Study of the mechanisms leading to mitochondrial fragmentation and mitophagy in Brucella-infected cells and analysis of its functional consequences

PhD thesis defended by Jérémy VERBEKE (Prof. Thierry ARNOULD & Prof. Xavier DE BOLLE) - 30/03/2023

Prof. Thierry ARNOULD, UNamur, Department of biology, Laboratory of Cellular and Molecular Biology (URBC)

Prof. Xavier DE BOLLE, UNamur, Department of Biology, Research Unit in Biology of Microorganisms (URBM)

  • Thierry Arnould, URBC-NARILIS-UNamur (promoter)
  • Xavier De Bolle, URBM-NARILIS-UNamur (co-promoter)
  • Michel Jadot, URPhyM-NARILIS-UNamur (president)
  • Patsy Renard, URBC-NARILIS-UNamur
  • Suzana Salcedo, IBCP-Université de Lyon
  • Carles Canto, School of Life Sciences-Ecole polytechnique fédérale de Lausanne

Brucella species are Gram-negative facultative intracellular bacteria responsible for brucellosis, a worldwide zoonosis affecting various hosts including humans. Along the infection in most host cells, Brucella first interacts with the endosomal pathway and eventually reaches its replicative niche inside the endoplasmic reticulum (ER), in which bacteria multiply massively. Regarding the physical and functional interactions between the ER and mitochondria, as well as the major role of mitochondria in inflammation, we put our efforts and interest to study the involvement of the host mitochondria in Brucella intracellular life cycle. Mitochondria are essential organelles at the basis of a plethora of cellular functions ranging from energy production to inflammation and cell death regulation. Mitochondria therefore constitute an attractive target of choice for invading pathogens to fulfil their infectious cycle. This involves the modulation to their advantage of the dynamic aspects of mitochondrial network morphology, as well as their controlled degradation through mitophagy. Previously, our team has shown that Brucella abortus induces the fragmentation of the mitochondrial network in infected macrophages and epithelial cells (Lobet et al., 2018). The global aim of this thesis was thus to better understand the effect of Brucella on the mitochondrial population of infected cells and the crosstalk existing between them which is still poorly understood. The precise objectives of this thesis were then 1) to identify and characterise the prokaryotic and/or eukaryotic effectors by which B. abortus induces the host mitochondrial fragmentation during the late steps of infection, and 2) to analyse the functional consequence(s) of B. abortus-mediated mitochondrial fragmentation for the bacteria and/or for the host. In this work, we successfully showed that B. abortus induces mitophagy through an increase in LC3/mitochondria colocalising events, as well as an increase in the number of acidified FIS1 mitochondrial fragments at 48 h post-infection in host cells. B. abortus-induced mitophagy is accompanied by a strong mitochondrial network fragmentation which is dependent on the mitophagy receptor BNIP3L as we demonstrated that a siRNA-mediated silencing of BNIP3L prevents the mitochondrial fragmentation and mitophagy in B. abortus infected cells. Our results also show that the expression of BNIP3L induced by B. abortus relies on the activation of the hypoxia-inducible factor HIF- 1α in an oxygen-independent way. Instead, HIF-1α activation appears to be iron-dependent since FeCl2 supplementation prevents HIF-1α nuclear translocation and BNIP3L expression in B. abortus infected cells even if the origin of a putative iron starvation response in the host cells remains to be elucidated. To better understand the functional role of Brucella abortus-induced BNIP3L-mediated mitophagy, and what would be the resulting advantage for the host cell and/or the bacteria, our results show that BNIP3L silencing, as well as iron supplementation, drastically reduced the number of reinfection events at 72 h post-infection, suggesting that bacterial egress from the host cell is impaired. Future research will be needed to decipher the dynamics and the role of mitophagy and mitochondrial membranes in the last steps of Brucella abortus intracellular cycle. Altogether, those results highlight the intricate link between Brucella and the host mitochondria during the critical steps involved in Brucella trafficking, replication, and dissemination through its host.