Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is responsible for more than 1.8 million deaths worldwide. Males are affected roughly twice as much as females by TB, both in terms of cases and deaths. It is tempting to speculate that such discrepancy between male and female susceptibility to TB relies at least in part on specific biological features. Indeed, sex differences are frequently observed in many infectious and non-infectious diseases and rely on biological, particularly immunological differences. The objective of our project is to explore the role and mechanisms of action of sex hormones in resistance vs. sensitivity to TB. To this end, we have assembled a consortium formed by the group of O. Neyrolles, (Partner 1, IPBS, Toulouse) specialist in immunity to TB, JC Guéry (Partner 2, CPTP, Toulouse) specialist in the action of steroid hormones on immunity and the group of L Balboa (Partner 3, non-eligible for funding, Buenos-Aires, part of an international laboratory with Partner 1) who provides access to samples from human TB patients. Beside its novelty, the strengths of our project are several: i) it will be developed both in humans, in which the differential sensitivity of males and females is well established but through unknown mechanisms, and in mice, in which the mechanisms of action of steroids on the immune response is the focus of intense investigation in general but not in the context of TB; ii) the models and genetic tools required for exploring the role of sexual hormones are already available in Partner 2 laboratory and can be easily adapted to the study of TB, thus securing the project’s feasibility. Based on previous work and our preliminary data, the project will address the following two questions: 1) What are the molecular mechanisms at work to explain sex differences in immune cells upon Mycobacterium tuberculosis infection? 2)To what extent the mechanisms identified for sex differences observed in mice apply to humans? These questions will be answered through experiments performed along three work packages. For WP1, we will use an unbiased approach to investigate the impact of sex and sex hormones on TB. Male and female mice either sensitive or resistant to TB will be infected with M. tuberculosis and we will analyze bacterial loads, histological and immunological parameters. To directly explore the role of steroids, those mice will be castrated and compared to sham-operated ones. Finally, we will restrict the expression of steroid receptors to the hematopoietic compartments using already available mice and generation of speed congenic when necessary. For WP2, we will employ existing murine models (already present in our laboratory or commercially available) on the C57BL/6 background, which display selective ablation of steroid receptors on chosen cellular compartments. Based on the literature on TB and our preliminar results we hypothesize that the following cell compartments will be key to analyze: 1) macrophages/dendritic cells, 2) T cells and, 3) innate-lymphoid cells. Here too, we will analyze bacterial loads, histological and immunological parameters after infection with M. tuberculosis. For, WP #3, we will study the impact of gender in immunity to TB in humans in two ways. First, we will have access to pleural fluid and blood samples from TB patients (and blood samples from healthy controls); second, we will analyze an in vitro assay measuring the formation of a structure induced M. tuberculosis, i.e. the granuloma using blood cells and will use of variables: the gender of blood donor, the addition of steroid or steroid receptor antagonists with the aim to measure the impact of gender/steroid on the formation of this important structure. Altogether, our project will provide knowledge on how steroid hormones impact anti-TB immunity and tissue damages and could find development beyond TB.

In collaboration with Alain Townsend's laboratory in Oxford, we have developed an extremely simple and very inexpensive serological test based on haemagglutination, which allows the detection of antibodies directed against the RBD domain of the SARS-2 virus. This test, called HAT, can be performed anywhere without any specialized equipment. The performance of this test has so far been documented in serum banks, and has shown a sensitivity greater than 90% and a specificity of 99%. The protocol has now been adapted and optimized so that HAT can be performed anywhere, on capillary blood drawn from the fingertip, and the central aim of this project is to validate this adapted protocol. In parallel, the possibility of using flow cytometry to extend the capabilities of HAT will be explored, in particular for the quantitative evaluation of different classes of serum antibodies reacting against the virus. Our ultimate goal is to be able to make this test available to all laboratories around the world who wish to use it and possibly to health agencies who are interested.

Bacteria have evolved a highly conserved chromosomally encoded recombination machinery, Xer, to resolve chromosome dimers. With the exception of a few species, Xer is composed of two tyrosine recombinases, XerC and XerD, which act on a specific chromosome site, dif. Diverse mobile elements harness XerC and XerD for their own benefit. Indeed, Xer was initially discovered because of its role in multicopy plasmid dimer resolution. Since then, numerous Integrative Mobile Elements exploiting Xer (IMEX) have been described. Phages and genetic islands harness Xer recombination to integrate into the dif site of one of their host chromosomes, while small genetic cassettes flanked by pseudo dif (pdif) sites harness Xer to disseminate on plasmids. IMEXs are generally associated with the evolution of pathogenic bacteria, including (in)famous human pathogens (Vibrio cholerae, Yersinia pestis, Neisseria meningitidis and gonorrhea, Acinetobacter baumannii), animal pathogens (a large panel of Vibrios) and plant pathogens (Xanthomonas campestris, Xylella fastidiosa). In particular, cholera toxin, which is responsible for the deadly pandemic diarrhea associated with the disease of the same name, is harbored in the genome of a V. cholerae IMEX, phage CTXΦ. Ecological interactions of CTXΦ with at least two other families of V. cholerae IMEX phage, VGJΦ and TLCΦ, are responsible for the constant rapid emergence of V. cholerae strains harboring new forms of the cholera toxin. The recent appearance of multidrug resistant strains of A. baumannii, a human opportunist pathogen that is responsible for nosocomial diseases, is linked the dissemination of carbapenem resistance genes by a fourth category of IMEXs, the pdif-modules. Thus, understanding how IMEXs exploit Xer recombination for their own benefit and unravelling their ecological interactions is crucial to prepare against new or re-emerging infectious diseases. Nevertheless, reports on IMEXs rarely go beyond the stage of the description of their genetic content and genomic context, due to the methodological challenges involved in probing the system from both a molecular-genetic and mechanistic viewpoint. XerC and XerD proteins cluster in two narrow closely-related phylogenetic groups and act on highly conserved dif sites, which denotes high evolutionary constraints. Remarkably, the core dimer resolution sites of multicopy plasmids exploiting Xer and the attachment sites of IMEXs significantly deviate from the target host dif site. Our preliminary data suggest that IMEXs rely on Integrative Xer recombination complex Stabilization (InXS) factors. The aim of this research program is to search for InXS factors and unravel the complex Xer recombination reactions that they promote. To this end, four teams combining a deep expertise in Xer recombination, bioinformatics, molecular-genetics, genomics, single-molecule biophysics, and structural biology join their forces to study the Xer exploitation strategies of the V. cholerae CTXΦ, VGJΦ and TLCΦ IMEXs and the A. baumannii pdif-modules, which represent the four categories of IMEXs so far described. The results of the InXS project will interest academic research scientists studying protein-DNA transactions and the microbiologist community in general. In addition, the results will be made available in an online database, thereby providing a new tool for the surveillance of pathogenic bacteria. The acquired knowledge could provide opportunities to take advantage and/or control of these 'natural genetic engineers'. In particular, we foresee the development of methodologies to enforce the excision of IMEXs that encode antibiotic resistance elements and/or toxins, which could serve as the basis of new prophylactic medical treatments.

Gram-negative bacteria represent a major public health concern due to their high resistance to antibiotics resulting in millions of human deaths world-wide each year. Their multilayered envelope contains an outer membrane (OM) that forms an effective permeability barrier shielding against noxious molecules, including several antibiotics. Being exposed to the cell surface, the OM represents a promising target for the development of new antimicrobials that can act from the exterior of the cell. The design of new antimicrobial stategies urges a better understanding of the molecular pathways of OM biogenesis. Integral OM proteins are crucial for envelope homeostasis. The beta-barrel assembly machinery (BAM) plays an essential role in OM protein assembly. The activity of BAM is regulated in space and time ensuring the constant supply of protein components to active sites of OM biogenesis. Many questions remain unresolved concerning the protein folding reaction mediated by BAM and the regulation of its activity throughout the OM. Motivated by the need to better understand the biogenesis of the bacterial OM, we have discovered that in the enterobacterium Escherichia coli, a member of gamma-proteobacteria, the lipoprotein DolP associates with the BAM complex and plays a critical role in OM homeostasis and integrity. DolP is widely conserved in gamma-, beta- and some alpha-proteobacteria contributing to the virulence of several pathogens, as well as to their ability to survive in the presence of some antibiotics. Our preliminary data reveal that DolP directly interacts with BamA, the catalytic subunit of BAM, promoting BamA folding and function. Inactivation of DolP phenocopies BamA depletion and makes cells sensitive to antibiotics that are normally excluded by Gram-negative bacteria. DolP localizes at active sites of OM biogenesis, ideally positioned to support BAM activity. The molecular mechanisms by which DolP contributes to OM assembly by the BAM complex and ensures OM integrity remain to be established. Our project uses an interdisciplinary approach to determine how DolP interacts with the BAM complex, influences its organization with partner complexes and regulatory factors, and supports its OM protein assembly activity. By employing a multiscale experimental strategy, we are investigating the molecular processes mediated by DolP i) at the cell envelope-wide scale, ii) in a chemically defined in vitro system, and iii) at the structural level. We will conduct these studies in the enterobacterial model organism E. coli and test our results in other pathogens of the gamma- and beta-proteobacterial classes. Our results will be important for the research of new antibacterial compounds that can interfere with OM integrity in Gram-negative bacterial pathogens.