Copper homeostasis in Bordetella pertussis
Copper is an essential micronutrient for most bacteria, serving notably as a co-factor of various enzymes and in electron transfer complexes, but it is also toxic and used as a killing agent by phagocytes. We have discovered that unlike other Gram-negative bacteria, the host-restricted pathogen Bordetella pertussis has few defenses against copper other than a custom-made system that fends off both metal and oxidative stresses. Conversely, this bacterium has acquired an original two-protein copper acquisition system that we are investigating, composed of a TonB-dependent transporter of a new subfamily and a heme-containing inner membrane protein. We have revealed a sophisticated mode of regulation of this system by copper involving a novel upstream Open Reading Frame (‘uORF’). Our results indicate that this system is involved in the provision of copper to the respiratory heme-copper oxidases. We are currently addressing the structure of the two proteins and their functions in B. pertussis.
We have also identified a new family of ribosomally produced, post-translationally modified peptides (‘RiPPs’) that we have called ‘bufferins’, in B. pertussis and other pathogenic and environmental bacteria. We have shown that model bufferins contribute to the protection against copper by chelating Cu(I) and Cu(II) ions. In the ANR CuRiPP grant we have discovered that conserved cysteines of bufferins are modified into thiooxazole by enzymes of the superfamily of multi-nuclear non-heme, iron-dependent oxidases (MNIO). In silico analyses have shown that bufferins represent a widespread family of MNIO-modified RiPPs. Our goals are to decipher the mechanisms of bufferin biogenesis, their structure and their mode of copper chelation.

Intracellular fate of respiratory pathogens within alveolar macrophages
Mycobacteria are predominantly intracellular pathogens and develop mechanisms to prevent intracellular degradation and to survive inside infected cells. Bordetella pertussis on the other side is a transient intracellular pathogen, the majority of which are killed by host cells within a couple of days.  
We recently became interested in studying molecular and cellular mechanisms involved in the degradation and/or survival of these two pathogens inside alveolar macrophages.  Once the respiratory pathogens are inhaled, they will encounter phagocytic cells which represent a first line of defense against the infection. Among them, alveolar macrophages play a key role in protection.  Xenophagy represents a highly conserved defense mechanism of eukaryotic cells involved in the clearance of invading pathogens. During this complex process, intracellular micro-organisms are targeted to the degradative lysosomal compartment. A non-canonical autophagic pathway through LC3-associated phagocytosis (LAP) also contributes to immune regulation and inflammatory responses.
A knowledge gap exists on the involvement of autophagy and/or LAP in the intracellular clearance of B. pertussis and its impact on innate immune responses. On the other hand, while the mechanisms leading to intracellular survival of M. tuberculosis are extensively studied, little is known on the intracellular fate of ancestral mycobacteria and the evolutionary mechanisms leading to intracellular persistence.
We established a novel partnership with Dr. Ghaffar Muharram (MCPI team, CIIL) to tackle this question.