the years, our team has deciphered in details the mechanisms of lysosomal toxicity of drugs accumulating in these compartments (successively aminoglycosides, macrolides, and lipoglycopeptides), which has as a common feature to trigger the accumulation of lipids. More recently, we turned our attention to oxazolidinones, which exert their antibacterial effect by inhibiting protein synthesis in bacteria. We evidenced a specific inhibition of the synthesis of protein encoded by the mitochondrial genome accompanied by an inhibition of the respiratory function and morphological alterations (swelling of mitochondria and disappearance of cristae). We are now exploring whether these changes may contribute to explain the thrombocytopenia and anemia reported in patients treated by these drugs.
Experimental approaches include cellular and molecular biology, electron microscopy, biochemistry.
Dose adjustment upon optimization of efficacy and safety
Today, we are far from the concept of ‘one size fits all’ when dealing with dosing of drugs. Personalized dosing is necessary in at-risk patient populations (intensive care, renal or hepatic insufficiency, geriatrics or pediatrics) in order to in crease the probability of achieving therapeutic concentrations while at the same time avoid excessive risk of toxicity. This holds also true for antibiotics.
Experimental approaches include analytic chemistry, pharmacokinetic modeling, statistics, collection of clinical data.
Beta-lactams are time-dependent antibiotics, for which efficacy is critically dependent on the time of exposure: the unbound antibiotic concentration needs to remain above the MIC of the infecting bacteria during the whole (or a substantial proportion; > 40 or 70% depending on the models) of the dosing interval. We are involved in a series of clinical trials with beta-lactams (temocillin, meropenem) in specific patient populations (renal insufficiency, intensive care, pediatrics) in which we measure unbound drug concentrations in the plasma or other biological fluids, establish population pharmacokinetic models and run Monte Carlo simulations in order to define adequate dosing insuring high probability of target attainments. These models are built in collaboration with PMGK or external experts. Our work has already led to the revision of the Summary of Product Characteristics of specific antibiotic with adjusted dose for specific patients.
As described in the page related to antibiotic toxicity, we study the molecular mechanisms of the thrombocytopenia induced by linezolid. In parallel, we ran a retrospective study in Belgian hospitals to better documents the clinical use of linezolid in our country (mainly off-label) and the associated prevalence of adverse events, which were much frequent than described in the Summary of Product Characteristics. On this basis, we performed a prospective study aiming at finetuning out data thanks to a more systematic collection of adverse events, and at relating the development of adverse reactions with the serum levels of the drugs. Our aim is to establish guidelines applicable in Belgium to help clinicians detecting the patients at risk of developing adverse effects.
Experimental approaches include analytic chemistry, pharmacokinetic modeling, statistics, collection of clinical data.
Ngougni Pokem P, Matzneller P, Vervaeke S, Wittebole X, Goeman L, Coessens M, Cottone E, Capron A, Wulkersdorfer B, Wallemacq P, Mouton JW, Muller AE, Zeitlinger M, Laterre PF, Tulkens PM, Van Bambeke F. Binding of temocillin to plasma proteins in vitro and in vivo: the importance of plasma protein levels in different populations and of co-medications. J Antimicrob Chemother 2022; 77(10):2742-2753
Thirot H, Briquet C, Frippiat F, Jacobs F, Holemans X, Henrard S, Tulkens PM, Spinewine A, Van Bambeke F. Clinical Use and Adverse Drug Reactions of Linezolid: A Retrospective Study in Four Belgian Hospital Centers. Antibiotics 2021; 10(5):530.
Epidemiological surveys
While resistance is increasing worldwide, it remains critical to collect local data regarding susceptibility pattern of pathogenic bacteria, in order to define the most appropriate therapeutic options for our country. It is also essential to understand the molecular mechanisms of resistance as well as the link between resistance development and antibiotic usage.
If you are interested in the different projects or if you wish to apply for an undergraduate/PhD or postdoc position related to one of the projects, please click on the corresponding subjects below to get more information.
Antibiotic failure can be related to resistance, but also to persistent forms of infection (see page dedicated to this concept). We showed recently that clinical isolates of Staphylococcus aureus collected from persistent or recurrent infections differ by their fraction of persistence when exposed to a bactericidal antibiotic like moxifloxacin, and that isolates with a high persister character gained more rapidly resistance to this drug when exposed to sub inhibitory concentrations. In the same line, we aim now at collecting Escherichia coli from recurrent urinary tract infection, in order to follow in successive isolates from the same patients the evolution of their persister character, their capacity to survive intracellularly or in biofilm, or to gain resistance.
Experimental approaches include molecular biology, models of intracellular infections and biofilms.
Nguyen TK, Peyrusson F, Dodémont M, Pham NH, Nguyen HA, Tulkens PM, Van Bambeke F.
The persister character of clinical isolates of Staphylococcus aureus contributes to faster evolution to resistance and higher survival in THP-1 monocytes: a study with moxifloxacin.
Frontiers in Microbiology (2020) 11:587364