Tn-seq on the pathogen Brucella abortus uncovers essential functions for culture and critical pathways for macrophages infection including pyrimidines biosynthesis

  • Jean-François Sternon

Student thesis: Doc typesDoctor of Sciences


Brucella abortus is a class III zoonotic bacterial pathogen able to survive and replicate inside host cells, including macrophages. Here we applied a highly saturating mutagenesis (Tn-seq) to identify genes necessary for growth on rich medium as well as genes required for the infection of RAW 264.7 macrophages, either for survival in the first step of intracellular trafficking, or for proliferation inside the endoplasmic reticulum. Our approach first allowed the functional annotation of conserved essential pathways for growth on plates. Time-resolved Tn-seq then enabled the discrimination between two categories of mutants, “ aggravated mutants” which display a growth defect on plates that is just amplified in the course of infection, such as the purine biosynthesis pathway, and “attenuated mutants” which show decreased infectiosity while being able to grow as the wild type strain on plates. We found that about a third the previously reported Brucella attenuated mutants are actually “aggravated mutants”. Nine genes generated increased invasion potential when mutated and all are predicted to be involved in lipopolysaccharide O chain synthesis. Besides virulence factors previously characterized such as the VirB secretion system, we identified 30 genes involved in early survival and 48 required for intracellular growth. In particular, we report the need of an intact pyrimidine nucleotides biosynthesis pathway in order for B. abortus to proliferate inside macrophages, but not for anterior infection steps or in vitro culture. Indeed, a B. abortus pyrB mutant is still able to reach the ER in HeLa cells, suggesting that its intracellular trafficking remains intact. We thus propose that hyper-saturating Tn-seq performed in a time- resolved manner is a useful strategy to identify genes required at different steps of an infection by a bacterial pathogen. In addition, we also advocate the use of our R200 analysis methodology as it strongly reduces analyses biases due to the non-requirement of anterior gene prediction and allow for deeper analyses such as essential domain mapping.
Date of Award18 Oct 2017
Original languageEnglish
Awarding Institution
  • University of Namur
SponsorsFund for Research Training in Industry and Agriculture (FRIA)
SupervisorXavier De Bolle (Supervisor), Thierry Arnould (President), JEAN-JACQUES LETESSON (Jury), Géraldine Laloux (Jury), Clayton C. Caswell (Jury) & Stephan Köhler (Jury)


  • Brucella
  • Tn-seq
  • Bacterial pathogenesis
  • infection

Attachment to an Research Institute in UNAMUR


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