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Updated: Jul 19, 2026
Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host
Michel S Zygmunt1, Sue D Hagius, Joel V Walker
1UR 1282, Unité de Recherche Infectiologie Animale et Santé Publique, Institut National de la Recherche Agronomique, 3738 Nouzilly, France. zygmunt@tours.inra.fr
Abstract:
Brucella species are gram-negative bacteria which belong to alpha-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified.
Insights
Signature-tagged mutagenesis identified key Brucella virulence factors. Seven attenuated mutants, including those affecting LPS, flagellar structure, and polyphosphate synthesis, were found in goats, revealing novel determinants of Brucella melitensis pathogenesis.
Area of Science:
- Microbiology
- Bacteriology
- Pathogenesis
Background:
- Brucella species are zoonotic Gram-negative bacteria causing significant disease in livestock and humans (Malta fever).
- Understanding Brucella virulence mechanisms, particularly host cell colonization and multiplication, is crucial for disease control.
- The genetic basis of Brucella virulence remains incompletely understood.
Purpose of the Study:
- To identify novel Brucella virulence factors using a genetic screening approach.
- To elucidate the genetic determinants enabling Brucella melitensis to colonize host tissues.
Main Methods:
- Utilized signature-tagged mutagenesis (STM) to generate a library of Brucella melitensis mutants.
- Employed PCR amplification for efficient identification of tagged mutants.
- Screened 288 mutants in goats, assessing colonization of spleen, lymph nodes, and liver post-infection.
Main Results:
- Identified 7 attenuated mutants (approx. 5%) that failed to colonize goat tissues.
- Confirmed known virulence genes (lpsA, virB operon) and identified novel candidates.
- Disruptions in genes homologous to flgF (flagellar basal body) and ppk (polyphosphate synthesis) were associated with reduced virulence.
Conclusions:
- STM is an effective tool for identifying Brucella virulence factors in a relevant animal model.
- Novel virulence determinants, including those involved in flagellar structure and inorganic polyphosphate metabolism, were identified.
- This study provides new insights into the genetic basis of Brucella pathogenesis.
