Histidine biosynthesis, cell separation and copper resistance in Brucella abortus

PhD thesis defended by Agnès ROBA (Prof. Xavier DE BOLLE) - 04/02/2022

Agnès ROBA


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

  • Prof. R. Martin Roop II, East Carolina University, USA
  • Dr. Coralie Fumeaux, Université de Lausanne, Switzerland
  • Prof. Laurence Van Melderen, Université Libre de Bruxelles, Belgium
  • Prof. Géraldine Laloux, Université Catholique de Louvain, Belgium
  • Prof. Jean-Yves Matroule, President, Université de Namur, Belgium
  • Prof. Xavier De Bolle, Promoter, Université de Namur, Belgium

Brucella spp. are collectively responsible for a worldwide zoonosis called brucellosis. Inside its bovine host, Brucella abortus invades and survives inside cells. In particular, Brucella is adapted to survive in a nutrient restricted environment. Indeed, this bacterium possesses many biosynthetic pathways, that allows Brucella to make up essential building blocks on its own. Some of these biosynthetic pathways become essential during macrophage infection, suggesting either that the end product of the pathway is limited, or that it is particularly crucial for intracellular survival. In this work, we investigated the requirement of Brucella for the histidine biosynthesis pathway.

First, we found that hisB, a histidine auxotroph mutant is defective for intracellular multiplication and also surprisingly displays a chaining phenotype. The intensity of this phenotype varies with the culture medium and is exacerbated inside host cells. We investigated how those chains were generated, and found out that chaining bacteria consist of contiguous peptidoglycan, and likely result from the defective cleavage of peptidoglycan during cell separation. Suppression of the chaining phenotype revealed two essential genes with a crucial role during cell division in B. abortus, dipM and cdlP. While DipM is strictly localized at the division site, CdlP is localized at the growth pole and the division site. Depletion of dipM and cdlP results in swelling at the division site. Altogether, the unexpected chaining phenotype of the hisB mutant allowed the discovery of new actors of cell division in B. abortus.

In the second part of this work, we investigated the impact of the deletion of histidine biosynthesis genes on copper homeostasis in B. abortus. Histidine is an amino acid that has the ability to coordinate different metal ions. Accordingly, most of the predicted proteins of B. abortus that are histidine-enriched are involved in metal homeostasis or use metals as cofactors. We found that four histidine auxotroph mutants (hisA, hisB, hisC and hisD) showed both lower replication inside macrophages and increased sensitivity to copper. We also found that B. abortus does not show this increased sensitivity to copper when actors classically described as involved in copper resistance, namely cueO and copA, are deleted. Furthermore, we were able to isolate suppressors of each histidine auxotroph mutant, in which spontaneous mutations restored copper resistance. Fifteen of the 22 mutations found in suppressors occurred inside the opp operon, coding for an oligopeptide transporter. Overall, our data suggest that histidine might have a much more important role than expected for copper resistance in B. abortus.