Smooth lipopolysaccharide biosynthesis, unipolar growth and disulfide bond formation in Brucella abortus cell envelope

Promoter

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

Jury

• Prof. Xavier De Bolle, UNamur (Promoter)
• Prof. Alessandra Polissi, Université de Milan
• Prof. Raquel Condez-Alvarez, University of Navarra
• Dr. Michaël Deghelt, UCLouvain
• Dr. Francesco Renzi, UNamur (President)

Summary

The Gram-negative cell envelope is an essential structure composed of an inner membrane (IM), a periplasmic space hosting the peptidoglycan (PG) and an outer membrane (OM), mainly containing lipopolysaccharide (LPS) in the outer leaflet. Similar to other Rhizobiales, the Gram-negative pathogen Brucella abortus grows in a unipolar manner, meaning that new envelope material is incorporated at one pole in non-divisional bacteria (i. e. the new pole) and at the constriction site in divisional bacteria. Several OM components including LPS were previously shown to be incorporated at the growth sites. Importantly, B. abortus is a “smooth” strain, meaning that it displays both rough (R-LPS) and smooth (S-LPS) at the surface, without and with the O-antigen respectively. During this thesis we first aimed to localize the proteins involved in LPS transport across the cell envelope, from the IM to the OM of Brucella abortus. We showed that the LPS incorporation sites are determined by the IM complex of the LPS transport pathway. Moreover, we identified a new class of bifunctional O-antigen ligase that is almost exclusively found in the Brucella genus.

While the general mechanisms governing unipolar growth are still unknown, several Rhizobiales growth and septation (Rgs) proteins were recently identified as required for unipolar growth in the plant-associated nitrogen-fixing Sinorhizobium meliloti. We investigated two Rgs homologs in B. abortus (RgsE and RgsS) and showed that their localization and function appeared conserved between B. abortus and S. meliloti. Overall, we proposed that similar mechanisms governing unipolar growth likely occur in the Rhizobiales order, and that expression of Rgs proteins must be tightly regulated in a spatiotemporal manner.

Finally, as disulfide bond formation (Dsb) is crucial for OM integrity and proper folding of virulence factors, we aimed to study the Dsb system in B. abortus. We identified DsbA and DsbB1 as main molecular actors and showed that Brucella Dsb system is likely involved in the biogenesis of the Type IV Secretion System (T4SS), which is required for full virulence inside the host.