PhD thesis defense in biological sciences by Charline Focant

Biogenesis of the VirB-type IV secretion system and role of histidine metabolism in copper tolerance in Brucella abortus

Candidate

Charline Focant

Promoter

Prof. Xavier De Bolle, UNamur, Department of biology, Research Unit in Biology of Microorganisms (URBM)

Jury

• Prof. Jean Celli, Larner College of Medicine, University of Vermont, USA
• Dr. Eric Cascales, LISM, CNRS/Aix-Marseille Université, France
• Prof. Laurence Van Melderen, Université Libre de Bruxelles, Belgium
• Prof. Jean-Yves Matroule, Université de Namur, Belgium
• Prof. Xavier De Bolle, Université de Namur, Belgium

Summary

The type IV secretion system (T4SS) of Brucella abortus, VirB is a major virulence factor required for the establishment of the replicative niche. This thesis investigated the localization and assembly of the VirB system in Brucella using fluorescent protein fusions. VirB3, VirB4, and VirB11 were found to localize at the new cell pole, with VirB3 and VirB4 forming subpolar ring-like structures and VirB11 localizing at the pole tip. VirB4 localization was independent of its ATPase activity but dependent on downstream virB genes, whereas VirB11 required a functional active site. Among the lytic transglycosylases studied, VirB1 was required for optimal intracellular replication and VirB4 ring formation. We also demonstrated that VirB4 and VirB11 localization are not affected by the absence of LPS O-chain and by a morphological defect. Finally, vacuolar acidification was shown to be critical for the proper localization and assembly of the VirB machinery.

The second part of this thesis explored the mechanisms underlying copper resistance in B. abortus. A genome-wide Tn-seq screen identified classical copper resistance key actors and histidine and purine biosynthesis, as key determinants of copper tolerance. We showed that, unlike copA and cueO mutants, histidine biosynthesis mutants are copper-sensitive. We isolated suppressor strains that regained copper resistance while remaining histidine auxotrophs. These suppressors likely overproduce OppA₂, a periplasmic component of an oligopeptide transporter. These strains revealed that impaired histidine biosynthesis, rather than copper sensitivity, is the main cause of the intracellular growth defect of B. abortus in J774A.1 macrophages.

Overall, this work provides the first characterization of the subcellular localization of VirB3, VirB4 and VirB11 in B. abortus. In addition, our findings reveal a connection between histidine metabolism and copper resistance. These results contribute to a deeper understanding of the factors that enable successful adaptation of B. abortus to the intracellular environment.