Comprehension of the mechanisms of action of antimicrobial molecules using nanobiotechnologies

Mon travail de thèse consiste à utiliser les techniques de Microscopie à Force Atomique (AFM) pour étudier les microorganisms pathogènes, et leurs interactions avec des antimicrobiens. Ces dernières décennies, la résistance microbienne a augmenté de façon dramatique. Les bactéries et levures pathogènes sont à l'origine d'infections mettant en jeu la vie de patients. Il y a donc deux urgences : la première est de trouver de nouveaux antimicrobiens; pour cela il faut acquérir des données fondamentales sur la paroi des microorganismes, afin d'identifier des cibles. La deuxième urgence est donc de développer des nouvelles techniques pour explorer la paroi des. Durant cette thèse nous avons donc utilisé l'AFM, adapté aux conditions biologiques. Un avantage de l'AFM est la possibilité de travailler en liquide, ce qui nous a permis d'imager l'élongation de cellules de P. aeruginosa traitées avec un antibiotique, ainsi que la disparition de la capsule de K. pneumoniae traitée avec de la colistine. L'AFM est aussi une machine de force qui enregistre des courbes de force permettant d'accéder aux propriétés nanomécaniques et d'adhésion des cellules. Nous avons ainsi observé les modifications des propriétés adhésives de la levure C. albicans traité avec de la caspofongine. Enfin il est possible de fonctionnaliser des pointes AFM avec des biomolécules ; cette stratégie nous a permis de localiser des protéines spécifiques à la surface de levures et cellules animales, et d'étudier la paroi de P. aeruginosa traité par un antibactérien innovant, le Cx1. Pour conclure, cette thèse a permis d'adresser spécifiquement la contribution de la biophysique en microbiologie clinique.My PhD work consists in using Atomic Force Microscopy (AFM) techniques to study pathogenic microorganisms, and to probe their interactions with antimicrobials. During the last three decades, microbial resistance has dramatically increased and spread around the world. Pathogenic bacteria and yeasts are the cause of life-threatening infections in some patients. There are therefore two emergencies; the first one is to find new antimicrobial molecules; for that, it is mandatory to get further knowledge on the microbial cell wall. Therefore the second emergency is to develop new techniques to explore microbial surfaces. During my PhD, we took advantage of a technology coming from physics, and adapted to biological conditions, AFM. An advantage of AFM is the possibility to work in liquid on living cells, which allowed us to image the elongation of cells of P. aeruginosa treated by ticarcillin, and the removal of capsular polysaccharides from K. pneumoniae upon treatment with colistin. AFM is also a force machine, able to record force distance curves that give access to nanomechanical and adhesive properties of cells. We could observe the modifications of the adhesive properties of the yeast C. albicans, treated by caspofungin. Finally it is possible to functionalize AFM tips with biomolecules; we used this strategy to localize specific proteins at the surface of living yeasts and mammalian cells, and to study the cell wall of P. aeruginosa treated by an innovative antibacterial, Cx1. In conclusion, during my PhD, we especially addressed the contribution of biophysics in clinical microbiology

Development of Polythiourethane/ZnO-Based Anti-Fouling Materials and Evaluation of the Adhesion of Staphylococcus aureus and Candida glabrata Using Single-Cell Force Spectroscopy

The attachment of bacteria and other microbes to natural and artificial surfaces leads to the development of biofilms, which can further cause nosocomial infections. Thus, an important field of research is the development of new materials capable of preventing the initial adhesion of pathogenic microorganisms. In this work, novel polymer/particle composite materials, based on a polythiourethane (PTU) matrix and either spherical (s-ZnO) or tetrapodal (t-ZnO) shaped ZnO fillers, were developed and characterized with respect to their mechanical, chemical and surface properties. To then evaluate their potential as anti-fouling surfaces, the adhesion of two different pathogenic microorganism species, Staphylococcus aureus and Candida glabrata, was studied using atomic force microscopy (AFM). Our results show that the adhesion of both S. aureus and C. glabrata to PTU and PTU/ZnO is decreased compared to a model surface polydimethylsiloxane (PDMS). It was furthermore found that the amount of both s-ZnO and t-ZnO filler had a direct influence on the adhesion of S. aureus, as increasing amounts of ZnO particles resulted in reduced adhesion of the cells. For both microorganisms, material composites with 5 wt.% of t-ZnO particles showed the greatest potential for anti-fouling with significantly decreased adhesion of cells. Altogether, both pathogens exhibit a reduced capacity to adhere to the newly developed nanomaterials used in this study, thus showing their potential for bio-medical applications

Clumping factor B promotes adherence of <i>Staphylococcus aureus </i>to corneocytes in atopic dermatitis

Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for interventio

Nanoplastic-Induced Nanostructural, Nanomechanical, and Antioxidant Response of Marine Diatom Cylindrotheca closterium

The aim of this study was to examine the effect of positively charged (amine-modified) and negatively charged (carboxyl-modified) polystyrene nanoplastics (PS NPs) on the nanostructural, nanomechanical, and antioxidant responses of the marine diatom Cylindrotheca closterium. The results showed that both types of PS NPs, regardless of surface charge, significantly inhibited the growth of C. closterium during short-term exposure (3 and 4 days). However, longer exposure (14 days) to both PS NPs types did not significantly inhibit growth, which might be related to the detoxifying effect of the microalgal extracellular polymers (EPS) and the higher cell abundance per PS NPs concentration. The exposure of C. closterium to both types of PS NPs at concentrations above the corresponding concentrations that resulted in a 50% reduction of growth (EC50 ) demonstrated phytotoxic effects, mainly due to the excessive production of reactive oxygen species, resulting in increased oxidative damage to lipids and changes to antioxidant enzyme activities. Diatoms exposed to nanoplastics also showed a significant decrease in cell wall rigidity, which could make the cells more vulnerable. Atomic force microscopy images showed that positively charged PS NPs were mainly adsorbed on the cell surface, while both types of PS NPs were incorporated into the EPS that serves to protect the cells. Since microalgal EPS are an important food source for phytoplankton grazers and higher trophic levels, the incorporation of NPs into the EPS and interactions with the cell walls themselves may pose a major threat to marine microalgae and higher trophic levels and, consequently, to the health and stability of the marine ecosystem

Compréhension des mécanismes d'action des molécules antimicrobiennes utilisant nanobiotechnologies

My PhD work consists in using Atomic Force Microscopy (AFM) techniques to study pathogenic microorganisms, and to probe their interactions with antimicrobials. During the last three decades, microbial resistance has dramatically increased and spread around the world. Pathogenic bacteria and yeasts have developed several ways to resist against almost all antimicrobials used. These pathogens can cause a wide range of superficial infections, but are also the cause of life-threatening infections in some patients. There are therefore two emergencies; the first one is to find new antimicrobial molecules, with an innovative mechanism of action. To reach this goal, it is mandatory to get further fundamental knowledge on the microbial cell wall, in order to identify original targets at the cell surface for new molecules. Therefore the second emergency is to develop techniques to explore microbial surfaces from a different angle, which requires an original experimental approach. In this context, biophysical approaches still remain underexploited in clinical microbiology. In my PhD, we took advantage of a technology coming from physics, and adapted to biological conditions, AFM. Its principle relies on the measure of a force between a sharp tip and a surface; by keeping this force constant while scanning the sample, it is possible to get a three dimensional image of it. An advantage of AFM is the possibility to work in liquid on living cells, which allowed us to image the elongation of live bacterial cells of P. aeruginosa treated by ticarcillin, a cell wall targeting antibiotic, and the removal of capsular polysaccharides from K. pneumoniae upon treatment with colistin, a last chance antibiotic. Nevertheless, sample immobilization is often a challenge that must be addressed for each kind of microorganisms studied. It represents an entire field of research, and led us to engineer a microstructured polydimethylsiloxane (PDMS) stamp to immobilize round cells of different sizes such as yeasts. Once this step was accomplished, AFM can be used in classic imaging and force spectroscopy modes (contact mode, oscillating mode, force volume mode), but also in advanced modes in order to acquire multiparametric sets of data on living cells. Indeed, AFM is also a highly sensitive force machine, able to record force distance curves that give access to nanomechanical and adhesive properties of living cells. We therefore used the multiparametric imaging mode, QITM from JPK Instruments, to image and quantify the nanomechanical/adhesive properties of microorganisms as well as mammalian cells and their isolated nucleus. We could then observe the modifications of the adhesive properties of the yeast C. albicans, treated or not by caspofungin, a last chance antifungal. Finally, to get further in the architecture of the cell wall of microorganisms, it is possible to functionalize AFM tips with biomolecules. A strategy that we have developed has consisted of grafting antibodies targeted against a peptide on the AFM tip while tagging proteins with the same peptide at the surface of living cells. This allowed mapping the localization of specific proteins at the surface of living yeasts and mammalian cells. Another strategy we developed has consisted in directly grafting on the AFM tip a biomolecule that naturally interacts with cell wall components. We used this last strategy to probe the bacterial cell wall of P. aeruginosa submitted to treatment by an innovative antibacterial, Cx1, and better understand the structure of its peptidoglycan. In conclusion, during my PhD, we especially addressed the contribution of biophysics in clinical microbiology. We developed original techniques to immobilize samples, to functionalize AFM tips, and used advanced multiparametric AFM modes to acquire original data on the surface of pathogenic microorganisms, in native conditions or in interaction with antimicrobials.Mon travail de thèse consiste à utiliser les techniques de Microscopie à Force Atomique (AFM) pour étudier les microorganisms pathogènes, et leurs interactions avec des antimicrobiens. Ces dernières décennies, la résistance microbienne a augmenté de façon dramatique et s’est répandue dans le monde. Les bactéries et levures pathogènes ont développé différents moyens pour résister à presque tous les antimicrobiens utilisés. Ces pathogènes peuvent être la cause d’une large gamme d’infections superficielles ; ils sont aussi à l’origine d’infections mettant en jeu la vie de patients. Il y a donc deux urgences : la première est de trouver de nouvelles molécules antimicrobiennes, avec un mécanisme d’action innovant. Mais pour atteindre cet objectif, il est nécessaire d’acquérir des données fondamentales sur la paroi des microorganismes, afin d’identifier des cibles originales à leur surface pour de nouvelles molécules. La deuxième urgence est donc de développer des techniques pour explorer la paroi des microorganismes d’un point de vue différent, ce qui nécessite une approche expérimentale originale. Dans ce contexte, les approches biophysiques restent sous-exploitées en microbiologie clinique. Durant cette thèse, nous avons utilisé une technologie provenant de la physique, l’AFM, adapté aux conditions biologiques. Le principe de l’AFM est basé sur la mesure d’une force entre une pointe et un échantillon ; en gardant cette force constante pendant le scan de l’échantillon il est possible d’en obtenir une image tridimensionnelle. Un avantage de l’AFM est la possibilité de travailler en liquide, ce qui nous a permis d’imager l’élongation de cellules bactériennes P. aeruginosa traité avec de la ticarcilline, un antibiotique à cible pariétale, ainsi que la disparition des polysaccharides capsulaires de la bactérie K. pneumoniae traitée avec de la colistine. Cependant, l’immobilisation des échantillons est souvent un challenge, différent pour chaque type de microorganismes. L’immobilisation d’échantillons biologiques représente un domaine de recherche à part entière, qui nous a conduit à développer un timbre de polydiméthylsiloxane (PDMS) microstructuré, pour immobiliser des cellules rondes de différentes tailles, comme des levures. Une fois cette étape d’immobilisation franchie, l’AFM peut être utilisé dans les modes classiques d’imagerie et de spectroscopie de force (mode contact, mode oscillant, mode force volume), mais aussi dans des modes avancés afin d’obtenir des données à haute résolution ou multiparamétriques. En effet, l’AFM est aussi une machine de force très sensible capable d’enregistrer des courbes de force qui permettent d’accéder aux propriétés nanomécaniques et d’adhésion des cellules. Ainsi nous avons pu imager et quantifier les propriétés nanomécaniques et adhésives de microorganisms, mais aussi de cellules de mammifères vivantes et de leurs noyaux isolés. Nous avons ainsi observé les modifications des propriétés adhésives de la levure C. albicans traité avec de la caspofongine. Enfin pour aller plus loin dans l’étude de l’architecture des parois des microorganismes, il est possible de fonctionnaliser des pointes AFM avec des biomolécules. Une stratégie développée a consisté à lier des anticorps dirigés contre un peptide sur la pointe, et de tagguer des protéines avec ce même peptide à la surface des cellules, Ainsi nous avons localisé des protéines spécifiques à la surface de levures et cellules de mammifères vivantes. Une autre stratégie développée consiste à directement lier une biomolécule sur la pointe, qui interagit naturellement avec un composé pariétal. Nous avons utilisé cette stratégie pour étudier la paroi de la bactérie P. aeruginosa traité par un antibactérien innovant, le Cx1, et ainsi mieux comprendre l’architecture de son peptidoglycane. Pour conclure, cette thèse a permis d’adresser spécifiquement la contribution de la biophysique en microbiologie clinique

Contribution of atomic force microscopy to the study of biofilm formation and antibiotic resistance

International audienc

Flocculation-flotation harvesting mechanism of Dunaliella salina: From nanoscale interpretation to industrial optimization

International audienceDunaliella salina is a green microalgae species industrially exploited for its capacity to produce important amounts of carotenoid pigments. However in low nitrogen conditions in which they produce these pigments, their concentration is low, which results in harvesting difficulties and high costs. In this work, we propose a new solution to efficiently harvest D. salina at the pre-industrial scale, using flocculation/ flotation harvesting induced by NaOH addition in the medium. We first show, using numerical simulations and nanoscale atomic force spectroscopy experiments, that sweeping mechanism in formed magnesium hydroxide precipitate is only responsible for D. salina flocculation in hypersaline culture medium upon NaOH addition. Based on this understanding of the flocculation mechanism, we then evaluate the influence of several parameters related to NaOH mixing and magnesium hydroxide precipitation and show that NaOH concentration, mixing, and salinity of the medium can be optimized to achieve high flocculation/flotation harvesting efficiencies in laboratory-scale experiments. We finally successfully scale-up the data obtained at lab-scale to a continuous pre-industrial flotation pilot, and achieve up to 80% of cell recovery. This interdisciplinary study thus provides original results, from the nano to the pre-industrial scale, which allow the successful development of an efficient large-scale D. salina harvesting process. We thus anticipate our results to be the starting point for further optimization and industrial use of this flocculation/flotation harvesting technique

The contribution of Atomic Force Microscopy (AFM) in microalgae studies: A review

International audienceAtomic force microscopy (AFM) has now become a major technology to study single cells in living conditions. It provides nanoscale resolution imaging capacities and is a sensitive force machine able to record piconewton-scale forces, thereby making it possible to gain insights into the nanomechanical properties and molecular interactions of cells. While an extensive number of studies on microorganisms have demonstrated the potential of AFM to understand complex phenomena at cell's interfaces, its use in microalgae studies remains limited. These recent years, microalgae have been the subject of a significant number of fundamental studies notably because of their capacity to convert light, water and inorganic nutrients into a biomass resource rich in value-added products. The existing literature reporting AFM use to understand microalgae cell morphology, their nanomechanical properties or their interactions with their environment give a large overview of the contribution AFM can bring into microalgae studies. In this review, we will first present the principles of AFM and the different possibilities it offers to characterize cells. Then in a second part, the contribution of AFM to understand the effects of environmental conditions and external stress on microalgae cells will be discussed. Finally, we will show how AFM can be used to probe the interactions of microalgae with their environment and how such fundamental studies can represent a basis to improve microalgae production systems. Overall, this review, the first on this topic, aims to highlight the opportunities that AFM technology can bring to this field of research

Cell biology of microbes and pharmacology of antimicrobial drugs explored by Atomic Force Microscopy

International audienceAntimicrobial molecules have been used for more than 50 years now and are the basis of modern medicine. No surgery can nowdays be imagined to be performed without antibiotics; dreadful diseases like tuberculosis, leprosis, siphilys, and more broadly all microbial induced diseases, can be cured only through the use of an-timicrobial treatments. However, the situation is becoming more and more complex because of the ability of microbes to adapt, develop, acquire, and share mechanisms of resistance to antimicrobial agents. We choose to introduce this review by drawing the panorama of antimicrobial discovery and development, but also of the emergence of microbial resistance. Then we describe how Atomic Force Microscopy (AFM) can be used to provide a better understanding of the mechanisms of action of these drugs at the nanoscale level on microbial interfaces. In this section, we will address these questions: (1) how does drug treatment affect the morphology of single microbes?; (2) do antimicrobial molecules modify the nanomechanical properties of microbes, or do the nanomechanical properties of microbes play a role in antimicrobial activity and efficiency?; and (3) how are the adhesive abilitites of microbes affected by antimicrobial drugs treatment? Finally, in a second part of this review we focus on recent studies aimed at changing the paradigm of the single molecule/cell technology that AFM typically represents. Recent work dealing with the creation of a microbe array which can be explored by AFM will be presented, as these developments constitute the first steps toward transforming AFM into a higher throughput technology. We also discuss papers using AFM as NanoMechnanicalSensors (NEMS), and demonstrate the interest of such approaches in clinical microbiology to detect quickly and with high accuracy microbial resistance

Forces guiding staphylococcal adhesion.

Staphylococcus epidermidis and Staphylococcus aureus are two important nosocomial pathogens that form biofilms on indwelling medical devices. Biofilm infections are difficult to fight as cells within the biofilm show increased resistance to antibiotics. Our understanding of the molecular interactions driving bacterial adhesion, the first stage of biofilm formation, has long been hampered by the paucity of appropriate force-measuring techniques. In this minireview, we discuss how atomic force microscopy techniques have enabled to shed light on the molecular forces at play during staphylococcal adhesion. Specific highlights include the study of the binding mechanisms of adhesion molecules by means of single-molecule force spectroscopy, the measurement of the forces involved in whole cell interactions using single-cell force spectroscopy, and the probing of the nanobiophysical properties of living bacteria via multiparametric imaging. Collectively, these findings emphasize the notion that force and function are tightly connected in staphylococcal adhesion