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The team is interested in the defense mechanisms of organisms against environmental aggressors and the pathophysiological consequences when these mechanisms fail. With a focus on host-pathogen interactions, we study the physiology of the intestine and the functions of intestinal stem cells in maintaining cellular homeostasis and innate immunity. Our biological models are Drosophila melanogaster and bacteria of the Bacillus cereus group, with a particular interest in Bacillus thuringiensis, which is used as a microbial insecticide in agriculture.

Context and Objectives

The Bacillus cereus (Bc) group is a family of Gram-positive, facultative anaerobic, spore-forming bacteria that are ubiquitous in the environment. It includes the pathogens Bc sensu stricto (Bc s.s.) and B. anthracis, and the bacterium B. thuringiensis (Bt), which is widely used as an insecticide throughout the world (second most widely used insecticide, including synthetic), whether in organic or conventional agriculture, forestry or mosquito control. Bc s.s. is an opportunistic food poisoning pathogen that causes diarrhoea (Nhe, Hbl and CytK enterotoxins) and/or emesis (cerulean toxin). It is the leading cause of foodborne outbreaks (FBO) in France and Europe. Some studies have also highlighted the lethal potential of Bc s.s. in people with compromised immune system.

In response to societal and governmental demands, Bt insecticides are increasingly being used to replace chemical insecticides, which are less environmentally friendly. Bacteria of the Bc group have a two-stage life cycle: the vegetative cell, which is capable of proliferation, and the spore, a dormant form that is resistant to unfavourable environmental conditions. Bt is characterised by the production of a protein crystal (of plasmid origin) during sporulation. This crystal is mainly composed of Cry toxins (δ-endotoxins), which give Bt its entomopathogenic activity. After spraying, Bt products containing spores and toxin crystals are ingested by pest larvae. Cry toxins, activated by digestive proteases, bind to receptors in the intestinal epithelium (ALP, APN, cadherins and ABCC transporters) and create pores, leading to the death of the insect (known as the target) by septicemia within 48-72 hours.

Bacillus-based products have a wide range of applications. They show promise as biocontrol agents, as many Bacillus spp. have exploitable properties such as promoting plant growth, activating plant defences and producing antibiotics, antifungals and entomopathogenic toxins. Bacillus strains are also used as probiotics for humans and animals. However, the classification of members of the Bc group is complex due to their very similar characteristics and highly conserved genomes. The virulence factors, or conversely the beneficial factors, are very poorly understood. Advances in genomics suggest that Bt and Bc belong to a single species: Bc s.s. The safety of Bt insecticides for the environment and humans is increasingly controversial. Recent studies have shown that several strains of Bt from commercial products have been found in FBO, suggesting that Bt insecticides may pose a real risk to food safety. Furthermore, there are no reliable data in the literature on the effects of chronic ingestion of Bt products. Finally, the ingestion of spores, unlike vegetative bacteria, has been little studied and their behaviour and effects in the gut are not well understood. All these data show that there is still much to be learned about Bc group bacteria.

The aim of our team is to study the mechanisms of intestinal homeostasis and to understand the host responses following ingestion of Bc/Bt/toxins through several lines of research:

• signalisation and intestinal homeostasis

• virulence/modes of action and immune response

• intestinal pathophysiology

A - Spore of Bacillus thuringiensis ; B - 3D modelling of Cry toxins ; C - Apico-basal view of the Drosophila midgut ; D - Drosophila adult midgut ; E - Coomassie blue staining of Cry toxin ; F - Cross section of the Drosophila posterior midgut ; G - Longevity curves of adult Drosophila ; H - Cell counting in the Drosophila midgut.

Biological models

Our study model is Drosophila melanogaster (The Interactive Fly) (Flybase), which allows us to combine genetic, cellular and molecular biology and physiology approaches, as well as omics and bioinformatics tools. Drosophila is a non-target insect model for Bt products and can therefore be used to detect non-lethal but potentially undesirable long-term physiological changes that would be much more difficult to detect using conventional toxicological and epidemiological approaches. In addition, the high degree of conservation of key physiological mechanisms and signalling pathways between Drosophila and vertebrates allows results to be rapidly translated to other animals.

In addition to possessing pathogenic properties, bacteria of the Bc group (Bacillus Genetic Stock Center) have a variety of properties that are exploited to stimulate plant growth, activate plant defenses and produce antibiotics, antifungals and entomopathogenic toxins. They are also used as probiotics in human and veterinary medicine. All these properties are poorly understood. In our team we use different Bc/Bt strains and associated mutants, giving us a range of tools for in-depth studies.

The scientific originality of the team

Our team is the first to develop systematic research into the unintended effects of Bt insecticides by combining two distant disciplines: Ecotoxicology and Cellular/Developmental biology. This is made possible by the use of Drosophila. We are able to simultaneously study :

1. a targeted approach to study the physiological and pathophysiological impacts of Bt insecticides and their toxins                                                                                                                                                                                                                                       
2. a fundamental approach to identifying the cellular and molecular mechanisms involved and impacted.

Collaborations

We have established interdisciplinary collaborations with teams working on Drosophila and mouse models, as well as with hospital and food safety laboratories. These collaborations complement our approaches to provide a more global view of the potential health and environmental impacts of Bt/Bc spores.

• team " Virulence microbienne et signalisation inflammatoire" of Laurent BOYER (C3M, Nice - France)

• team "Bacillus & Clostridium"  of Olivier FIRMESSE and Mathilde BONIS (Anses, Maisons-Alfort - France)

• team "Mucosal microbiota in chronic inflammatory diseases" of Benoit CHASSAING (Institut Cochin, Paris - France)

• bacteriology laboratory of Raymond RUIMY (CHU de l'Archet II, Nice - France)

• team "Chromatine et Biologie Cellulaire - Dynamique du génome" of Giacomo CAVALLI (IGH, Montpellier - France)

• team "Stem cell immunity and vibrio pathogenesis" of Edan FOLEY (University of Alberta, Canada)

• team "Mécanismes mOléculaires et Cellulaires des Agents biologiques infectieux (MOCA)" of Dani OSMAN (PIMT, La Réunion - France Overseas)

• team "Interaction hôte-pathogène dans le modèle de la drosophile" of Julien ROYET (IBDM, Marseille - France)

Projects

I – Virulence/modes of action of Bt/Bc/toxins and host immune response

I-1. Virulence of Bt/Bc

In collaboration with the "Bacillus & Clostridium" team of Olivier FIRMESSE and Mathilde BONIS (Anses, Maisons-Alfort - France)

I-1.1 Implications of Bt in FBO

Bt is not currently subject to any regulatory food safety criteria in France or Europe, whereas Bc has been implicated in numerous FBO. The French Food Safety Authority (Anses) and the European Food Safety Authority (EFSA) stress that the involvement of Bt in FBO is likely to be underestimated (Bt and Bc are not distinguished by microbiological laboratories), as are the long-term environmental and health effects.

A recent retrospective analysis by Anses of 250 episodes of FBO associated with the Bc group revealed the presence of Bt in 20% of cases, and in 8% of cases Bt was the only microorganism detected. More than 95% of the Bt identified was indistinguishable from certain strains in commercial use, suggesting an agricultural origin of the contamination (Bonis M. et al., 2021. Comparative phenotypic, genotypic and genomic analyses of Bacillus thuringiensis associated with foodborne outbreaks in France. PLoS One). A collaboration has been initiated between our two teams to use the Drosophila model to assess and compare the pathogenicity of different Bt strains in vivo (FBO and commercial products). We are also developing experiments to study intestinal permeability and to detect and quantify gastrointestinal symptoms (diarrhoea/vomiting). These approaches will allow us to assess the virulence potential of a given strain in terms of its ability to cause food poisoning.

II-1.2. In silico research on virulence factors and in vivo study of their expression/function

This part consists of searching for virulence markers associated with Bt/Bc by analysing the complete genomes of 50 selected strains and adding about thirty public genomes of interest. In parallel, we are carrying out an in vivo bacterial transcriptome analysis in the Drosophila gut. The level of expression of all bacterial genes will be compared with virulence. The project described in this section will shed light on the involvement of Bt/Bc in FBO and on the causes of virulence (virulence factors, expression and function).

I.2. Host immune response

In collaboration with the " Virulence microbienne et signalisation inflammatoire" team of Laurent BOYER (C3M, Nice - France), the "Bacillus & Clostridium" team of Olivier FIRMESSE and Mathilde BONIS (Anses, Maisons-Alfort - France) et the bacteriology laboratory of Raymond RUIMY (CHU de l'Archet II, Nice - France).

We have shown in Drosophila and mice that group Bc spores persist in the gut for up to 10 days (Hachfi S. et al., 2023. Ingestion of Bacillus cereus spores dampens the immune response to favor bacterial persistence. bioRxiv. https://biorxiv.org/cgi/content/short/2023.03.16.532769v1). In addition, we show in both models that ingestion of spores, unlike vegetative bacteria, does not trigger a defense response in the anterior part of the intestine (the site of a strong and effective immune response). As a result, the spores reach the posterior part, where the immune response is limited, and germinate. This biological process is found in Drosophila and mice, showing a strong conservation of the intestinal response to spore ingestion.

We are continuing this study by analysing the nature of the intestinal innate immune response and the host defense mechanisms. To this end, we performed a transcriptomic analysis at different times (from 4 hours to 10 days) after ingestion (96 samples; in collaboration with the bioinformatics platform of our institute and the France Génomique platform in Sophia Antipolis). We are continuing to validate a number of potentially interesting genes/signaling pathways and to analyse their functions in the immune response set up after germination.

I.3. Mode of action of Cry toxins and receptors in non-target organisms

The team observed that Bt insecticides significantly increased the number of enteroendocrine cells (EEC) to the detriment of the appearance of new enterocytes (EC) (Jneid R. et al., 2023. Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila. eLife). The strength and duration of cell adhesion, in particular through Cadherin-dependent adherens junctions, between intestinal stem cells (ISC) and progenitor cells is important in defining the cellular fate of progenitor cells. Our results indicate that Cry1A toxins disrupt the intercellular adhesion between ISC and progenitors, leading to insufficient activation of the Notch signalling pathway and causing progenitors to differentiate into EEC rather than EC. We now aim to identify the intestinal proteins targeted by Cry1A toxins (immunoprecipitation and mass spectrometry). Once the target proteins have been identified, we will validate their functional involvement in vivo using Drosophila genetic tools (mutant, RNAi, overexpression), which will help us to understand the damaging mode of action of Cry1A toxins.

II – Bt spores and intestinal pathophysiology

In collaboration l’with the "Mucosal microbiota in chronic inflammatory diseases" team of Benoit CHASSAING (Institut Cochin, Paris - France)

The impact of chronic ingestion of Bt containing foods on adult non-target organisms has not been studied. Our team has developed a protocol in which animals are exposed to a dose in their diet equivalent to the amount of Bt spores applied in the field. Using these rearing conditions, we aim to assess the effects of Bt ingestion on gut physiology and the development of 1) inflammatory (Crohn's disease, ulcerative colitis), 2) metabolic (metabolic syndrome) and 3) tumour (colon cancer) pathologies. These experiments were carried out in healthy adults. However, chronic ingestion of Bt, even at low doses, could be a factor that increases the risk of developing intestinal pathophysiology in vulnerable individuals. Conditions of genetic predisposition (inflammation, tumours), immaturity (young individuals) and ageing (older individuals) are also being tested.

II.1. Inflammatory diseases

Here we assess the effects of chronic Bt consumption in healthy adult flies using longevity assays and markers of gut structure/physiology/aging. In particular, we will analyse cellular alterations in the gut (permeability, oxidative damage, dysplasia, inflammation), which are recurrent symptoms of inflammatory bowel disease (IBD). Next, we will assess the impact of Bt products on individuals at risk (predisposition in inflammatory pathways). Finally, the role of the intestinal microbiota will be assessed.

To determine the cellular responses induced, we carried out an analysis of the intestinal transcriptome. The project as a whole will therefore allow us to investigate whether chronic ingestion of Bt containing foods can promote the onset or development of IBD, a question that has not been addressed in any other study to date.

II.2. Metabolic diseases

The increase in the number of peptide hormone-secreting EEC by Cry1A toxins (Jneid R. et al., 2023. Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila. eLife) could have deleterious consequences for the maintenance of intestinal cellular homeostasis, feeding behaviour and metabolism, especially in individuals predisposed to the development of intestinal pathophysiologies. We intend to continue this study by measuring cellular bioenergetics (sugars, lipids, respiration, etc.) to assess the metabolic parameters affected by Bt consumption. In addition, the tools available in Drosophila will allow us to analyse different processes such as ISC proliferation, progenitor mis-differentiation, dysplasia, aging and feeding behaviour. This work will allow us to identify the factors that are produced in excess (or in deficiency) by the excess number of EECs and that are involved in the appearance of symptoms associated with Bt ingestion.

II.3. Tumor diseases

The pro-inflammatory state induced by Bt ingestion could promote the development of diseases such as cancer. The aim of this part of the project is to investigate the role of Bt bacterial intoxication in the development of intestinal cancer in individuals at risk.

III - Intestinal homeostasis

In collaboration with the "Chromatine et Biologie Cellulaire - Dynamique du génome" team of Giacomo CAVALLI (IGH, Montpellier - France) and the "Stem cell immunity and vibrio pathogenesis" team of Edan FOLEY (University of Alberta, Canada)

The Polycomb group (PcG) family is involved in the negative regulation of gene expression and includes the PRC1 and PRC2 ("Polycomb Repressive Complex") complexes. PRC1 generally acts in conjunction with the PRC2 complex to cause chromatin compaction at gene promoters. Because of their role in chromatin remodelling, PcG proteins are important for the development and homeostasis of many tissues, particularly stem cells. For example, Bmi1 of the PRC1 complex is involved in stem cell renewal and the development of human cancers. PRC1 has recently been shown to be critical for the renewal of ISC in mice. Our aim is to characterise the role of PRC1 and PRC2 in the Drosophila intestine. Important questions to be addressed are: Do PRC1/PRC2 play a role in ISC, and if so, what role? Do PRC1/PRC2 play a role in other cell types (EC lineage, EE lineage)? Which members of the PRC1/PRC2 complexes are involved? What are the target genes of PRC1/PRC2?

Taken together, these results will provide a better understanding of the epigenetic mechanisms that control the proliferation and differentiation of ISC in Drosophila. They should also allow us to draw parallels with the mammalian PRC1 complex, which maintains the undifferentiated state of ISC.

ISC = intestinal stem cell; EB = enteroblast; EC = enterocyte; EEP = enteroendocrine precursor; EEC = enteroendocrine cell; Classes I and II: hormone peptides.

Conclusion

The work of the team as a whole will enable us to assess the responses to ingestion of spores from the Bc group in the gut, to elucidate the underlying cellular and molecular mechanisms, and to analyse the consequences for the whole organism. It will also increase our fundamental knowledge of the relationship between spore-forming allochthonous bacteria, which are widely distributed in the environment, intestinal physiology and health. The Drosophila model will allow us to assess the impact of Bt insecticides on the health of insects, which account for 85% of animal biodiversity and whose importance for plant health (and therefore the environment) is well known.