Soutenance de thèse
Thesis defense - Salma HACHFI

Thesis defense - Salma HACHFI

21 March 2019

Visioconférence par ZOOM & A010

Salma HACHFI : "The fate and behavior of spores of the Bacillus cereus group in Drosophila and mice"

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devant le jury composé de :

Présidente du jury :                      

Christine Coustau

Rapporteurs:                    

Bruno Lemaitre
Phillipe Gérard                                

Examinateur:

Bernard Charroux  

Directeur/trice de Thèse :

Armel Gallet
Laurent Boyer

Abstract:

The Bacillus Cereus (B cereus) group consists of Gram-positive bacteria, commonly isolated from the environment. Members of this group are spore-forming bacteria commonly associated with food poisoning and represent the third cause of foodborne outbreak in Europe and the second in France. The most studied members of this group are B. anthracisB. cereus sensu stricto (Bc), and B. thuringiensis (Bt) that are well known for their pathogenic activity. Bt is characterized by the production, during its sporulation, of a crystal containing insecticidal Cry toxins. Thanks to this entomopathogenic activity, bioinsecticides based on spores and crystals of Bt are used to specifically kill lepidopteran pests. Upon ingestion of Bt bioinsecticides (spores and toxin crystals), Cry toxins are released from the crystal and destroy the insect gut epithelium allowing spores to invade the internal milieu. Then spore germination in the hemolymph leads to the pest death by septicemia in less than 3 days. However, the increasing environmental dispersion of Bt products raises the question about their potential risks for non-target animals.

Using Drosophila melanogaster (a non-target organism), it has been previously shown that Bt vegetative cells are rapidly cleared from the intestine. This elimination is due to the activation of the local innate immune response. However, few studies suggest that Bt spores can persist in human and rodent intestines. To understand why and how spores persist in the gut of non-target organisms and the implication of the innate immune response in fighting spores, I have used two powerful models: Drosophila melanogaster and mice. First, I have shown that spores can persist, after acute ingestion, up to 10 days in the Drosophila midguts and at least 5 days in mice small intestines. Then, I have shown, using a fluorescence microscopy approach on whole intestines that spores accumulated mainly in the posterior midgut of Drosophila and the posterior small intestine of mice where they could further germinate. I confirmed these data using quantitative monitoring of spores vs. vegetative bacteria accumulation in vivo in the intestines of Drosophila and mice. Interestingly, I have demonstrated that spores from many species of the cereus group behave similarly to the Bt spores. Altogether, my data suggest that, in the posterior regions of the Drosophila midgut (and the small intestine of mice), the innate immune response is inefficient to promptly eliminate spores and germinated bacteria.

In Drosophila midgut, the local innate immune response (i.e. the production of Reactive Oxygen Species (ROS) and Anti-Microbial Peptides (AMPs)) occurs in the anterior regions. I have shown that, unlike vegetative cells, spores do not trigger ROS and AMPs production. Strikingly, I have even observed that spores inhibit AMPs production. Interestingly, I have found a transcriptional induction of negative regulators (e.g. PGRP-SC1, -SC2, and -LB) of the immune signaling pathways in the posterior region of the midgut in response to spore ingestion. Then, using Drosophila genetics, I have been the first to demonstrate that both immune pathways, Imd and Toll, coexisted in the Drosophila midgut, and ultimately participated to the intestinal persistence of the spores.

Finally, I have studied the inflammatory response to spores using in vitro culture of mouse macrophages. The NLRP3 inflammasome is a key platform of this response. I have shown that Bt/Bc vegetative cells were phagocytosed by macrophages triggering the NLRP3 inflammasome activation. Conversely, although spores were still phagocytosed, they did not activate the inflammasome pathway. Moreover, spores were able to persist alive within the macrophages.

My work provides scientific data on the behavior and fate of Bt spores in the gut of non-target organisms and will help food safety authorities to implement measures to improve the use of Bt biopesticides to limit their unintended effects.

 

Keywords : Bacillus cereus group, Drosophila, mice, spores, innate immune response

Contact: changeMe@inrae.fr

Publication date: 13 September 2023