In detail

Bruxelles Woluwe

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,…).

 

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.

 

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).

 

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.

Electron microscopy images of cells exposed to 20 mg/L oritavancin (lipoglycopeptide) and showing signs of phospholiposis (left) or of cells incubated with 15 mg/L linezolid (oxazolidinone) and showing signs of mitochondrial toxicity (right). Reproduced from van Bambeke et al, 2005 and Milosevic et al, 2018 doi: 10.1128/AAC.49.5.1695-1700.2005 ; 10.1128/AAC.01599-17

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.

 

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.