Characterization of immune effector mechanisms controlling Brucella growth in a murine intranasal infection model

Abstract

Brucellosis is one of the most widespread bacterial zoonoses worldwide. Four strains of Brucella, including B. melitensis and B. abortus, can accidentally infect humans, mainly via aerosols or ingestion.
Our aim was to identify the effector mechanisms controlling the early stages of intranasal infection by Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main infected cells in the lungs. Using an unbiased approach by RNA sequencing, we identified the acod1 gene as being among the 20 most upregulated genes in murine AMs in response to a primary intranasal infection with B. melitensis. An upregulation has also been confirmed by qRT-PCR in lungs of mice infected by B. melitensis and B. abortus. Indeed, acod1-deficient C57BL/6 mice display a higher bacterial load in their lungs than wild type (WT) mice following a primary B. melitensis or B. abortus infection. However, acod1-/- mice are able to control a secondary infection as efficiently as WT mice, suggesting that acod1 does not seem to be required to establish this secondary defence.
The Acod1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite from the Krebs cycle, into itaconate. In order to better understand the mechanism of action of Acod1 contributing to the control of Brucella infection, we analysed the effect of itaconate on B. melitensis and B. abortus growth in vitro. We observed that dimethyl-itaconate (DMI) and 4-octyl-itaconate (4OI), chemically modified membrane- permeable forms of itaconate, have a dose-dependent bacteriostatic effect on B. melitensis and B. abortus in rich medium. Several studies suggest that itaconate might inhibit isocitrate lyase, an enzyme of the bacterial glyoxylate cycle, hence possibly leading to energy deprivation. Interestingly, we observed that in minimal medium, 4OI only inhibits multiplication of the wild type B. abortus, but not of the isocitrate lyase deletion mutant B. abortus ∆aceA. Hence, the aceA gene might play a role for the bacterium for growth in minimal medium, but not in rich medium. Preliminary results have shown that B. abortus ∆aceA displays a more efficient multiplication than WT B. abortus in lungs of WT mice, but not in lungs of acod1-/- mice, suggesting that the bacterial isocitrate lyase might be a target of itaconate in AMs.

Date of Award	16 Jan 2020
Original language	English
Awarding Institution	University of Namur
Supervisor	Eric MURAILLE (Supervisor) & Xavier De Bolle (Co-Supervisor)