CtrA-dependant cell cycle regulation of Brucella abortus in culture and during infection

PhD thesis defended by Nayla FRANCIS (Prof. Xavier DE BOLLE) - 01/12/2015
Promoters

Prof. Xavier DE BOLLE, UNamur, NARILIS, URBM (promoter)

Jury
  • Xavier De Bolle, UNamur (Promoteur),
  • Patricia Renard, UNamur (Président),
  • Sean Crosson, University of Chicago (Jury),
  • Emanuele Biondi, Aix Marseille University (Jury)
  • Jean-François Collet, UCLouvain (Jury)
Summary

Investigating the CtrA/mediated cell cycle regulation of Brucella abortus

How a conserved transcription factor is adapted to the intracellular lifestyle of a pathogen

Coordinating growth with chromosome replication and division is a basic condition for the survival of any living organism, be it unicellular or multicellular, prokaryote or eukaryote. For this coordination to occur correctly, several external and internal cues have to be sensed and integrated into the complex system that is a living cell. During evolution, bacteria have selected a conceptually simple but robust system to sense signals and respond accordingly, the twoCcomponent system (TCS). As the name implies, a TCS is composed of two actors, a sensor and an effector. The sensor, often a histidine kinase, is activated by recognizing various physical or chemical stimuli. It transmits the signal to the effector component, often a transcription factor that initiates a specific gene expression program in response to the signal sensed by the histidine kinase. In Caulobacter crescentus, a model organism for studying bacterial cell cycle and differentiation, a TCS regulating the phosphorylation status of a central transcription factor called CtrA was thoroughly studied. Together with other essential regulators, CtrA coordinates, among other processes, chromosome replication with division and polar morphogenesis. Most interestingly, this TCS and CtrA are conserved among Alphaproteobacteria members, to which C. crescentus belongs, even in those devoid of polar organelles. This class brings together bacteria with diverse lifestyles, ranging from freeCliving organisms to pathogens of plants and mammals, as well as symbionts of plants and arthropods.

During this thesis, we aimed at investigating the role of CtrA and the TCS regulating its phosphorylation in Brucella abortus, a pathogen of mammals. We performed kinase assays and showed that the PdhS sensor kinase can modulate the phosphorylation status of the response regulator DivK. CtrA being essential, we constructed a depletion strain and analysed its growth, morphology, DNA content and virulence in a HeLa cell infection model. We showed that CtrA is essential for B. abortus division and intracellular survival, but not for bacterial elongation. Indeed, in absence of CtrA, B. abortus cells continue elongating and form branched morphologies. A ChIPCseq assay allowed the mapping of CtrA binding sites on the whole genome of B. abortus and the identification of two main functional categories for CtrA potential target genes, cell cycleCrelated genes and genes involved in envelope biogenesis and homeostasis. The latter putative function of CtrA was of particular interest for us, as it distinguishes CtrA of the pathogen B. abortus from the one of the freeCliving bacterium C. crescentus.

All along this manuscript, we attempt to compare the TCS and CtrA of C. crescentus, B. abortus and Sinorhizobium meliloti, an alphaproteobacterium symbiont of legume plants. A substantial amount of data has been published during the last few years concerning the events regulating the cell cycle of S. meliloti. These data together with those obtained along this thesis give a more extensive insight of how a highly conserved phosphorylation cascade was exploited and adapted by microorganisms with different lifestyles.