Molecular mechanisms of pathogenicity
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 chains, but it is also toxic and used as a killing agent by phagocytes. We are investigating the mechanisms of copper homeostasis in the whooping cough agent Bordetella pertussis. Unlike model Gram-negative bacteria, we found that this host-restricted pathogen has few defenses against copper other than a custom-made system for protection against both metal and oxidative stresses. Conversely, we have discovered that this bacterium has an original, tightly regulated copper acquisition system to feed its respiratory heme-copper oxidoreductase complexes. We have thus characterized a new type of TonB-dependent transporter that scavenges copper complexed with small chelating molecules and are investigating another protein of this system which appears to be a metal reductase. We have also identified a new family of ribosomally produced, post-translationally modified peptides that wehave called bufferins in B. pertussis and other pathogenic and environmental bacteria. Model bufferins contribute to the protection against copper by chelating Cu(I) and Cu(II) ions in the periplasm. In the ANR CuRiPP grant (2022-2026) we have determined the modifications and the biogenesis pathway of bufferins and performed in silico analyses of their modifying enzymes, that belong to the superfamily of multinuclear non-heme iron-dependent oxidases. We are currently investigating the mode of copper chelation by bufferins in collaboration with biophysicists.
Project leaders are :
Carine Rouanet & Nathalie Mielcarek
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.
Implication of protein-protein interactions in mycobacterial pathogenesis
Deciphering protein-protein interactions (PPIs) in pathogenic bacteria can help to understand cell physiology and elucidate host-pathogen interactions in which proteins play a crucial role. In addition, the study of PPIs can facilitate the discovery of protein function(s) through the 'guilty by association' principle.
As PPIs are key factors in the physiology and virulence of Mycobacterium tuberculosis, we are particularly interested in dissecting the PPI network in mycobacteria. For example, mycolic acid biosynthesis, which is the target of several anti-tuberculosis drugs, relies on specialised and interconnected protein complexes. Therefore, the identification and characterisation of PPIs may be an attractive approach for the development of new drugs and/or peptidomimetics capable of destabilising the formation of such complexes.
In addition, deciphering the PPI network of M. tuberculosis will identify interconnected pathways and critical steps required for mycobacterial infection, allowing a better understanding of TB pathogenesis.
