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

Brucella abortus is a facultative intracellular pathogen able to traffic inside
host cells to reach a replication niche, most often inside the endoplasmic
reticulum. Several studies suggest an interplay between Brucella and
mitochondria leading to an alteration of the metabolism of mitochondria in
Brucella-infected cells, as well as a putative interaction between this
organelle and the Brucella-containing vacuole (BCV). Recently, in the host
laboratories, Brucella has been shown to trigger mitochondrial
fragmentation at 48 h post-infection. Preliminary results suggest that this
phenotype might be due to a deficit of mitochondrial fusion as the
abundance of mitofusins 1 and 2 (MFN1/2) is decreased in mitochondrial
fractions prepared from Brucella-infected RAW264.7 macrophages.
However, the molecular mechanisms (including putative Brucella effectors)
responsible for this phenotype are still unknown. In this work, we intend to
achieve four goals. (1) We will analyse the interaction between BCV and
mitochondria by performing co-immunoprecipitation. (2) We will test whether
the reduced abundance of MFN1/2 is the driver of mitochondrial
fragmentation in Brucella-infected cells by studying this phenotype in
infected cells that overexpress genes encoding these proteins. The
subcellular localization of MFN1/2 will also be analysed by super-resolution
microscopy. (3) We will next identify potential Brucella effectors involved in
the mitochondrial fragmentation by testing several types of mutants and
performing a screen without a priori using a library of B. abortus
transpositional mutants. (4) Finally, we will study the functional impact of the
mitochondrial fragmentation induced by B. abortus on host cells by
exploring ATP and mtROS production, sensitivity to apoptotic stress and
mitophagy. This research will bring a better understanding of the hostpathogen
relationship during Brucella infection, especially of its trafficking in
the host cells.