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Updated: Sep 27, 2026
Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Nramp1 is not a major determinant in the control of Brucella melitensis infection in mice
Laurence A Guilloteau1, Jacques Dornand, Antoine Gross
1Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, Nouzilly, France. Laurence.Guilloteau@tours.inra.fr
Abstract:
Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1(s)) and C.CB congenic mice (Nramp1(r)) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by BRUCELLA: However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-alpha), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-gamma), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-gamma and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-alpha mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene appears to be of limited importance for the natural resistance of mice to Brucella.
Insights
The Nramp1 gene influences early mouse susceptibility to Brucella melitensis infection, but its role in long-term resistance is limited. This suggests other immune factors are more critical for controlling Brucella.
Area of Science:
- Immunology
- Microbiology
- Genetics
Background:
- Brucella species cause brucellosis, infecting humans and animals by proliferating within macrophages.
- The Nramp1 gene is crucial for macrophage-mediated resistance against various pathogens.
- Understanding Nramp1's role in Brucella infection is vital for developing control strategies.
Purpose of the Study:
- To investigate the role of the Nramp1 gene in controlling Brucella melitensis infection in mice.
- To determine if Nramp1 expression affects Brucella replication within macrophages.
- To analyze the immune response, including cytokine and chemokine expression, in relation to Nramp1 function during infection.
Main Methods:
- Comparative infection of BALB/c (Nramp1(s)) and C.CB congenic (Nramp1(r)) mice with Brucella melitensis.
- Quantification of Brucella organisms in spleens at different time points post-infection.
- In vitro replication assays of Brucella in macrophages and RAW264 transfectants.
- Analysis of inflammatory responses, including splenomegaly and phagocyte recruitment.
- Gene expression analysis using Reverse Transcription-PCR (RT-PCR) for various immune mediators.
Main Results:
- C.CB mice (Nramp1(r)) exhibited higher Brucella loads in spleens during the first week post-infection compared to BALB/c mice (Nramp1(s)).
- This Nramp1-linked susceptibility was temporary, with similar bacterial loads observed by two weeks post-infection.
- No significant differences in in vitro Brucella replication rates were found in macrophages from either mouse strain.
- Enhanced inflammatory responses, including splenomegaly and phagocyte recruitment, were observed in C.CB mice.
- Differential expression of chemokine KC mRNA was noted in C.CB mice, while other inflammatory markers were similarly induced or showed strain-specific dynamics over time.
Conclusions:
- Nramp1 expression contributes to early, but not sustained, resistance against Brucella melitensis infection in mice.
- The temporary susceptibility in Nramp1(r) mice may be linked to increased neutrophil recruitment.
- Unlike infections with Salmonella, Leishmania, and Mycobacterium, Nramp1 plays a limited role in the natural resistance to Brucella.
- Further research is needed to identify the primary host factors governing long-term Brucella resistance.
