During co-evolution with their hosts, bacterial pathogens have evolved an impressive variety of strategies to take control of cellular pathways and avoid host defence mechanisms in order to establish a productive infection. Among the range of tools dedicated to host manipulation, pathogenic intracellular bacteria employ effector proteins that are secreted into the host cytoplasm during infection. These bacterial effectors localize to specific cellular compartments where they exert their function, mostly by modifying eukaryotic proteins, reshaping cellular organelles or hijacking signaling pathways. Over the last decade, several mechanisms of action have been described for secreted bacterial effectors ranging from innate immune suppression, evasion from autophagy, apoptosis inhibition, subversion of membrane trafficking, and manipulation of cytoskeleton dynamics. However, the majority of virulence factors described to date have been shown to target eukaryotic proteins. In contrast, direct targeting of host nucleic acids remains poorly explored and mainly focused on host DNA. Although RNA targeting would offer a bacterium a wide spectrum of host processes to manipulate for its own benefit, bacterial effectors able to target RNA have not been reported. L. monocytogenes is a clinically important, food-borne human pathogen that can lead to serious sepsis, encephalitis or meningitis in immunocompromised individuals and to abortion in pregnant women. To be virulent, L. monocytogenes uses an arsenal of virulence factors that are able to subvert several host cell activities. We have recently identified Zea, the first secreted bacterial RNA-binding protein (RBP) that regulates L. monocytogenes virulence. Zea binds secreted L. monocytogenes RNA and stimulates the the type-I interferon response during infection. Zea localizes to the nucleus during infection and is found enriched at the nuclear speckles, the sites where the splicing machinery accumulates. Immunoprecipitation experiments revealed that Zea binds splicing factors in an RNA-dependent manner. We have also identified another L. monocytogenes secreted effectors, LntB, that is able to bind splicing factors and possibly host RNA. These results indicate that, by secreting RBPs, Listeria can potentially target mammalian RNA and affect RNA fate for its own benefit Our research program intends to identify the RNA targets of both Zea and LntB, characterize their interaction with the splicing factors and determine the functional role of this binding in the context of L. monocytogenes pathogenesis. Finally, by applying the interactome capture method, we plan to identify novel secreted RNA-binding effectors in L. monocytogenes. This project will pinpoint a still-uncharacterized strategy used by a bacterial pathogens to subvert its host that is via the secretion of RNA-binding effectors. We anticipate our findings to have a great impact in several research fields comprising infection biology, basic microbiology and fundamental cell biology.

As the size and complexity of synthetic genetic circuits increase, they progressively become too burdensome for a single cell. Consequently, many toy-model genetic circuits are easily lost to negative selection when the engineered organisms are exposed to less controlled environments. In contrast, natural systems are able to carry much larger genetic programs by using complex regulatory mechanisms to keep the costs of expression under check. They do this by a combination of temporal control over gene expression and additionally by spatial distribution of functions in multicellular systems. In this project, we plan to develop experimental and theoretical methods to measure the cost of maintaining and executing synthetic genetic circuits inside cells. Quantitative models will be built to calculate the cost of expression, and the accompanying growth effect, of single proteins and multi-protein circuits over time. The designs will be studied under different growth and stress conditions to assess the relationship between the calculated costs and the long-term evolutionary stability of the circuits. The analyses will inform design choices relevant for building the most cost-efficient unicellular circuits, and determine the upper size-limit at which it would be more efficient to distribute them across multiple cells in a consortium. We expect the results of this work to have key implications not only for the scale-up of synthetic genetic circuits but also for the understanding of ecosystem functions in microbial communities and division of labour in multicellular organisms.

Overweight and obesity have become global epidemics in recent decades, affecting over 2.5 billion and 890 million people, respectively, in 2022. These conditions are associated with metabolic disorders, including cardiovascular diseases, type 2 diabetes, non-alcoholic fatty liver disease (NAFL), musculoskeletal diseases, chronic kidney disease, and predisposes to an increased risk of certain cancers and autoimmune diseases. Obesity-related comorbidities contribute to 2.8 million deaths annually worldwide and have significant implications for public health and healthcare systems. As obesity and related metabolic diseases have reached epidemic proportions, understanding their causes and developing new approaches for prevention and treatment is now a top priority. However, this endeavour is made challenging by the complex aetiology of these diseases, which includes factors such as host genetics, diet, and the microbiota. The gut microbiota, predominantly composed of commensal bacteria, plays a crucial role in maintaining host health. Emerging evidence suggests that pattern recognition receptors (PRRs) involved in sensing microbiota-derived molecules also regulate obesity-related inflammation, insulin resistance, metabolic dysregulation, and barrier dysfunction. Recently, alpha kinase 1 (ALPK1) has been identified as a novel PRR sensing the small metabolite ADP-heptose produced by Gram-negative bacteria. Our research has shown that commensal bacteria can activate the ALPK1 pathway. Moreover, human genome-wide association studies (GWAS) and longitudinal genetic epidemiological studies have linked single nucleotide polymorphisms (SNPs) in the ALPK1 locus to obesity, body weight index, and type 2 diabetes. Despite advances in understanding the microbiota and PRRs in metabolic homeostasis, the role of ALPK1 in this context remains unexplored. Our preliminary unpublished data indicate a significant impact of ALPK1 deletion on weight gain, glucose response, and insulin tolerance. In light of this evidence, we hypothesize that ALPK1 is a central signaling pathway activated by microbiota-derived cues that regulate host metabolic homeostasis. The overarching objective of the Ob-ALPK1 project is to characterize how ALPK1 regulates obesity and diabetes. To achieve this, we will use transdisciplinary approaches, combining unique animal models with state-of-the-art primary murine and human cell in vitro assays, metabolic profiling, and genomic/transcriptomic analysis in humans. In this project, we will address the mechanisms and cellular targets involved in ALPK1-dependent modulation of weight gain, glycemia, and metabolic dysregulation. We will also assess the clinical relevance of our findings in humans and explore the therapeutic potential of the ALPK1 pathway in vivo. Overall, this innovative project aims to uncover new mechanisms and bioactive molecular signals from the microbiota that can be targeted to prevent and treat obesity and metabolic syndromes.

The project aims at discovering the fundamental principles governing the adaptation of multi-species communities to disturbance on a 4-species (4S) bacterial biofilm of natural origin. In the nature, these systems play a crucial role in the biogeochemical cycles of carbon, nitrogen and water. The disturbance of their balance can only come along with striking consequences at a global scale. However, we currently ignore how these communities will respond to climate change. The examination of this question at the natural ecosystem scale is hardly feasible due to the impossibility to rationally vary and control the environmental conditions. Besides the laboratory studies are mostly mono-species while it is increasingly becoming obvious that the inter-species interactions are crucial for the assembly and the development of these communities. To better understand the factors which support the adaptation of these communities to disturbances, we propose gathering biophysicists and microbiologists who will examine the global and molecular responses of the model 4S community to controlled environmental changes. In a first phase, we will build a quantitative phenotypic and genetic description of the 4S biofilm established in a microfluidic platform enabling to control the applied physical and chemical conditions as well as to monitor in real time the development of the community. Through a combinatorial approach — all biofilms from mono- to 4-species will be examined in parallel — we will first identify interspecies interactions and their environmental and genetic background in a reference state. Then, in a second phase, we will carry out the perturbation program consisting in completing series of controlled disturbances of various natures — chemical, physical and social — to detect characteristic adaptive trajectories (resistance, resilience or redundancy) and select remarkable time-points — climax or plateau — which will then be studied from a genetic point of view in the third phase. In this third phase, we will study the transcriptional and genomic alterations having occurred at the selected time points of the adaptive trajectory. Through this approach, we aim at identifying the genes and the interspecies interactions involved in the adaptation to a given perturbation, and isolate potentially emerging mutants. On the other hand, we will conduct a theoretical analysis to model the population dynamics induced by disturbances. This program holds several methodological and technological challenges such as the development of a quantitative method for describing the community phenotype, the development of the experiment automation required by the combinatorial approach and the disturbance screening step; as well the genetic analyses that will be performed in the multi-species context, thus needing the implementation of the latest technical advances in the field. Our approach aims at overcoming the difficulty in linking phenotypic and genetic information. Our strategy is to pre-select a limited number of relevant trajectories and to perform correlated analyses — phenotypic and genetic — on defined time points of the adaptive path to bring over adaptation mechanism features in this 4S adherent community. The completion of our program should provide a first clarification on the role of interspecies interactions in the specific architecture of the mixed community and its capacity to adapt to a given stress. We also expect other benefits such as the advance of new experimental tools to analyze adherent bacterial communities and new strategies to control bacterial biofilms, potentially new avenues to artificially assemble useful multispecies communities with defined function. Finally, our work will allow to evaluate the potential of multispecies simplified models, grown in the laboratory conditions for understanding and predicting the dynamics of natural systems.

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal (GI) disorders for which there are limited treatment options, yet which results in significant impairment of quality of life and societal costs. The severity of IBS correlates in multiple paradigms with sugar, and in particular fructose, intake. The current fructose consumption often exceeds the absorption capacity of the small intestine leading to its malabsorption. Previously we showed in rodents that malabsorbed fructose spills over to distal GI tract where it modifies the microbiota composition and an unexpected enteroendocrine cell (EECs) secretory pattern resulting in increased in cholecystokinin (CCK) in the distal region of the gut. Since CCK has been associated with pain signaling, here we propose to investigate the relationships between the microbiota changes resulting from fructose malabsorption, the CCK expression and visceral hypersensitivity. Based on the complementary expertise of the 2 partners involved in the project (intestinal physiology, microbiota, IBS and EEC biology), we will first, investigate, in both rodents and humans, the relationship between the microbiota and the visceral hypersensitivity in the context of fructose malabsorption. Then, we will examine whether CCK mediates visceral hypersensitivity . Finally,we will examine the mechanisms by which malabsorbed fructose induces changes in the EEC features that result in the increase in CCK synthesis. We will use complementary approaches of microbiota analysis (in mice and humans), transfer of intestinal microbiota (mouse to mouse and human to mouse), as well as new mouse genetic models and organoids to decipher the mechanisms involved in the regulation of CCK in distal gut and its role in the development of visceral hypersensitivity. Our results will provide important clinically-relevant information about the mechanisms of hypersensitivity in IBS in the context of fructose malabsorption that may lead to better stratification of IBS patients.