PhD defense of Clément Caffaratti from TIMC TrEE on January, the 20th at 2pm:

" Development of engineered lived biotherapeutics with controlled metabolism for innovative immunotherapy. "
 

• Place : Amphithéâtre du Bâtiment Boucherle, Faculté de Médecine et Pharmacie de Grenoble, La Tronche
   

Jury

• Audrey LE GOUELLEC, Maîtresse de conférence des Universités - Praticienne Hospitalière, Université Grenoble Alpes, Supervisor
• Bertrand TOUSSAINT, Professeur des Universités - Praticien Hospitalier, Université Grenoble Alpes,  Co-Supervisor
• Hans GEISELMANN,  Professeur des Universités, Laboratoire Liphy, Univ. Grenoble Alpes, chairman
• Justine BERTRAND MICHEL, Ingénieure de recherche, INSERM Occitanie Pyrénées, Reporter
• Nicolas CENAC, Directeur de recherche, INSERM Occitanie Pyrénées, Reporter
• Muriel THOMAS, Directrice de recherche, INRAE, Ile de France, Examiner
• Nicolas MATHIEU, Professeur associé, Univ. Grenoble Alpes & CHU Grenoble Alpes, Examiner

Keywords

biotherapeutics, medical bioengineering, microbiota, immunotherapy, synthetic biology

Abstract

The intestinal microbiota is, by its functional character, an important actor for various physiological or pathological processes of its host. In particular, it is, from birth, one of the main regulators of the intestinal immune response. In this context, the gut mucosal immune system receives a certain number of microbial signals involved in its activation, to protect its host from an infection, or in the maintenance of tolerance. In recent years, numerous studies have shown that Microbiota-derived metabolites are part of these signals. Currently, many therapeutic strategies offer to modify the immune response of the host by modifying the composition or the functionality of the microbiota.

In this thesis, we propose to modulate the host immunity via the in situ delivery of a metabolite, produced and secreted, by a genetically modified probiotic strain. This strategy is based on the rational engineering of the metabolism of the bacterium Escherichia coli Nissle 1917 (EcN), a probiotic known for its anti-inflammatory effects. Based on the knowledge of the host-microbiome dialogue, we selected spermidine, as a metabolite of interest, for its anti-inflammatory properties and its demonstrated effects in human health. This work started with the rational design of a genetic and metabolic engineering strategy put in place into a range of different variants to increase the EcN’s spermidine production capacity. Then, we used an LC-MS/MS approach to monitor the amount of spermidine present in the extracellular space of the engineered EcN strain. We found that increasing the expression of genes involved in polyamine biosynthesis, namely the S-adenosylmethionine synthase speD and the spermidine synthase speE, resulted in an increase in spermidine produced and excreted by our engineered bacteria.

Metabolomic analyses performed showed that some of the spermidine produced by the strains was acetylated. To solve this problem, we increased the expression of the spermidine exporting system MdtI/MdtJ. This second strategy had a major impact on the spermidine profile by maximizing the amount of spermidine found in the culture supernatant and limiting its acetylation. This work demonstrated for the first time that EcN bacteria could be used for spermidine production.

In order to evaluate if the increase of spermidine production and secretion could increase the immunomodulatory capacities of the EcN strain, we performed an in vivo characterization in a mouse model. The results we obtained suggest that, for one of the variants (EcN pCC31), its intake allows to increase the proportion of regulatory lymphocytes in the lamina propria of mice. However, we also observed, in healthy and antibiotic-treated mice, that the ingested strains have a different viability than the wild type EcN. We also found that spermidine concentrations in the intestinal lumen were similar to those of the controls, regardless of the strain used.

This work demonstrates the feasibility of using the EcN strain as a chassis for the production of a metabolite of interest in human health and lays the groundwork for understanding the effects of engineered probiotics.