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Deletion of purE attenuates Brucella melitensis infection in mice
R M Crawford1, L Van De Verg, L Yuan
1Department of Infectious and Parasitic Diseases, Armed Forces Institute of Pathology, Washington, D.C. 20306-6000, USA.
Abstract:
We previously showed that a purE mutant (delta purE201) of Brucella melitensis 16M is attenuated for growth in cultured human monocytes (E. S. Drazek, H. H. Houng, R. M. Crawford, T. L. Hadfield, D. L. Hoover, and R. L. Warren, Infect. Immun. 63:3297-3301, 1995). To determine if this strain is attenuated in animals, we compared the growth of the delta purE201 mutant with that of strain 16M in BALB/c mice. The number of bacteria in the spleen and spleen weight peaked for both strains between 1 and 2 weeks postinfection (p.i.), though the number of delta purE201 cells was significantly less than the number of 16M cells recovered from the spleens of infected mice. During the next 6 weeks, delta purE201 was essentially eliminated from infected mice (three of five mice sterile; < 100 CFU in two of live mice at 8 weeks p.i.), whereas bacteria persisted at a high level in the spleens of 16M-infected mice (about 106 CFU per spleen). The number of bacteria in the livers and lungs of mice infected with either strain paralleled those in the spleen. Mice infected with 16M had a strong inflammatory response, developing dramatic and prolonged splenomegaly (five to eight times normal spleen weight) and producing serum interleukin-6. In contrast, mice infected with delta purE201 developed only mild, transient splenomegaly at 1 week p.i. and produced no interleukin-6 in their serum. We further characterized the host response to infection by measuring changes in immune spleen cell populations by flow cytometry. CD4- and CD8-positive lymphocytes declined by I week in both experimental groups, while MAC-1-positive cells increased. T-cell subpopulations remained low or declined further, and MAC-1 cells increased to three times normal levels during 8 weeks of infection with 16M but returned to normal by 4 weeks after infection with delta purE201. These results document infectivity and attenuation of delta purE201 and suggest that it should be further evaluated as a potential vaccine.
Insights
A Brucella melitensis purE mutant (delta purE201) shows significant attenuation in mice, with rapid clearance and reduced inflammatory responses compared to the wild-type strain. This suggests its potential as a live vaccine candidate.
Area of Science:
- Immunology
- Microbiology
- Vaccinology
Background:
- Brucella melitensis is a significant human pathogen.
- A previously identified purE mutant (delta purE201) demonstrated attenuated growth in human monocytes.
- The in vivo attenuation and host response to this mutant were not previously characterized.
Purpose of the Study:
- To evaluate the in vivo attenuation of the Brucella melitensis delta purE201 mutant in a murine model.
- To compare the growth kinetics and host immune response elicited by the mutant versus the wild-type strain (16M).
- To assess the potential of the delta purE201 mutant as a live vaccine candidate.
Main Methods:
- BALB/c mice were infected with either Brucella melitensis 16M or the delta purE201 mutant.
- Bacterial loads were quantified in spleen, liver, and lungs post-infection.
- Spleen weights, serum interleukin-6 levels, and immune cell populations (CD4+, CD8+, MAC-1+) were analyzed via flow cytometry.
Main Results:
- The delta purE201 mutant exhibited significantly reduced bacterial numbers in spleens, livers, and lungs compared to strain 16M.
- Infection with delta purE201 resulted in mild, transient splenomegaly and no detectable serum interleukin-6, unlike the severe inflammatory response to 16M.
- Immune cell analysis showed distinct patterns: MAC-1 cells returned to normal levels by 4 weeks post-infection with delta purE201, whereas they remained elevated with 16M.
Conclusions:
- The Brucella melitensis delta purE201 mutant is significantly attenuated in vivo, demonstrating reduced infectivity and immunogenicity in mice.
- The attenuated phenotype and reduced inflammatory response suggest a favorable safety profile.
- The delta purE201 mutant warrants further investigation as a potential live attenuated vaccine for brucellosis.