FEMS Microbiology Letters 124 (1994) 157-166 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier 157 FEMSLE 06269 Isolation of a Francisella that is sensitive to serum and is avirulent in mice: of tularensis mutant and oxidative killing Correlation with the MinD homologue expression loss Lawrence S.D. Anthony, Siobhfin C. Cowley, Khisimuzi E. Mdluli and Francis E. Nano * Department of Biochemistry and Microbiology and Canadian Bacterial Disease Network, University of Victoria, P.O. Box 3055, Victoria, British Columbia, V8W 3P6 Canada (Received 15 September 1994; accepted 21 September 1994) Abstract: We constructed mutant strains of Francisella tularensis biotype novicida by insertional mutagenesis with a kanamycm resistance (Kin R) cassette. One mutant, KEM7, was defective for survival in macrophages in comparison with the wild-type (WT) strain and a random insertion strain, KEM21. While all three strains exhibited intracellular growth, the number of viable KEM7 present after 24-48 h of infection was approximately 10 times less than that of WT or KEM21. This observation was apparently due to a reduced number of viable KEM7 associated with the macrophages one hour after phagocytosis. KEM7 was approximately 3 times more susceptible than WT or KEM21 to killing by the products of the xanthine-xanthine oxidase reaction or by hydrogen peroxide. KEM7 was also found to be susceptible to killing by serum, whereas WT and KEM21 were resistant. Upon intravenous inoculation of C57BL/6 mice, the number of KEM7 in the livers and spleens 48 h post-infection was found to be 1000- to 10000-times less than that of either KEM21 or WT. DNA sequence analysis at the Km R insertion site suggested that the F. tularensis homologue of minD had been interrupted. Western immunoblot analysis confirmed the presence of a MinD homologue in F. tularensis WT and KEM21, and demonstrated its absence in KEM7. Key words: Francisella; MinD; Macrophage; Tularemia Introduction Francisella tularensis, the Gram-negative bacte- rial etiologic agent of tularemia, is a facultative intracellular pathogen. It has been shown that F. * Corresponding author. Tel: + 1 (604) 721-7074; Fax: + 1 (604) 721-8855; e-mail: fnano@sol.uvic.ca. tularensis multiplies intracellularly in macro- phages, probably within a phagosome which has not fused with lysosomes [1]. Resolution of murine tularemia was found to be dependent upon the activity of class II-restricted T cells [2,3] as well as the macrophage activating lymphokine, gamma interferon [4,5] and the macrophage product, tu- mour necrosis factor [6]. Recently, it has been demonstrated that the mechanism of action of gamma interferon on the inhibition of intracellu- SSDI 0378-1097(94)00422-6