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Deletion of purE attenuates Brucella melitensis 16M for growth in human monocyte-derived macrophages
E S Drazek1, H S Houng, R M Crawford
1Department of Infectious and Parasitic Diseases, Armed Forces Institute of Pathology, Washington, D.C. 20306-6000, USA.
Abstract:
We constructed a defined purine-auxotrophic mutant of Brucella melitensis 16M by chromosomal gene replacement. We electroporated B. melitensis 16M with suicide plasmids containing a kanamycin resistance cassette that replaced 226 bp at the carboxyl end of purE, the intergenic region, and 18 bases of the purK open reading frame. Recombinant B. melitensis delta purE201 required exogenous purines for growth on minimal media. Purine auxotrophy was complemented by electroporation of B. melitensis delta purE201 failed to grow in human monocyte-derived macrophages, while the growth of wild-type 16M and the complemented strain, delta purE201 (pSD5), increased by nearly two logs. These results suggest that B. melitensis delta purE201 will be attenuated in animals and humans and thus may be useful as a live attenuated vaccine.
Insights
A new Brucella melitensis mutant lacking purine synthesis genes cannot grow independently. This purine-auxotrophic strain showed reduced growth in macrophages, suggesting potential as a live attenuated vaccine.
Area of Science:
- Microbiology
- Vaccinology
- Genetics
Background:
- Brucella melitensis is an important zoonotic pathogen.
- Developing live attenuated vaccines requires understanding bacterial metabolic requirements.
- Purine biosynthesis is essential for bacterial survival and virulence.
Purpose of the Study:
- To construct and characterize a purine auxotrophic mutant of Brucella melitensis 16M.
- To evaluate the in vitro growth and intracellular survival of the mutant.
- To assess the potential of the mutant as a live attenuated vaccine candidate.
Main Methods:
- Chromosomal gene replacement was used to create a purine auxotrophic mutant (delta purE201) by disrupting purE and purK genes.
- Suicide plasmids with a kanamycin resistance cassette were employed for gene replacement.
- Growth assays on minimal media and in human monocyte-derived macrophages were performed.
Main Results:
- The Brucella melitensis delta purE201 mutant required exogenous purines for growth.
- The purine auxotrophy was successfully complemented.
- The mutant exhibited significantly reduced growth within macrophages compared to wild-type and complemented strains.
Conclusions:
- The constructed purine auxotrophic Brucella melitensis mutant is attenuated in its ability to replicate within macrophages.
- This attenuated phenotype suggests that Brucella melitensis delta purE201 may serve as a promising live attenuated vaccine candidate for brucellosis.