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The team is interested in the mechanisms of defense of an organism against environmental aggressors and the pathophysiological consequences when those mechanisms failed. Focusing on host-pathogen interactions, we are studying gut physiology, intestinal stem cell functions in the maintenance of cellular homeostasis and innate immunity in Drosophila melanogaster.

Background

The use of conventional "synthetic" pesticides is controversial, mainly because of their detrimental effects on both environment and human health. Public opinion, along with governmental policies, encourage reductions in their use. However, because of global population expansion, the use of pesticides will still be essential to produce enough food to feed the expected 9.5 billion humans by 2050 (www.insee.fr). An emerging solution to decrease the use of conventional pesticides is to replace them with biopesticides. Biopesticide sales currently represent only 5 to 7% of the global market of pesticide products, but the objective is to reach 50% by 2050. This objective is supported by various government incentives, for example, Plan Ecophyto 2 in France, which aims for a 50% reduction in the use of chemical pesticides by 2025. This will lead to a marked increase in the use, and therefore dispersion, of biopesticides in the environment. Such growth is expected to continue, and thus the health and environmental consequences of acute and chronic exposure to biopesticides are of utmost interest.

Bioinsecticide products based on the spore forming bacterium Bacillus thuringiensis (Bt) are widespread in both organic farming and conventional agriculture. Bt products are the main bioinsecticides with more than 50% of the global market share and they are applied to 69% of the certified organic farming cultivated areas. Moreover, Bt products are also the second most used insecticides (including chemical pesticides) in the world with 32 000 tons sold in 2015. Bt was first identified and characterized for its specific entomopathogenic properties due to the presence of crystals made of δ-endotoxins (mainly Cry toxins), produced during the sporulation of the bacteria. Upon ingestion of Bt bioinsecticides (made of spores, crystals and additives), Cry toxins are released from the crystals and destroy the insect gut epithelium. The spores germinate and, thanks to the Cry toxin-induced holes in the gut lining, invade the internal body cavity and induce septicemia. The targeted pest dies in 2 to 3 days. Each strain of Bt produces between 1 and 6 different Cry toxins. The cocktail of Cry toxins produced by Bt strains confer a specificity of action towards different phylogenetic orders (e.g. lepidopteran, coleopteran, rhabditida, etc.). Bt ssp kurstaki and Bt ssp aizawai are the most used bioinsecticides in farming and forestry owing to the specificity of their Cry toxins, which target one of the most problematic pests: lepidopteran larvae. Therefore, using Drosophila melanogaster as system model, our team is studying the putative unintended impacts of Bt spores and toxins.

Objectives

- Deciphering the innate immune response mounted by the host upon oral or systemic infection by Bacillus spores and vegetative cells

- Identifying the signaling pathways involved in the intestinal integrity maintenance

- Characterizing the developmental and pathophysiological consequences of ingesting low doses of Bt products or pathogenic bacteria

- Studying the behavior and fate of Bacillus spores, toxins and vegetative cells in the intestine

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-Transversal section of the Drosophila posterior midgut ; G-Longevity curves of adult Drosophila ; H-Cell counting of the Drosophila midgut.

Biological models

Drosophila melanogaster being a non-target insect to Bt bioinsecticides (that specifically kill lepidopteran), our team use this powerful laboratory model (https://www.sdbonline.org/sites/fly/aimain/1aahome.htm) (http://flybase.org/) to study the putative adverse effects of Bt spores and toxins on both development and adult gut physiology. We are combining genetics, developmental, cellular and molecular biology tools as well as omics and bioinformatics to detect long-term non-lethal, but potentially harmful effects that would be much more difficult to figure out by classical epidemiological or toxicological approaches. Moreover, the high conservation of the main physiological mechanisms and signaling pathways between Drosophila and vertebrate allows to rapidly transpose the results to other animals (Pasco et al., 2015; .DOI: 10.14670/HH-30.277).

Bacillus  cereus and thuringiensis are  Gram-positive sporulating bacteria belonging to the Bacillus cereus  sensu lato group. Bacillus-based products are of promising way as biocontrol agents because many Bacillus spp. display exploitable properties such as plant growth promotion, activation of plant defense as well as the production of antibiotics, antifungals and entomopathogenic toxins. Moreover, Bacillus strains are also used as probiotics for humans and animals. Finally, Bacillus spp. are endospore-forming bacteria easy to produce and suitable for long-lasting storage and transport.

Projects

1. Maintenance of intestinal cell homeostasis upon oral infection of allochthonous bacteria

The gastrointestinal tract is a vital organ involved in digestion and absorption of nutrients, as well as in defense against pathogens, toxins or chemicals ingested with food. The cellular and physiological balance (homeostasis) of the intestinal epithelium is ensured by the rapid replacement of damaged, aged or dead cells, while limiting the risk of developing pathophysiologies such as cancers. This renewal is supported by intestinal stem cells (ISC), present within the tissue itself, which adapt their proliferation to daily needs or during aggression. The rate of ISC proliferation is controlled by several signalling such as for instance JNK, Hippo, Wg/Wnt, EGFR, or JAK/STAT pathways. Due to its structural and functional similarities with the small intestine of vertebrates, the midgut of Drosophila melanogaster serves as a model for the study of intestinal homeostasis (Pasco et al., 2015). In Drosophila as in vertebrates, there are different types of cells that make up the midgut: the ISC, the precursor cells (from the asymmetric division of ISC) which themselves differentiate into enterocytes (EC) or secretory cells (Joly and Rousset, 2020).

Mimicking environmental conditions, we have shown that the ingestion of low levels of opportunistic bacteria was sufficient to launch an accelerated cellular renewal program despite the brief passage of bacteria in the gut and the absence of cell death and this is due to the moderate induction of the JNK pathway that stimulates stem cell proliferation. Consequently, the addition of new differentiated cells to the gut epithelium, without preceding cell loss, leads to enterocyte overcrowding. Finally, we show that a couple of days later, the correct density of enterocytes is promptly restored by means of a wave of apoptosis involving Hippo signaling and preferential removal of old enterocytes. (Loudhaief et al., 2017)

2. Bt and the local immune response

The immune response is essential to fight against pathogens (bacteria, viruses, fungi, etc ...). Any impact on its functioning alters its effectiveness and thus the ability of an individual to defend itself in a hostile environment.

The midgut of Drosophila (and insects in general), like the intestines of mammals, is endowed of a local immune response capable of fighting against pathogens present in the food bowl. This response is essential for the survival of an organism without which its life would be in peril systematically after food ingestion. This local defense results in the expression and secretion of antimicrobial peptides (AMP) by the enterocytes in insects or Paneth cells in mammals, which will kill the pathogen.

Bt is considered as entomopathogenic because when it penetrates inside the body of an insect (whatever it is), it causes septicemia and kills its host quickly. When Bt is ingested by a "target" (or "sensitive") organism, Cry toxins are necessary to damage the intestinal epithelium of the host. Consequently, the intestine no longer fulfills its role as a barrier and Bt enters the inside the body, spreads out via the hemolymph, proliferates and kills its host in 2-3days. In non-target organisms (such as Drosophila or human), Cry toxins can not damage the intestine either because Cry toxins are ineffective or because the intestine is repaired faster than toxins damage it. In parallel, the secretion of PAM by the enterocytes eliminates Bt. The bacterium does not penetrate inside the body and therefore does not kill its host. However, Bt is considered to be an opportunistic bacterium in mammals (Bt belongs to the group of B. cereus and B. anthracis), meaning that it can cause diseases (pneumonia and eye infections for examples) in immune-weakened individuals (immunocompromised).

3. Assessing Bc/Bt virulence

Metazoan are in contact during their lifespan to many environmental bacteria that can penetrate within their body through ingestion along with food. Opportunistic bacteria become pathogenic when the defenses of the host are diminished. Sporulating bacteria belonging to the Bacillus cereus (Bc) group are environmental widespread opportunistic agents involved both in foodborne outbreaks (2^nd cause in France) and sporadically in nosocomial diseases such as pulmonary infection or bacteremia.

Scientific and social issues

• Scientific issues: Although the acute targets of Bt bioinsecticides and the underlying cellular/molecular mechanisms are fairly well characterized, targets and modes of action during chronic intoxication remain unexplored. Our complementary ecotoxicological and mechanistic approaches will allow to link finalized and fundamental researches. The more we understand the foundations of a problem, at best we will assess the risks to anticipate them.
• Social and economic issues: Bioinsecticides will be increasingly used and Bt currently represents the majority of the indications ("organic" farming; mosquito control, transgenic crops). Sources of contact with these bioinsecticides toxins are multiple for human (food, air, water). Anticipating the risks associated with an increasing use of Bt bioinsecticides should avoid the renewal of a posteriori disappointments encountered with the chemical pesticides. Our work will help to establish preventive measures for users and consumers and allow adapting the mode of use of Bt bioinsecticides. Our work will also allow to optimize the compositions and production of Bt bioinsecticides. As Cry toxins are also broadly used in transgenic plants, our work will help to expertise the sanitary and environmental impacts of such crops.