Investigation of the surface lipoprotein export machinery of Bacteroidetes

Student thesis: Doc typesDoctor of Sciences

Abstract

This work focuses at exploring the mechanisms underlying lipoprotein surface localization in Gram-negative bacteria of the phylum Bacteroidetes using as model organism the dog commensal and human pathogen Capnocytophaga canimorsus (Cc). While unusual in most studied bacteria, this lipoprotein localization is crucial both for commensalism and pathogenicity of many Bacteroidetes.

By in silico analyses of Cc surface exposed lipoproteins, we identified an N-terminally conserved motif (QKDDE). We show that this motif is sufficient for cell surface localization when introduced in an intracellular lipoprotein and thus represents the Cc lipoprotein export signal (LES). We further demonstrate that the overall negative charge of the LES is essential for protein transport. We also determined the minimal composition for a functional LES as well as its optimal positioning. Finally, an in silico analysis performed on the lipoproteins of two other Bacteroidetes species, namely Bacteroides fragilis and Flavobacterium johnsoniae, revealed that the LES is broadly distributed among the phylum. The derived LES of each species was tested and found to be functional in Cc, indicating strong conservation of the signaling and the putative lipoprotein transport mechanisms in Bacteroidetes.

We also focused at identifying the underlying lipoprotein transport machinery. We first searched for LolA interaction partners, the periplasmic chaperone of lipoproteins, which led to the identification of several candidates. We found all of them to be involved to some extend in outer membrane biogenesis and/or to be required for growth in liquid medium. In particular, we could show that an Skp homolog is essential in Cc and that its depletion leads to early growth arrest. However, their exact function remains to be clarified.
In parallel, we investigated highly conserved proteins unique to Bacteroidetes, i.e. putative candidates for the lipoprotein transport machinery. We found that most Bacteroidetes genomes encode more than one BamA homolog. This additional copy of BamA (Ccan_17810) turned out to be essential in Cc and to require an N-terminal lipid anchor for its functioning. Furthermore, when expressed in E. coli, Ccan_17810 led to rapid growth arrest and formation of ghost cells. Due to the lack of efficient regulatable expression systems in Cc and despite our efforts to generate new ones, the precise function of this protein could not be determined.

Finally, we also investigated the function of surface exposed lipoproteins in Cc. Following in silico analyses, we identified and characterized a new type of iron acquisition system essential for growth of Cc in human serum. This was of particular interest due to the broad substrate specificity of the system, targeting several iron carrying proteins found in humans and other mammals, as well as its pathogen specific distribution among Bacteroidetes. Interestingly, this system was found to have the classical architecture of Sus-like systems, outer membrane anchored complexes usually devoted to polysaccharide degradation. These systems being mostly composed of surface exposed lipoproteins, this study thus showed for the first time that Sus-like systems can target other substrates than carbohydrates, in this case iron, and that surface exposed lipoproteins can be virulence factors in the phylum Bacteroidetes.
Date of Award10 Oct 2016
Original languageEnglish
Awarding Institution
  • University of Namur
SponsorsERC (European Union)
SupervisorGuy Cornelis (Supervisor), Francesco Renzi (Co-Supervisor), Xavier De Bolle (President), Johan Wouters (Jury), Jean-François Collet (Jury) & W Bitter (Jury)

Attachment to an Research Institute in UNAMUR

  • NARILIS

Keywords

  • Lipoprotein
  • Bacteroidetes
  • Surface transport
  • Capnocytophaga canimorsus
  • Surface exposed
  • Gram-negative
  • Outer membrane

Cite this

Investigation of the surface lipoprotein export machinery of Bacteroidetes
Lauber, F. (Author). 10 Oct 2016

Student thesis: Doc typesDoctor of Sciences