Intestinal microbiota plays a major role in human health. Horizontal gene transfer (HGT) between bacteria brought by food and resident bacteria can threaten its equilibrium by conferring novel properties to digestive bacteria (antibiotic resistance, novel catabolic properties…). Conjugation, the main mechanism of HGT, is encoded by several classes of mobile genetic elements, in particular chromosomal Integrative and Conjugative Elements (ICEs), which are widespread but still poorly known.
This project aims at evaluating gene transfers occurring through ICE between bacteria brought by dietary intake and commensal bacteria of the digestive tract. Work focuses on gene transfers within Streptococcus, an interesting bacterial genus since it includes various species that live or transit in the human gastrointestinal tract (commensal i.e. Streptococcus salivarius, pathogenic or food bacteria i.e. Streptococcus thermophilus) but also with Enterococcus faecalis, a main component of the digestive flora. Using ICEs belonging to the ICESt3 family as models, our objectives are to (i) characterize the environmental stimuli and cellular contacts (role of surface molecules, impact of biofilm) required to initiate their transfer, (ii) decipher the molecular mechanisms of their regulation and (iii) gather data on the occurrence of gene transfers via these ICEs in a human digestive ecosystem. This project will help to propose strategies to fight against bacterial gene transfers mediated by conjugation.
ICE transfer will be studied in different conditions: (i) in vitro on filter (ii) in situ in biofilm, (iii) in vitro in a dynamic artificial digester to integrate physical and biochemical parameters of stomach and small intestine, and to get closer to conditions encountered by bacteria in their ecosystem (iv) in a dynamic artificial colon maintaining a complex microbiota and (iv) in vivo in mice. We will decipher the regulation pathways (interplay between regulators, control by an anti-repressor) controlling ICE excision and transfer in both inducing and non-inducing conditions. This will be made using molecular approaches (construction of mutants, transcriptional fusions, protein expression, and analysis of protein and protein-DNA interactions). We will also characterize the surface molecules required for cell-cell contacts as a prerequisite to DNA transport into the recipient cell. Finally, we will evaluate the impact of biofilm on the frequency of ICE transfer and go further to explore ICE transfer in 4D scale. A fluorescence tool will be developed at this occasion.
We demonstrated autonomous transfer of ICEs from Streptococcus salivarius to other S. salivarius but also to Streptococcus thermophilus, a bacterium used in dairy industry and to Enterococcus faecalis that belongs to the human digestive flora. The frequency of transfer observed in filter experiments was very low (<10 8 transconjugants/donor cell) suggesting a tight regulation of excision and transfer of the ICEs found in S. salivarius.
The composition of the bacterial cell wall (presence of lipoproteins, teichoïc and lipoteichoïc acids and synthesis of exopolysaccharides) has an impact on the frequency of ICE transfer. A large increase of the frequency of ICE transfer was observed in recipient strains affected in the production of these surface molecules but not in donor strains.
Horizontal gene transfer contributes to the spread of genes in particular antibiotic resistance genes that compromise human health. Prevention strategies are thus urgently needed to control this phenomenon. Results obtained in this project will enable to explore several strategies to fight HGT: (i) use of probiotic bacteria not prone to gene transfers as a colonizer of digestive tract (flora displacement), (ii) blocking of the activation of ICE transfer (control of induction of SOS system or targets in the regulation cascade), (iii) blocking of DNA transport across membrane by exploiting natural bacterial defenses against gene transfers or targeting the surface molecules involved in the establishment of the conjugative apparatus and (iv) an anti-biofilm strategy that can be combined with the other strategies. Further work will be needed to develop and optimize these strategies.
-N. Dahmane, V. Libante, F. Charron-Bourgoin, E. Guédon, G. Guédon, N. Leblond-Bourget and S. Payot (2017). Diversity of integrative and conjugative elements of Streptococcus salivarius and their intra- and interspecies transfer. Appl Environ Microbiol do
Intestinal microbiota is now recognized as playing a major role in human health by conferring a protection against pathogens and contributing to human nutrition. A disequilibrium of this ecosystem (dysbiosis) can lead to the development of diseases (inflammatory bowel diseases, obesity or colorectal cancer). Throughout life, the total consumption of food by an individual will reach 60 tons on average, corresponding to a large ingestion of bacteria (bacteria used in food industry or pathogens). These bacteria brought by food will be able to interact with symbiotic and commensal bacteria and in particular exchange genes (horizontal gene transfer) with them. This acquisition of genes can confer novel properties to bacteria (antibiotic resistance, colonization factors, novel catabolic properties, synthesis of bacteriocin, stress responses…). This can threat the equilibrium of digestive microbiota or lead to the emergence of new pathogens. Conjugation is the main mechanism of gene acquisition and can lead not only to gene transfers between strains belonging to the same species but also between very distantly related bacteria. It is encoded by several classes of mobile genetic elements, in particular chromosomal elements called Integrative and Conjugative Elements or ICEs, a widespread but poorly known class of elements. This project aims at evaluating gene transfers occurring through ICE between bacteria brought by dietary intake and commensal bacteria of the digestive tract. Work will mainly focus on gene transfers within Streptococcus, an interesting bacterial genus since it includes various species that live or transit in the human gastrointestinal tract (commensal, pathogenic or food bacteria). The objectives of the project are to characterize the factors and the mechanisms of induction of transfer of ICEs and to give a first insight on the incidence of gene transfers mediated by these ICEs in the human digestive ecosystem. Approaches will include in vitro analyses, biorelevant in vitro studies (in artificial digester and in artificial colon inoculated with human microbiota) and in vivo experiments (axenic mice inoculated with strains of interest). This project will help to propose strategies to fight against bacterial gene transfers mediated by conjugation.
Madame Sophie PAYOT-LACROIX (UMR1128 Dynamique des génomes et adaptation microbienne)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
EA CIDAM Equipe d'Accueil Conception, Ingénierie et Développement de l'Aliment et du Médicament
UMR1319 MICALIS UMR1319 Microbiologie de l’Alimentation au service de la Santé
UMR1128 DynAMic UMR1128 Dynamique des génomes et adaptation microbienne
Help of the ANR 438,062 euros
Beginning and duration of the scientific project: September 2015 - 36 Months