Microbial virulence and inflammatory signaling in disease (VIRINFLAM)

We are looking for the molecular bases that control the activation of the NLRP3 inflammasome in response to the CNF1 toxin and other virulence factors activating RhoGTPases.

Evidence of an immune response in animals directed against RhoGTPases-activating virulence factors has recently emerged. The major role of the NOD-Like Pyrine Receptor in sensing cellular changes triggered by RhoA GTPase-inhibiting toxins, indicates the involvement of NLR in sensing virulence factors (Xu et al 2014). Our work showed the critical role of caspase-1 and ASC in response to the CNF1 toxin produced by uropathogenic Escherichia coli, indicating the central role of inflammasomes in sensing the virulence potential of microbes (Diabate et al., 2015, Dufies et al, 2021).
More recently, our work has highlighted the molecular bases controlling the activation of the NLRP3 inflammasome in response to the CNF1 toxin and other virulence factors activating RhoGTPases. Our work has also highlighted the role of Pak1/2 kinases and phosphorylation of NLRP3 by Pak on T659 in IL-1beta secretion (Dufies et al., 2021; Dufies and Boyer, 2021).
We will now determine how this modification regulates the NLRP3 complex but also identify other regulators of this pathway responsible for the secretion of cytokine IL-1beta in the context of infection and in inflammatory diseases.

By comparing isogenic strains of Esherichia coli expressing either the wild-type form of CNF1 or the inactive catalytic mutant of CNF1, we showed that CNF1 activity is sensed by the immune system, leading to rapid elimination of bacteria during bacteremia. We also observed an increase in IL-1beta in the blood of mice infected with E. coli expressing CNF1 compared to the inactive catalytic mutant of CNF1.
We also verified that the NLRP3 inhibitor, MCC950, blocks the elimination of bacteria during bacteremia. In addition, our bacteremia results performed on NLRP3 KO mice allowed us to genetically demonstrate the importance of this inflammasome in the host response during bacteremia.
Following this observation, we used Pak1 inhibitors to block the immunity triggered by CNF1 and the elimination of bacteria during bacteremia. The aim now is to determine the immune cells involved in this phenomenon and more generally to study the involvement of the NLRP3 inflammasome in the elimination of bacteria during bacteremia and sepsis. To this end, we have set up a clinical study and a cellular test allowing us to measure the state of activation of the NLRP3 inflammasome in bacteremia patients.

When the COVID-19 outbreak started, we adapted our test. Specifically, we investigated the importance of the NLPR3 inflammasome in COVID-19 by building a patient cohort and analyzing circulating myeloid cells.

This study allowed us to highlight the involvement of non-classical monocytes and immature neutrophils in severe forms of COVID-19. Inflammasome activation is increased in nonclassical monocytes from patients with severe forms of COVID-19 because it collapses in immature neutrophils. The results obtained allowed us to develop a prognostic score with good predictive value on the outcome of COVID-19 patients (Courjon et al., 2021).

In our team, we use several experimental models ranging from invertebrate to mammalian systems to understand the molecular bases of the innate immune response during an infection. My group focuses on the importance of ubiquitination, a post-translational modification altering the stability, localization or activity of proteins in the innate immune system. In fact, ubiquitination is a reaction positively and negatively regulated by hundreds of enzymes and involved in virtually all physiological processes. Its involvement in innate immunity has been particularly well described for the regulation of the Toll/NFkB pathway. However, less is known about the role ubiquitination plays in other immune signaling pathways and in the response to infection. The nematode Caenorhabditis elegans has developed an innate immune system independent of the NFkB factor and ubiquitination appears to be an important factor in host defense against bacterial and viral infections (Garcia-Sanchez et al., 2021). Our research aims to identify and characterize conserved enzymes that modify ubiquitin and regulate innate immunity and host defense. Using RNAi-Seq analysis and fluorescent reporter-based RNAi screening, we identify regulators and effectors of the immune pathway controlling host defense in C. elegans. Using experimental mammalian systems, we aim to identify conserved immune enzymes that are also required for host defense against infection in vertebrate models.

Our team includes 8 clinical doctors specialized in infectiology, parasitology, bacteriology and virology. Drawing on their expertise in human pathogens and related diseases, we develop research at the interface between fundamental research and clinical research to dissect the mechanisms of pathogenicity and detection of pathogens by the innate immune system. This work benefits from the proximity of C3M and the Archet Hospital in Nice and allows emulation between the various research projects, all centered on the study of the interaction between pathogenic microorganisms for humans and the immune system.
With the Parasitology laboratory of the Nice University Hospital, we have identified the importance of the interaction of the leishmania parasite with adipose tissue and we are currently developing tools to study the impact of this interaction on the immune system in humans ( Schwing et al., 2019; Schwing et al., 2021). We are studying this interaction in the context of leishmaniasis and asymptomatic carriage of Leishmania infantum in the context of PHRC.
With the Bacteriology laboratory, we demonstrated the emergence of the pathogenicity of strains of Bacillus cereus and Aeromonas. Using infection models (Drosophila and mouse), we determined the role of bacterial virulence and host innate immunity in this interaction (Lotte et al., 2017; Barraud et al., 2020; Ben Khedher et al., 2021). Work on B. cereus has made it possible to highlight the emergence of B. cereus infections in premature newborns. We are continuing this study with a national recruitment of bacterial strains and a transversal project in collaboration with INRAE ​​and ANSES to determine the origin of this emergence, the role of virulence factors and the innate immune response.