Thèse Transfert Horizontaux de Gènes Inter-Espèces au Sein de Biofilms Pulmonaires chez les Patients Atteints de la Mucoviscidose H/F - Doctorat.Gouv.Fr

Établissement : Université Paris-Saclay GS Life Sciences and Health École doctorale : Structure et Dynamique des Systèmes Vivants Laboratoire de recherche : Infection et inflammation Direction de la thèse : Nicolas MIROUZE ORCID 0000000210380495 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-07-31T23:59:59 ContexteLes patients atteints de la mucoviscidose sont sujets à des infections respiratoires sévères causées notamment par des biofilms, communautés bactériennes formées dans le mucus à la surface des cellules épithéliales respiratoires. On retrouve dans ces biofilms les deux principaux pathogènes opportunistes Staphylococcus aureus and Pseudomonas aeruginosa. Ces deux espèces pathogènes posent également de graves problèmes de santé publique due à l'apparition de souches multi-résistantes aux antibiotiques, représentant de réelles impasses thérapeutiques. Ces résistances sont principalement acquises via le Transfert Horizontal de Gènes (THG) permettant l'acquisition de nouvelles séquences présentes chez d'autres espèces bactériennes co-existant au sein des poumons infectés.Or, il a récemment été démontré que S. aureus et P. aeruginosa sont capables d'induire, au sein de biofilms, la compétence pour la Transformation Naturelle (TN), l'un des trois mécanismes de THG chez les bactéries.Objectif du projetNotre hypothèse est donc que les biofilms formés dans le poumon des patients atteints de la mucoviscidose représentent malheureusement un environnement favorable au THG, via la compétence pour la TN, chez S. aureus et P. aeruginosa. Compétence et TN permettraient donc à ces deux pathogènes opportunistes de s'adapter, de persister et de résister aux antibiotiques. Nous proposons donc (i) d'étudier comment la formation de biofilms induit le développement de la compétence chez S. aureus et P. aeruginosa, (ii) de démontrer en quoi les biofilms représentent l'environnement adéquat pour le développement de la compétence et (iii) l'acquisition de nouveaux gènes de résistance aux antibiotiques par la TN, et enfin (iv) d'explorer, à travers un modèle ex vivo, la possibilité pour ces deux pathogènes d'induire compétence et TN dans des biofilms formés à la surface de cellules épithéliales respiratoires en présence de mucus.Perspectives dans le cadre de la mucoviscidoseLa démonstration de la capacité de ces deux grands pathogènes humains d'acquérir des gènes de résistance aux antibiotiques dans les poumons des patients atteints de la mucoviscidose représentera une étape essentielle pour la limitation voire la prévention de l'apparition de souches multi-résistantes aux antibiotiques. Par la suite, nous pourrons cribler des collections de composés naturels permettant d'inhiber compétence et/ou TN. On pourra également envisager de développer de nouvelles approches conduisant à la perturbation ou la dissociation des biofilms au sein des poumons, permettant de modifier les conditions environnementales perçues par les bactéries et ainsi prévenir le développement de la compétence. Position of the project as it relates to the state of the artDuring evolution, the ability of bacteria to adapt to evolving environments often resulted from the acquisition of new genes, and therefore new functions, through Horizontal Gene Transfer (HGT). HGT, defined as the transmission of genetic material between organisms that are not in a parent-offspring relationship, is an important means by which bacteria ensure genomic plasticity and acquisition of antibiotic resistance [11]. HGT may occur via three main mechanisms: transduction, conjugation and Natural Transformation (NT). The latter mechanism ensures the internalization and homologous recombination within the chromosome of high molecular-weight exogenous DNA [5]. Importantly, NT requires that bacterial cells enter a differentiated state called competence that has been studied in a number of different bacteria, particularly Bacillus subtilis and Streptococcus pneumoniae [6]. However, new bacterial species displaying such ability are often characterized, two of the latest being the opportunistic pathogens Staphylococcus aureus [12] and Pseudomonas aeruginosa [13].Competence for NT in historical model organisms.Competence is considered as a highly regulated bacterial differentiation [6]. Early' central competence regulatory pathways are activated in response to environmental signals (usually stresses, Fig. 1A). These pathways activate dedicated central competence regulators (Fig. 1A) among which are found alternative sigma factors (such as ComX in S. pneumoniae, [14]), transcription activators (such as ComK in B. subtilis, [15]) and transcription co-regulators. Once activated, these regulators induce the expression of the late' competence genes encoding the proteins essential for NT (Fig. 1A, [16], [17]).While the steps and actors involved during the NT process are widely conserved (DNA binding, uptake and recombination), competence regulation varies considerably among species. Interestingly, despite global similarities between these pathways, competence regulation has probably evolved independently in different species in order to adjust to a large diversity of ecological niches and stimuli [6].Over the past decades, the host laboratory has greatly participated to a better understanding of competence for NT in various model organisms. In S. pneumoniae, we particularly showed that the transformation-dedicated DNA processing protein A (DprA), allows the loading of RecA to form presynaptic filaments during NT [18], [19] but is also involved in the shutoff of pneumococcal competence by directly interacting with early regulators [20]. In addition, we also investigated the development of competence in B. subtilis by showing that Spo0A, a major regulator, imposes a bimodal expression of competence [21], [22]. Finally, we demonstrated that the growth arrest imposed during competence in B. subtilis requires the spatial regulation of the cytoskeleton [23] and identified the structure responsible to the initial DNA binding at the surface of B. subtilis competent cells [24].Competence regulation in S. aureusOver the past fifty years, S. aureus strains have acquired resistance to a broad spectrum of antibiotics to become a major cause of hospital-acquired infections but also of outbreaks among healthy individuals outside from the hospital. Antibiotic resistance genes are usually located on horizontally acquired mobile genetic elements, such as transposons, plasmids, genomic islands and ICEs. The genomic island responsible for the spread of methicillin resistance among S. aureus is termed the staphylococcal cassette chromosome mec' (SCCmec), which often harbors a number of additional genes conferring resistance to a wide variety of antibiotics [25]. Strikingly, the fact that competence for NT is, to date, the only mechanism able to transfer a complete SCCmec, even considering the longest subtypes, strongly reinforces the potential of this mode of HGT to generate resistant S. aureus isolates [8]. Indeed, the possibility to transfer mobile genetic elements through NT has been confirmed in Acinetobacter baylyi [26]. This result led the authors to conclude that NT provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits even from divergent species.Furthermore, if considering S. aureus high adaptability to its environment, especially during virulence, competence for NT could be considered as a mechanism of choice to improve its general fitness. It was recently shown that HGT in Staphylococcus extends beyond functions usually associated with mobile genetic elements [27]. Indeed, analysis of 13 genomes of 4 Staphylococcus species has provided evidence of HGT in almost 50% of the analyzed genes, influencing functions such as membrane transport, information processing and metabolism. Importantly, such functions could be, directly or indirectly, associated to highly adapted and virulent S. aureus strains and transferred by NT.For S. aureus, the ability to induce competence and NT has only been described 10 years ago [12]. The S. aureus competence-specific machinery combines early regulators originally found in the two main model organisms [28], [29]: a cryptic secondary sigma factor, SigH (comparable to ComX in S. pneumoniae), and two genes, comK1 and comK2, encoding putative homologs of the transcription factor ComK present in B. subtilis. Interestingly, the use of multiple regulators might have evolved to allow S.aureus to be able to use a wider range of cues to decide whether or not to become competent for genetic transformation.Indeed, in addition to planktonic cultures, the formation of biofilm has been shown to induce the development of competence for NT in S. aureus [30]. Indeed, it seems that biofilm growth conditions enhance the efficiency of the transfer of SCCmec and therefore the spread of methicillin resistance and MRSA clones [30].Competence regulation in P. aeruginosaRegarding competence for NT in P. aeruginosa, data are scarcer. The absence of environmental conditions leading to the development of competence questioned for a long time the ability of P. aeruginosa to perform NT. However, the very recent report that P. aeruginosa is capable of NT in biofilms completely changed our way of thinking and opened new lines of investigation [13]. Indeed, recombination has been identified as a major means of genetic diversity in many P. aeruginosa clinical isolates although the source of DNA and the mechanism of HGT was not determined [13]. Therefore, NT may be an important mechanism for the acquisition of antibiotic resistance and virulence genes in this pathogen and a significant contributor to the rapid increase in the number of multidrug-resistant P. aeruginosa strains [13]. Published and preliminary data from the host laboratoryIn S. aureusThe host laboratory has already generated important published and preliminary data describing the complex regulatory pathways at play in S. aureus and the possible environmental stimuli allowing the development of competence for NT in vivo. Published data regarding the identification of competence regulators and NT actorsNicolas Mirouze's (NM) group (ANR JC/JC GenTranSa grant, CES35, 2019-22, NM) has recently demonstrated through genetic studies that SigH and ComK1 are both essential, probably through a direct protein-protein interaction, for the control of the NT genes expression under microaerobic conditions in planktonic cultures [8]. Using flow cytometry analysis of GFP-based transcriptional reporters (compatible with low-oxygen concentrations as shown in [8]) as well as global transcriptomics (RNA-seq), we also identified two classes of NT genes characterized by specific regulations: class I genes are controlled by both SigH and ComK1 while class II genes are exclusively controlled by ComK1 [8]. NM's group has also shown, using high-resolution microscopy, that NT occurs in the vicinity of the division septum and proposed that S. aureus competent cells initiate and then block cell division to ensure the success of NT before the final constriction of the cytokinetic ring [31].Published data regarding competence regulation and oxygenFirst, NM's group recently discovered that microaerobic conditions represent an important environmental signal inducing the development of competence in S. aureus [8]. Indeed, we found that an important decrease in the concentration of environmental oxygen is sensed by the SrrAB two-component system, potentially activating SigH (Fig. 1B, [8]). In addition, we also characterized a new regulatory pathway, modulating the development of competence under strict anaerobic conditions (Fig. 1C, [32]). We particularly showed that the absence of oxygen is sensed by another two-component system, NreBC that inhibits the expression of the NT genes. We also found that this inhibitory pathway involves ComK2, the third central competence regulator, and SA2107, a protein with no predicted function. We detected a putative protein-protein interaction between ComK2 and SA2107 (using a yeast two-hybrid screening), essential to inhibit the expression of the NT genes. These findings demonstrate the necessity for S. aureus to regulate its need for HGT through the modulation of competence development. They also corroborate published results demonstrating the development of competence in S. aureus biofilms, cellular communities associated with oxygen gradients [9].Preliminary data regarding competence regulation and biofilm formationThese results demonstrate our ability to accurately monitor the development of competence in a wide variety of experimental setups. This also true in the context of biofilm formation. Indeed, Yannis Arab (second year PhD in NM's team) has demonstrated and studied the development of competence for NT in S. aureus biofilms (In the context of endocarditis, Not published yet). First, Yannis has characterized the expression of NT genes in colonies grown on solid surfaces (i.e. agar, Fig. 2). These experiments allowed him to confirm the development of competence in biofilms, to visualize and monitor through time the cells inducing the expression of NT genes and to study the impact of the environmental oxygen concentration.Furthermore, Yannis has also shown, in preliminary experiments, that pulmonary epithelial cells (BCi-NS1.1 immortalized bronchial basal cells, cultivated in a air-liquid interface system) provided a conducive environment for the development of competence in S. aureus (Fig. 3). Even though these results need to be repeated and optimized, they represent a milestone in our project linking biofilm formation in vivo and S. aureus genomic plasticity through HGT.Preliminary data regarding methodologies developmentNM's team recently developed a reporter system, involving the luciferase from Photinus pyralis as a powerful transcriptional reporter (already validated by NM in several model organisms, [20], [21]), in order to study the temporal dynamic, the succession of events as well as new regulatory pathways involved during competence development in S. aureus grown under planktonic conditions (manuscript currently under review in Plos Pathogens, Fig. 4). Importantly, Yannis Arab has also demonstrated in preliminary experiments, our ability to use the luciferase to follow our genes of interest expression in biofilms (Fig. 5).Preliminary data regarding the S. aureus collection of clinical isolatesFinally, Anne Jamet (AJ) set-up over the past years, at the microbiology laboratory of the Necker hospital, a collection of S. aureus clinical isolates biobanked at -80°C for subsequent analysis. The AJ group has a specific interest in cohorts of patients with chronic S. aureus infections of the lung (CF patients) or wounds, whose isolates are routinely investigated to assess the virulence and antimicrobial resistance-associated genes as well as within-host acquisition of adaptive genetic and phenotypic polymorphisms. Importantly, such genomic adaptations of S. aureus clinical isolates could be associated with HGT and involve competence for NT. P. Poirette (third year PhD in NM's team) has already observed a wide range of competence induction in 30 clinical isolates chosen from AJ's collection.In P. aeruginosaImportantly, since the last application in 2024, we have now reproduced the data published by C. Whitchurch laboratory [13]. Indeed, we have confirmed that P. aeruginosa becomes competent for NT in biofilms. Indeed, Yannis Arab (a 2nd year PhD student from the host laboratory) has reproduced the acquisition of plasmids and chromosomal sequences by the P. aeruginosa strain PAK, grown in biofilms (colonies on agar plates and biofilm in static liquid cultures in 96-well plates). Strikingly, by optimizing pre-culture and culture conditions, Yannis even improved the transformation efficiencies by a 10-fold factor (reaching 10-6) in comparison to what was previously published [13]. Yannis is currently working on the protocol as we identified several key points that could be improved, leading to even better transformation efficiencies and definitely demonstrating the potential of competence for NT in P. aeruginosa genomic plasticity.Project's hypothesisBased on all the published and preliminary results presented above, and our knowledge of the chronic bacterial infections occurring in the viscous mucus present in the lungs of cystic fibrosis patients, we propose a strong hypothesis: the development of biofilm in the CF patients lungs could unfortunately represent the perfect environment to promote the genomic plasticity, the acquisition of new antibiotic resistance genes and global adaptation of S. aureus and P. aeruginosa through competence and NT. Ultimately, these published and preliminary data also demonstrate our knowledge of this scientific field, our ability to construct powerful genetic tools and to create adequate experimental setups to accomplish the present project. Cystic fibrosis (CF) is an autosomal recessive genetic disorder characterized by recurrent and chronic infections of the lung. Contrary to common intuition, the lungs are not entirely aerobic, especially the airways of patients with CF. The combination of thickened mucus and decreased clearance further facilitates the formation of mucus plugs that can obstruct the airways and form a protected niche for microbes [1]. Within this thick mucus, and particularly within the plugs, a steep oxygen gradient forms with hypoxic (low oxygen) or anoxic (no oxygen) regions [2]. Additionally, mucus hypoxia or anoxia may be further enhanced by biofilms, defined as structured consortia of bacteria, embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA in which nutrient and oxygen gradients are established.Chronic infections in the lungs of CF patients are predominantly controlled by the opportunistic pathogens Staphylococcus aureus and Pseudomonas aeruginosa. While S. aureus is the main colonizing species of the CF lungs during early childhood, its incidence later declines and infections by P. aeruginosa become more prominent [3]. The competition and cooperative interactions displayed by these two pathogens influence their survival, antibiotic susceptibility, persistence and, consequently the disease progression [3]. Indeed, while for years P. aeruginosa was though to only exhibit aggressive behavior toward S. aureus, more recently, more tolerant P. aeruginosa strains have been isolated from chronic CF infections [3]. The establishment of such coexisting interaction between the two species seems to arise from their co-evolution in the lung ecosystem.Interestingly, with the advent of improved culture methods and deep sequencing technology, it is now clear that the airways of patients with CF are chronically colonized, with complex polymicrobial communities that go beyond these two opportunistic pathogens. Indeed, core genera, including Streptococcus, Prevotella, Veillonella, Neisseria, Porphorymonas, and Catonella are also detected in abundance in the majority of adult sputum samples [2]. In addition to this core, deep sequencing typically identifies 50-200 unique operational taxonomic units in a single CF respiratory sample [2]. When considering genome's evolution and adaption through Horizontal Gene Transfer (HGT), such complexity of the CF lungs microbiota could be seen as the perfect environment for its diversity in terms of new genetic sequences. Importantly, in addition to improving the general fitness of the bacteria in a specific environment such as CF infected lungs, HGT events can also lead to multi-antibiotic resistance. This is particularly true for S. aureus and P. aeruginosa, which are part of a priority list of 12 families of pathogenic bacteria, multi-resistant to antibiotics, drawn by the World Health Organization (WHO) to find new effective treatments [4].Natural Transformation (NT), one of the three main HGT mechanisms present in bacteria, ensures the binding, uptake and homologous recombination within the chromosome of exogenous DNA present in the environment [5]. NT is entirely controlled by the recipient bacterial cell that needs to enter a genetically encoded differentiated state called genetic competence [6]. Early' competence regulatory cascades are triggered in response to diverse environmental or cellular cues ranging from quorum sensing, cellular nutritional status and stressful conditions. These pathways activate central competence regulators which control the expression of the late' competence genes encoding the proteins essential for NT. Importantly, over the past years, NT has gained even more interest in the scientific community because of its over-representation among pathogenic bacteria accumulating antibiotic resistances. Indeed, all the antibiotic multi-resistant pathogens listed by the WHO, have the ability to promote competence for NT [7].Importantly, we have recently shown that S. aureus induces competence for NT in response to decreasing oxygen concentrations, mimicking CF lungs environmental conditions [8]. Furthermore, very recent reports have demonstrated the ability of both S. aureus and P. aeruginosa to promote NT within biofilms [9], [10], an environment characterized by hypoxic or anoxic regions and also found in CF lungs. Therefore, we hypothesize that CF lungs could unfortunately represent the perfect environment for the two opportunistic pathogens, S. aureus and P. aeruginosa, to promote their genomic plasticity, coexistence and resistance. The main objective of the present grant application will be to understand and characterize genetic competence for NT in S. aureus and P. aeruginosa in the context of biofilms. Such knowledge should allow us to understand how biofilms present in CF lungs provide the environmental conditions (signals or stresses) to induce such processes and contribute to the proposition of therapeutic solutions to prevent them. Ultimately the demonstration that competence for NT participate in the genome plasticity and acquisition of new antibiotic resistances in these two opportunistic pathogens present in CF patients' lungs should initiate new research to prevent these HGT events.This multidisciplinary project will give the opportunity to the selected PhD student to continue the work currently carried out in the host team and will be divided in four main Work Packages (WP):WP1. Characterize the environmental stimuli and transcriptional regulations responsible for the development of genetic competence within in vitro S. aureus or P. aeruginosa mono species biofilms WP2. Determine, within single-species biofilms, the localization and local environmental conditions leading to the apparition of competent cells for NT WP3. Demonstrate HGT via NT within multi-species biofilms in vitro. WP4. Prove the development of competence for NT ex vivo, in S. aureus or P. aeruginosa mono-species biofilm formed in mucus produced by respiratory epithelial cells, using Air-liquid interface (ALI) models. Scientific programAs presented above, this project multidisciplinary project will give the opportunity to the selected PhD student to continue the work currently carried out in the team and will be divided in four main work packages (WP). Importantly, within this scientific program, we tried to answer all the weaknesses raised following our 2024 application. We particularly demonstrated that we could reproduce and even improve P. aeruginosa natural transformation in our laboratory. We also tried to narrow down the overall scientific program to ensure its feasibility in the context of a PhD project. Finally, we attempted to clarify and precise some of the experimental approaches proposed.This project will last 3 years and will involve, in addition of the PhD student, a number of scientists from the host team (Nicolas Mirouze, CRCN INSERM for the project management; Yannis Arab, 2nd year PhD student, who generated the preliminary data regarding S. aureus and P. aeruginosa natural transformation in biofilms; Vincent Pennaneach, CRCN INSERM, expert in microscopy and Sabine Blouquit-Laye, expert in the air-liquid interface model). The details of the Tasks assignment and timing of the project are summarized in a Gantt's chart.WP1. Characterize the development of genetic competence within in vitro S. aureus or P. aeruginosa mono-species biofilms WP1.1: Deciphering the role played by candidate regulators. We will first need to construct reporter strains harboring the gene encoding the luciferase under the control of promoters that respond to biofilm formation (adhesion, exopolysaccharide production, ...), competence regulation (early and central competence regulators) or NT (binding, transport and recombination of exogenous DNA) (WP1.1). The expression of these genes of interest will be compared, in the context of biofilms, in wild-type (wt) and mutant strains in which genes encoding expected or putative regulators will be deleted (WP1.2). Expected regulators have been previously identified in preliminary experiments (host laboratory) or are present in other competent model organisms (Gram-positive or -negative). In parallel, putative regulators will be proposed based on RNA-sequencing experiments (WP1.3). Since we currently have all the strains already available in S. aureus [8], the first part this WP, dedicated to the construction of reporter and mutant strains, will only concern P. aeruginosa (WP1.1). Importantly, in this first WP, we will focus on laboratory strains for which NT has been demonstrated (i.e. N315 for S. aureus and PAK for P. aeruginosa)The reporter strains, will be then grown to form biofilms in 96 well plates incubated in a temperature-controlled plate reader equipped for highly sensitive luminometry measurements. Biofilms could be generated on agar (i.e. forming colonies, see preliminary data) or in liquid medium at the bottom of the well. Optical density (reflecting the biofilm thickness and therefore growth) and luminometry measurements will be conducted every 10 minutes in order to accurately characterize the regulations involved in the development of competence within biofilms (WP1.2). Importantly, we know from numerous model organisms, that the development of competence is a sequential process, defined by successive waves of regulations. In order to identify early competence regulators, we propose to use Time-series transcriptomic profiling as a powerful approach to understand dynamic biological processes (WP1.3). Indeed, comparison of the global transcriptomic profiles (RNA-sequencing) of a wild type strain between different time points along biofilm formation should allow us to identify differentially expressed genes (DEGs) and groups of dynamic expression patterns, bringing us new insights into the early competence regulatory pathways in biofilms (we already have successfully performed this kind of experiments with S. aureus planktonic cultures).Milestones: We expect WP1.1 to last for the first two years of the project. This will first require us to construct the luciferase reporter strains in P. aeruginosa (since we already have them in S. aureus) in order to analyze and characterize genes' expression in our plate reader. In parallel, we aim to perform Time-series transcriptomics (RNA-seq) during Year 1 that could allow us to identify new pathways or regulators that will be tested using the luciferase. The PhD candidate will be mentored and assisted during the first year by Yannis Arab (2nd year PhD in the host laboratory) who masters all the techniques presented here and worked on all the preliminary data linked to biofilms. It is important to mention that Yannis Arab's PhD project is focused on biofilms in the context of endocarditis. Therefore, there will be no overlap between the two projects.WP2. Determine, within mono-species biofilms, the localization and local environmental conditions leading to the apparition of competent cells for NT The aim of this second WP will be to characterize the structure and environmental conditions allowing, within mono-species biofilms (S. aureus or P. aeruginosa), the development of competence for NT.WP2.1: Constructing competence-reporter strains and experimental setupWe will first need to construct reporter strains harboring the gene encoding the GFP under the control of promoters of genes of interest, essential for the NT process itself. Again, we already have all these fusions available in S. aureus (see preliminary results and [8]), therefore these constructions will exclusively concern P. aeruginosa. We will focus on the construction of two transcriptional fusions with the promoter of the genes comG (involved in the early transforming DNA transport steps) and ssb (involved I the late recombination steps). Then, we will need to establish S. aureus and P. aeruginosa mono-species biofilms in liquid or on agar (both in 96-well plates, see S. aureus colonies in preliminary data), compatible with microscopy observations. WP2.2: Microscopic analysis of competent cells within biofilmsWe have already implemented various microscopy techniques in our laboratory to study the general structure, cellular differentiation and genes expression within S. aureus (see preliminary results and Fig. 1). Binoculars, as well as real-time Confocal laser scanning microscopy will be available both at the single cell and biofilm levels. Ultimately, we will be able to identify competence-developing cells, pinpoint the part of the biofilm where they appear (edge, center or mushroom caps in P. aeruginosa case) and deduce the environmental conditions leading to this environmental adaptation. Mutant strains (constructed in WP1) in which the gene encoding potential regulators have been deleted will also be tested her. Indeed, it will be interesting to confirm that NT genes expression is abolished (absence of GFP-expressing cells) or modified (new regions of the biofilm become competent).Milestones: This second WP will be conducted during year 2 and 3 of the project. We will first construct the reporter strains (in P. aeruginosa) and transfer them in wt and mutant strains. GFP-expressing competent cells will be then localized within the different types of biofilms by various microscopy techniques (Binocular, real-time Confocal laser scanning microscopy). The PhD candidate will be mentored by Vincent Pennaneach (CRCN INSERM, host laboratory) who is an expert in microscopy. We will use the Cymages platform present in the host institute.WP3. Demonstrate HGT via NT within multi-species biofilms in vitro. In addition of our two model organisms, S. aureus and P. aeruginosa, we will also implement other species (both Gram-positive and -negative) found in the CF lungs microbiota in order to prove that genetic exchanges occur through NT in multi-species biofilms in vitro. WP3.1: Constructing donor and recipient strains and experimental setupIn order to be able to distinguish donor from recipient strains we propose to use the constitutive expression of different fluorophores (YFP for donor and CFP for recipient cells) and natural antibiotic resistance genes (different in the donor and recipient cells). The genetic sequences transferred (i.e. antibiotic resistance genes) will be present either in the chromosome of the donor strain or on a replicative plasmid, autonomous in both donor and recipient strains. As NT requires sequence homology, we will consider Gram-positive donors for S. aureus (such as S. pneumoniae) and Gram-negative donors for P. aeruginosa (such as Neisseria) (Fig. 2).WP3.2: Demonstrating HGT in multi-species biofilmsFollowing growth of the donor and recipient strains in the same biofilm (colony or liquid), the transfer of genetic sequences will be verified by plating the entire biofilm on agar plates containing the two antibiotics. The polarity of the transfer will be ultimately verified by confirming that the cells growing on the two-antibiotic plates, express the CFP by flow cytometry. Ultimately, we could attempt to transfer DNA sequences between S. aureus and P. aeruginosa by forcing the homology that could lack between Gram-positive and -negative bacteria (Fig. 2). We could for example use a S. aureus strain as a donor in which we cloned, in its chromosome, a P. aeruginosa gene interrupted by an antibiotic resistance gene (compatible with P. aeruginosa selection), providing the sequence homology necessary to recombine in P. aeruginosa' genome, or vice versa.If successful, these experiments could ultimately be performed using CF clinical isolates of S. aureus (Karine Moreau, CNR, Lyon) or P. aeruginosa (Laboratoire Chrono-environnement, UMR 6249, Besançon)Milestones: WP3 will be performed spanning over year 3. Again, we will first need to construct the donor and recipient strains (constitutive expression of YFP or CFP, first three months) to finally test the ability for S. aureus and P. aeruginosa to acquire DNA sequences from other species within biofilms (6 months).WP4. Prove the development of competence for NT in mono-species S. aureus or P. aeruginosa biofilms, ex vivo using Air-liquid interface (ALI) models (Fig. 3). The main goal of this last WP will be to demonstrate that competence for NT develops in an ex vivo model, mimicking the environmental conditions found in CF patients' lungs. Epithelial cells from mucociliary portions of the airways can be readily grown and expanded in vitro. When grown on a porous membrane at an air-liquid interface (ALI) the cells form a confluent, electrically resistive barrier separating the apical and basolateral compartments. ALI cultures replicate key morphological, molecular and functional features of the respiratory epithelium, including mucus secretion. Therefore, ALI cultures are now considered as the reference model to study the pathogenesis of CF [33]. We propose here to take advantage of the ALI model, in the presence of mucus, to test the establishment of mono-species biofilms formed by S. aureus or P. aeruginosa at epithelial cells surface (CFBE41o- Human CF bronchial epithelial cell line or 1HAEo- Human Airway Epithelial Cell Line). Such experiments will be performed with reporter strains expressing GFP under the control of the NT gene promoter (and already constructed in WP2). The organization of the cells into biofilms could be analyzed by microcopy while the presence of competence-inducing cells could be visualized by microscopy or measured by flow cytometry. We are conscious that the PhD student's project is already very ambitious. We would consider this last Task as the cherry on the cake if all the other tasks have been completed. However, our preliminary results already show that S. aureus is able to induce competence for NT in a biofilm formed in the mucus present at the surface of a respiratory epithelium. These results will need to be repeated and optimized. The same kind of protocol will be tested with P. aeruginosa.Milestones: Finally, WP4 we be realized over the last year of the project. The ALI model is already available and mastered in our laboratory (Sabine Blouquit-Laye, MCU UVSQ, host laboratory). The competence reporter strains used here will be the same as in WP2.

Nous recherchons un candidat créatif possédant :- une expérience pratique et des connaissances en microbiologie ; à défaut, ces compétences pourront être acquises durant le doctorat ;- une forte motivation pour cette thèse de doctorat à l'intersection de la microbiologie fondamentale et interdisciplinaire ;- la capacité de travailler de manière autonome et proactive ;- un fort esprit d'équipe ;- une excellente maîtrise de l'anglais, à l'oral comme à l'écrit, est fortement souhaitable.Une expérience de manipulation génétique de S. aureus et/ou P. aeruginosa sera considérée comme un atout, mais ne sera pas un critère éliminatoire.