Interactions Between the Outer Membrane and the Peptidoglycan in Brucella abortus

Abstract

The Gram-negative bacteria have an envelope made of three layers. The inner membrane (IM) and the outer membrane (OM) delimit the periplasmic space where the peptidoglycan (PG) is found. Escherichia coli, the Gram-negative model bacteria, has an abundant lipoprotein called Lpp, or Braun’s lipoprotein, embedded in the inner leaflet of the OM. Lpp is covalently bound to the PG through the amino group of its C-terminal lysine. This linkage is established by enzymes called L,D-transpeptidases (LDts). Brucella abortus is a mainly intracellular pathogen and is the etiological agent of bovine brucellosis, a worldwide neglected zoonosis.
In B. abortus, there is no homolog for Lpp nor any known structure
establishing a covalent link between the OM and the PG.
In this thesis, we first aimed to identify OM proteins (OMPs) that would be covalently linked to the PG. Mass spectrometry analysis proved that, in Brucella, the PG is anchored via several β-barrel proteins. While these OMPs have no C-terminal lysine, the link occurs between the N-termini of these proteins and the PG. We also showed that the aspartate 2 has a crucial role in the anchoring, as its punctual mutation to alanine completely abolished the linkage of the two major OMPs to PG. Doing so, we showed an OM instability in the mutant, highlighting the physiological relevance of this system. Finally, we have also shown that the β-barrel anchoring system is conserved in Agrobacterium tumefaciens and bioinformatic analysis suggests that it could also be broadly found in Rhizobiales.
In a second time, we focused on the eight putative LDts encoded in the B. abortus genome to identify those involved in the anchorage of OMPs to the PG. We established a heterologous E. coli system where co-expression of one OMP (Omp25) and one LDt of B. abortus lead us to identify three LDts able to anchor B. abortus OMP to the PG of E. coli. The role of one of these LDts (Ldt4) was further confirmed in Brucella. We also investigated the remaining LDts and found clues that one of them (Ldt7) is likely to be involved in the PG remodelling.
During this study, we identified an additional putative LDt (BAB1_2034) by homology with A. tumefaciens. Surprisingly, BAB1_2034 deletion lead to the opposite expected phenotype as more OMPs were attached to PG. Conversely, over-expression of the protein decreased the amount of OMPs linked to PG. Moreover, infection of murine macrophages with the deletion and the over-expression strains lead to a virulence defect at 24h post-infection. This suggests that not only the anchorage has a role – direct or not – in the virulence but also that the balance between attached and detached OMPs could be important.
Overall, we characterised a new OM-PG anchorage system and we provided new insights into the mechanisms behind the maintenance of the tethering. These data could be of major significance in the understanding of the envelope structure and resistance towards stress in Rhizobiales.

Date of Award	17 Dec 2021
Original language	English
Awarding Institution	University of Namur
Sponsors	University of Namur
Supervisor	Xavier De Bolle (Supervisor), Francesco Renzi (President), Axel Cloeckaert (Jury), Jean-François Collet (Jury) & Waldemar Vollmer (Jury)