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Identification and characterization of in vivo attenuated mutants of Brucella melitensis
P Lestrate1, R M Delrue, I Danese
1Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d'Immunologie et de Microbiologie, University of Namur, rue de Bruxelles 61, 5000 Namur, Belgium. Pascal.Lestrate@fundp.ac.be
Abstract:
Brucella melitensis 16M is a Gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes. Signature-tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella. A library of transposon mutants was screened in a murine infection model. Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice. The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild-type strain. The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone-like protein and proteins of unknown function. The mutants selected in this study provide new insights into the molecular basis of Brucella virulence.
Insights
Signature-tagged mutagenesis identified 18 Brucella melitensis 16M mutants with impaired virulence in mice. These mutants, affecting various cellular functions, offer new insights into the molecular basis of Brucella pathogenesis.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Brucella melitensis 16M is a Gram-negative bacterium causing goat abortions and human Malta fever.
- It is a facultative intracellular pathogen capable of surviving within host phagocytes.
Purpose of the Study:
- To identify genes essential for Brucella melitensis 16M virulence in vivo using signature-tagged mutagenesis.
- To characterize the functional roles of identified virulence genes.
Main Methods:
- Screening of a transposon mutant library (672 mutants) in a murine infection model.
- In vivo competition assays against the wild-type strain to confirm attenuation.
- Replication studies in murine macrophages and HeLa cells.
Main Results:
- 20 out of 672 mutants showed reduced recovery after 5 days in BALB/c mice.
- 18 attenuated mutants were confirmed via competition assays.
- Disrupted genes were associated with transport, metabolism, transcriptional regulation, peptidoglycan synthesis, and unknown functions.
Conclusions:
- This study identified novel genes crucial for Brucella melitensis 16M pathogenesis.
- The identified genes provide new molecular insights into Brucella virulence mechanisms.