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Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice
Georges Potemberg1,2, Aurore Demars1, Emeline Barbieux1,2
1Unité de Recherche en Biologie des Microorganismes (URBM)-Laboratoire d'Immunologie et de Microbiologie, NARILIS, University of Namur, Namur, Belgium.
Abstract:
Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B. melitensis. Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B. melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B. melitensis in RAW 264.7 macrophages in vitro. As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B. melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and β oxidation of fatty acids are no longer required in Interleukin (IL)-17RA-/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B. melitensis in vivo.
Insights
This study reveals how Brucella melitensis adapts to host environments. Gene requirements for bacterial survival differ between macrophages and lungs, influenced by the host immune status.
Area of Science:
- Microbiology
- Infectious Diseases
- Bacterial Pathogenesis
Background:
- Brucella melitensis causes brucellosis, a common zoonotic bacterial infection.
- Understanding B. melitensis survival mechanisms is crucial for controlling this disease.
Purpose of the Study:
- To identify genes essential for B. melitensis survival and growth in different host environments.
- To investigate the impact of host immune status on bacterial gene requirements.
Main Methods:
- Transposon sequencing (Tn-seq) was used to analyze gene fitness.
- B. melitensis survival was assessed in vitro (RAW 264.7 macrophages) and in vivo (mouse lungs).
- Gene requirements were compared between wild-type, IL-17RA-/-, and asthmatic mouse models.
Main Results:
- 861 genes are essential for B. melitensis growth in rich medium, and 186 for macrophage survival.
- 94 additional genes are critical for lung survival at 48 hours post-infection.
- Gene requirements varied between macrophages and lungs, with 52 genes unique to lung survival, including those for LPS biosynthesis and metabolism.
- Host immune status (e.g., IL-17RA deficiency, asthma) altered gene requirements for B. melitensis survival.
Conclusions:
- B. melitensis employs distinct genetic strategies for survival in different host compartments.
- Host immune status significantly shapes the selective pressures on B. melitensis, dictating essential genes for pathogenesis.
- These findings provide insights into bacterial adaptation and potential targets for therapeutic intervention.
