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Infection and Immunity, December 2001, p. 7736-7742, Vol. 69, No. 12
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.12.7736-7742.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Brucella abortus Genes Identified following Constitutive Growth and Macrophage Infection

Linda Eskra, Aurea Canavessi, Merriann Carey, and Gary Splitter*

Department of Animal Health and Biomedical Sciences, University of Wisconsin---Madison, Madison, Wisconsin 53706

Received 16 April 2001/Returned for modification 2 July 2001/Accepted 20 August 2001

The chronicity of Brucella abortus infection in humans and animals depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. Although no human vaccine exists for Brucella, vaccine development in other bacteria has been based on deletions of selective nutritional as well as regulatory systems. Our goal is to develop a vaccine for Brucella. To further this aim, we have used a green fluorescent protein (GFP) reporter system to identify constitutively and intracellularly induced B. abortus genes. Constitutively producing gfp clones exhibited sequence homology with genes associated with protein synthesis and metabolism (initiation factor-1 and tRNA ribotransferase) and detoxification (organic hydroperoxidase resistance). Of greater interest, clones negative for constitutively produced gfp in agar were examined by fluorescence microscopy to detect promoter activity induced within macrophages 4 and 24 h following infection. Bacterial genes activated in macrophages 4 h postinfection appear to be involved in adapting to intracellular environmental conditions. Included in this group were genes for detoxification (lactoglyglutathione lyase gene), repair (formamidopyrimidine-DNA glycosylase gene), osmotic protection (K+ transport gene), and site-specific recombination (xerD gene). A gene involved in metabolism and biosynthesis (deoxyxylulose 5' phosphate synthase gene) was also identified. Genes activated 24 h following infection were biosynthesis- and metabolism-associated genes (iron binding protein and rhizopine catabolism). Identification of B. abortus genes that are activated following macrophage invasion provides insight into Brucella pathogenesis and thus is valuable in vaccine design utilizing selective targeted deletions of newly identified Brucella genes.

* Corresponding author. Mailing address: Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Dr., Madison, WI 53706. Phone: (608) 262-1837. Fax: (608) 262-7420. E-mail: Splitter{at}ahabs.wisc.edu.

Infection and Immunity, December 2001, p. 7736-7742, Vol. 69, No. 12
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.12.7736-7742.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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