Isolation of a Francisella tularensis mutant that is sensitive to serum and oxidative killing and is avirulent in mice: Correlation with the loss of MinD homologue expression

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. tularensis multiplies intracellularly in macrophages, 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, tumour necrosis factor [6]. Recently, it has been demonstrated that the mechanism of action of gamma interferon on the inhibition of intracellu-

Genetics and Genetic Manipulation in Francisella Tularensis

“…The ability to construct libraries of random insertional mutants in Francisella was initially approached utilizing techniques developed for Haemophilus influenza. 28,45,46 To apply this strategy, F. novicida chromosomal DNA is digested to completion and the resulting restriction fragments are self‐ligated. A second restriction enzyme is then used to partially cut circularized fragments and a kanamycin resistance gene with ends compatible to the second enzyme is inserted randomly.…”

Spatial control of the cell division site by the Min system in Escherichia coli

“…The mechanisms that regulate the division site placement in these bacteria will require further investigation. Besides being of academic interest, the Min proteins have been correlated to the pathogenicity of Francisella tularensis, the causative agent of tularaemia (rabbit fever) (Anthony et al, 1994;Su et al, 2007) and N. gonorrhoeae, the causative agent of gonorrhoea (Parti et al, 2011a,b). In addition to the fact that homologues of MinCDE can be found in a range of human, animal and plant pathogens, they are also identified from representative species with environmental, biomedical and biotechnological significance (Table 1 and Table S1).…”

Dissecting the role of conformational change and membrane binding by the bacterial cell division regulator MinE in the stimulation of MinD ATPase activity

“…Nonetheless, pattern formation by ecMin proteins on planar lipid bilayers was found to occur over a range of lipid headgroup compositions (26), suggesting that the E. coli lipids serve as a reasonable substitute for those from N. gonnorhoeae. More importantly, the role of the MinE MTS in the Min cycle, as elucidated from our work on ngMin proteins, should have implications for Min protein cycles in general, including those linked to the pathogenicity of Francisella tularensis (55,56) and enterohemorrhagic E. coli (57) as well as the diverse range of eukaryotic species that are predicted to use mitochondrial and plastid Min proteins (58,59).…”