The GME team studies the pathogenic and adaptive properties of the spore-forming bacteria of the Bacillus cereus sensu lato group, as well as Clostridioides difficile. To reach our objectives we rely on molecular genetics and biochemistry approaches combined with single-cell imaging technologies and the use of the versatile infection model Galleria mellonella. As a direct result of our fundamental research, biotechnological applications in the fields of human health, food safety and crop protection also form a significant part of the GME team’s activities.
B. cereus sensu lato group comprises taxonomically closely related spore-forming bacteria capable of colonizing hosts as diverse as mammals and insects. We study in particular Bacillus thuringiensis (Bt), an entomopathogenic species used as a biopesticide due to its production of Cry insecticidal proteins, and B. cereus sensu stricto (Bc), a species responsible for human food poisoning. The ability of these bacteria to form biofilms and highly-adherent spores, which are widely distributed in the environment, leads to recurrent contamination of foodstuffs and equipment used by the agri-food industry. These bacteria are also human opportunistic pathogens that can cause local or systemic nosocomial infections (endocarditis, pneumonia, septicemia, endophthalmitis). In the frame of insect as a new source for feed and food, the insect health and the microbial safety of insect-based productshave been investigated with particular focus on the impact and avoidance of Bt and Bc.
C. difficile (Cd) is an anaerobic and spore-forming entero-pathogen that is able to jump across host species and represents a One Health problem. This opportunist behavior is controlled by the host microbiota: it can be eliminated or asymptomatically carried in a healthy host, but, after an antibiotic treatment, microbiota dysbiosis favors Cd emergence as a pathogen. Its secreted toxins cause diarrhea that are difficult to treat and recurrent. Like those of many pathogens, Cd biofilms could be involved in antibio-tolerance, persistence and infection recurrence.
Bt, Bc and Cd can trigger various developmental pathways that enable them to persist in their niche. In particular, these bacteria can produce biofilms, form spores or enter a state of non-sporulated persistence. These processes are activated when bacteria encounter unfavorable environmental conditions during infection or outside the host.
The different stages of the Bt/Bc infectious cycle (virulence, necrotrophism and sporulation) are controlled by cell-cell communication or quorum-sensing (QS) systems that enable bacteria to coordinate gene expression in response to cell density and environmental stimuli (doi: 10.3390/toxins6082239). Our research has led to the characterization of the RRNPP family of quorum sensors, initially comprising Rap, NprR, PlcR and PrgX (doi: 10.1073/pnas.0704501104). Work is currently underway to study how QS functions in vivo at the single cell level. We are also characterizing molecules that can inhibit QS (via quorum-quenching) and thus reduce bacterial virulence or sporulation in collaboration with Pr Z. Hayouka (The Hebrew University of Jerusalem). In addition, the ecological role of QS systems will be investigated using evolutionary biology approaches in collaboration with Pr. B. Raymond (University of Exeter).
Although quorum-sensing controls different stages of the Bt/Bc infectious cycle, not all bacteria engage in each process, resulting in phenotypic heterogeneity, in biofilms as well as during infection. In addition to spores, considered for many years to be the only mode of survival for these bacteria, a non-sporulating subpopulation capable of persisting through the late stages of infection of G. mellonella was identified (doi: 10.1128/mbio.00371-23). We are interested in characterizing the genetic basis of this state, and its importance for the fitness of the bacteria, using global and targeted approaches (Tn-Seq and single-cell gene expression analysis) combined with genetics methods. This study extends to other members of the B. cereus group such as emetic and clinical Bc, as well as Bacillus anthracis and Bc biovar anthracis, to determine the importance of non-spore-forming forms during infection for these closely related bacteria that present different life cycles.
Canonical Bt strains produce insecticidal parasporal crystals during sporulation in the same cell as the spore. A few rare strains however, can differentiate into two distinct subpopulations of spore-formers and crystal-producers and we showed that this division-of-labor phenotype provides the bacterium with a fitness advantage in competition with a canonical Bt strain (doi: 10.1038/ismej.2014.122). The CpcR transcription regulator was characterized as being responsible for this phenotype (doi: 10.1111/mmi.14439). Ongoing work aims at understanding the mechanisms underlying this phenotypic heterogeneity. This work illustrates the diversity of strategies employed by these bacteria to control their sporulation and enable their dissemination in various ecological niches. These studies were performed in collaboration with the laboratory of Pr. F. Song (IPP CAAS Beijing) and contributed to the creation of the International Associated Laboratory focused on bacteria-plant-insect interactions for disease biocontrol (LIA BIPI).
A major component of the biofilm is the matrix, a gel-like substance produced by bacteria themselves and in which they are embedded. In Bt, we have shown that the biofilm matrix is made up of two proteins (TasA and CalY) and two exopolysaccharides (Cps and Eps). In addition to their structural role in matrix biosynthesis, CalY and Cps are also involved in the bacterium pathogenicity. CalY can function as an adhesin, and Cps forms a capsule surrounding the bacterium and able to bind inert or living surfaces. Eps and Cps, are distributed very heterogeneously in the biofilm. Cps is localized only at the periphery, in contact with solid surfaces, while Eps is localized only at the center. We would like to understand how the switch to Cps-only or Eps-only production is regulated.
The persistence of the biofilm (adhesion, protection against external agents) and the resistance of the spore present in the biofilms make Bc a serious problem in the agri-food industry. We are particularly interested in determining the influence of factors met in production units (medium, pH, temperature, presence of other organisms) on biofilm sporulation.
We are investigating the formation and structure of Cd biofilms and their role in the infection cycle. First, in a model human strain, biofilm was found to be heterogeneous with cell aggregates and to under-express toxin genes (doi: 10.3389/fmicb.2018.02084). Biofilm architecture and cell morphology under different conditions is analysed, together with biofilm induction by antimicrobial peptides in collaboration with the Micalis BaPS team and the Institut Pasteur. Second, in collaboration with ANSES, undomesticated Cd strains were collected from Equidae and characterized for their molecular diversity and phenotypes, while animals were diagnosed as infected or carriers. As a PhD project, the equine strain biofilms are being studied for their biomass, architecture, antibio-tolerance, production of toxins and spores. In parallel, with CNR Cd and CHUs, the genomes of equine and clinical strains of the same region are being compared to monitor intra- and inter-species transmission. This will help understanding the contribution of Cd biofilms to persistence, virulence and spread, in a One Health perspective.
Iron is an essential element for all organisms. However, iron is not free and is bound to different molecules in the host (ferritin, transferrin, hemeprotein) and in the pathogen (bacterioferritin, heme, etc). Pathogenic bacteria express factors to acquire iron from the host proteins. In Bc, the surface protein IlsA and the siderophore Bacillibactin are important for iron acquisition from host ferritin and for full insect virulence and are expressed in iron depleted condition (doi: 10.1371/journal.ppat.1003935). Using various approaches, we have been interested in determining the bacterial iron status, notably during insect gut infection. Laser-assisted microdissection on G. mellonella whole larva cryo-sections was used to collect bacteria in situ for mRNA extraction. Targeted qPCR showed that genes involved in iron homeostasis and several virulence genes and regulators were expressed roughly at the same level in the early (3 h) and late (16 h) infection steps of the gut colonization indicating that the analyzed genes are tightly regulated in the gut environment. (doi: 10.1080/21505594.2021.1959790).
Insects possess effective mechanisms that enable them to detect and neutralize microbial infections. We are interested in characterizing the mechanisms of resistance of Bt/Bc to the humoral or cellular components of host innate immunity (especially antimicrobial peptides) and the host immune and physiological responses activated at the level of the epithelial barrier in G. mellonella and in Drosophila melanogaster during infection (in collaboration with the University Saint Joseph in Lebanon). We found that, in addition to its role in the resistance to cationic antimicrobial peptides (CAMPs), the dltXABCD operon of Gram-positive bacteria, which is required for the incorporation of D-alanine esters into cell wall-associated teichoic acids, limits the release of immunostimulatory peptidoglycan fragments and their subsequent detection by the receptors of the host’s innate immune system (doi: 10.3389/fmicb.2020.611220). We have also identified a new bacterial factor (FliK) involved in the resistance of Bt to the humoral or cellular components of Drosophila innate immunity and in the modulation of its immune response (doi: 10.3389/fmicb.2020.611220 and doi: 10.1016/j.resmic.2023.104089). This work could lead to the development of new insect control strategies aimed at improving the effectiveness of biocontrol agents by reducing host immune competence, as well as search for new therapeutic targets as an alternative to antibiotics.
The B. cereus group includes closely related species that produce virulence factors, such as enterotoxins, hemolysins, phospholipases, proteases and adhesins (doi: 10.1128/microbiolspec.GPP3-0032-2018). The master virulence regulator PlcR, which belongs to the RRNPP family of quorum sensors, controls the expression of most of these factors. However, while some strains are responsible for food-borne gastroenteritis, there is no correlation between the presence and expression level of virulence genes and the pathogenicity of strains. Distinguishing enteropathogenic strains is challenging due to the absence of an available animal model. Our objective is to investigate the regulation of virulence factor production in diarrheal versus environmental strains under conditions similar to those found in the intestine. A better understanding of the regulation of Bc pathogenicity will enable the identification of specific genetic determinants that differentiate enteropathogenic strains from the others.
Our group has identified a new actor of the virulence of Bt: the transcriptional regulator VipR (doi: 10.1128/microbiolspec.GPP3-0032-2018). This regulator is autoregulated and controls the expression of the exported insecticidal protein Vip3A, a toxin highly active towards the insect Spodoptera frugiperda, a devastating crop pest. In addition to Vip3a, VipR activates several other genes at the beginning of the stationary phase, including amidase and Cry toxin genes. Together, these results lead to reconsider the regulation and role of Cry toxins during infection, that 40 years of study had definitively classified as proteins specifically produced during sporulation to form spore-associated crystals. We are currently investigating the role of this regulator and of the amidases, as well as the potentially unexpected functions of the early expressed insecticidal toxins during the infectious cycle of the bacteria in an insect model. Part of this work will be conducted in the frame of the LIA BIPI focused on bacteria-plant-insect interactions for disease biocontrol.
Most emetic strains of Bc harbor a megaplasmid called pCER270. This plasmid carries the ces locus involved in the production of the emetic toxin, in addition to numerous genes of unknown function or coding for regulators. We have shown that pCER270 is involved in phenotypes specific to emetic strains, such as high spore resistance to heat or the formation of atypical biofilms, and we hypothesize that these specific phenotypes make the emetic strains well-adapted to human food and therefore contribute to their pathogenicity. As a consequence, transfer of pCER270 to non-pathogenic strains could result in the emergence of new pathogens. Although pCER270 is not a conjugative plasmid, we succeeded in transferring it to Bt and B. weihenstephanensis by a conduction process and found that transfer of the plasmid impacted the transcriptome of the new host (doi: 10.1016/j.resmic.2023.104074). We also found that pCER270 impacts both sporulation and biofilm formation and we identified pCER270-encoded regulators involved in the pCER270-induced phenotypes. We are currently determining the effect of pCER270 on the fitness of its host in various environments.
Some insects are considered to be a new sustainable source of protein for human and animal consumption among which are the mealworm Tenebrio molitor and the Black Soldier Fly larvae, Hermetia illucens. Galleria mellonella is included as a model to study interactions of microbiota, pathogen and the host responses. Our research is dealing with two main aspects: 1) the microbial safety of insect-based food/ feed, and 2)the role of gut microbiota and feeding substrate quality in insect resistance against pathogens.
With respect to “novel” food/feed sources like insects, research related to the microbial safety is needed (doi: 10.3920/JIFF2014.0022). The feeding substrates of insects reared for feed and food are often various food leftovers potentially rich in microbes. Especially, the larvae of Black soldier fly H. illucens can consume waste from households and manure making this species particularly interesting when looking at insects from circular economy and sustainable food-chain perspectives. Safety issues are both on potential risk of specific entomopathogens like Bt or opportunistic human pathogens (Serratia, Bc and Cd) to infect and persist in the insect and the rearing environment. In the FLY4WASTE project of the INRAE metaprogram Better, we tested the putative barrier effect of entomoconversion on Bt or Cd. After initial contamination of the larvae feeding substrate by spores of Bt or Cd, spore counts were monitored as a function of time in both the substrate and the insect larvae without revealing any decrease. Our work is also dealing with detection methods. We recently determined the detection threshold of Bc and Cd in different waste and identified other aerobic and anaerobic pathogen species. Results from an INRA/CIRAD GloFood metaprogram study with the meal worm T. molitor indicated the capacity of S. marcescens to persist in the live insect and in the frass for more than two weeks after removal. A specific growth medium proved to be the best way to detect the pathogen (doi: 10.3390/insects13050458).
The gut microbiota is increasingly emerging as a decisive factor to keep insects healthy in mass rearing environments. However, we lack knowledge about the molecular and cellular mechanisms involved in the interaction between the host, its microbiota and a pathogen during an infectious process and their contribution to the overall maintenance of the health of reared insects. Our aim is to help solve the problems associated with infectious diseases in mass rearing of insects in order to mitigate and prevent the occurrence of epidemics. More specifically, we want to understand how the commensal or dietary microbiota of insects affects their resistance and/or resilience to infection and stress, and to analyze how and to what extent the host’s immune response can be mediated by the microbiota. The topic has been part of the H2020 ITN (2019-2024) project Insect Doctors (https://www.insectdoctors.eu/en/insectdoctors.htm). Main results from one of the two PhD projects supervised by the GME team show that the addition of a probiotic strain to the feed of T. molitor decreased its susceptibility to Bt and to a fungal pathogen, and only little impact was found on the gut microbiota composition. Furthermore, the addition of egg-white increased the insect resistance to pathogens (doi: 10.3389/finsc.2024.1334526). We have also examined the interactions between the gut microbiota, immune response, and host susceptibility to various pathogens in G. mellonella larvae. We found that larvae maintaining Enterococci symbionts were less susceptible to oral Bt infections than their axenic counterparts. These Enterococci species exerted a marked influence on the basal gene expression linked to immunity, emphasizing the gut microbiota’s pivotal role in stimulating G. mellonella‘s immune response (doi: 10.3389/finsc.2023.1260333). This work could lead to new strategies to improve the control of feed-borne pathogens in mass rearing insect facilities by making insects more resistant.
In order to propose an alternative to chemical pesticides, we have developed a new type of biopesticide strains for use against insect pests, such as crop pests and disease vectors (BIOSAFE, MOSKO, BT-VIP). With financial support from SATT Paris-Saclay and INRAE, these strains were developed using the results generated by our work on stationary-phase regulators such as CpcR and VipR, mentioned above, as well as patents (EP17305011 and PCT/EP2023/061003).
Bt is largely used in crop protection because of its massive production of insecticidal Cry proteins, stored in large crystalline parasporal bodies. We have used Bt molecular tools to create a new platform for heterologous proteins production and in vivo-crystallization. Using GFP as a proof-of-concept for our platform, we found that this protein was stored in fluorescent crystals and represented up to 60% of the total bacterial protein content. We are currently improving the process to make it usable with a large range of proteins. The outcomes of this project are multiple. In vivo crystallization will facilitate the 3D-protein structure determination by avoiding the long and tedious in vitro protein crystallization process. In addition, proteins in crystals are stable, and display low intramolecular motion, thus allowing the production of unstable or cytotoxic proteins. Finally, crystals can be used as a drug-delivery vehicle for slow release of pharmaceuticals. This technology is based on patented results (PCT/EP2023/061003 and PCT/EP2023/061001).
Although most strains of Bc are innocuous and largely distributed in the environment, a number of strains are involved in food poisoning. Indeed, Bc is one of the most frequently isolated microorganisms in foodborne outbreaks. However, molecular markers are available only for emetic strains producing the cereulide toxin or for strains carrying the cytK1 gene coding for cytotoxin K1, which both make less than 1/3 of the food poisoning cases. All the other virulence factors are found in environmental as well as in pathogenic strains. As a consequence, almost all isolates inducing diarrheal symptoms cannot be currently detected or identified. We hypothesize that the amount of toxins produced, rather than the presence of a specific virulence factor, is the one of the critical factors responsible for the pathogenicity of diarrheal strains. We are therefore engaged in setting up immune-enzymatic assays in order to quantify the production of virulence factors by suspicious strains of Bc. We already successfully developed two ELISA assays, one directed against the phospholipase sphingomyelinase, the second against the hemolysin HlyII.
The larval stages of the lepidopteran insect G. mellonella are increasingly used in many laboratories as an infection model for bacterial and fungal pathogens. As it can be used for preclinical studies, it meets the 3R concept (reduce, replace, optimize) in order to limit the use of mammal models, notably for screening novel antibiotics but also for pathogen-host interaction studies. This insect has been reared in our insectarium for many years and has been used to identify genes involved in virulence, adaptation and survival and to visualize their expression in situ, particularly for Bc and Bt (see results in Axes 1, 2 and 3). Host-pathogen-microbiota studies were also undertaken as we are able to rear axenic larvae and conduct infection by oral force-feeding. We produce and sell last instars G. mellonella to several scientific laboratories in France. We set up collaborations to notably study bacteria-digestive tract interactions with respect to the role of gut microbiota, and the peritrophic matrix, using dissection and histology approaches. For instance, by using axenic larvae we showed that the gut microbiota played no role in the growth of G. mellonella on its natural feed (bees wax) and that the larva was not able to assimilate polyethylene plastic (PE) as shown in collaboration with Synchrotron using infrared microscopy FTIR on sections of whole larva fed with labelled PE (see figure) (doi: 10.1021/acs.est.1c03417).
Selected publications since 2018. For an exhaustive list since the creation of the Micalis UMR in 2010, click here
