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Journal of Bacteriology, October 2009, p. 6447-6456, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00534-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species ^,

Amanda A. Melillo,^# Manish Mahawar,^# Timothy J. Sellati, Meenakshi Malik, Dennis W. Metzger, J. Andres Melendez,^* and Chandra Shekhar Bakshi^*

Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York 12208

Received 21 April 2009/ Accepted 3 August 2009

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O₂^–). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (sodC) and a F. tularensis sodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB sodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-)-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis sodC and sodB sodC mutants showed attenuated intramacrophage survival in IFN--activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the sodC mutant or inhibiting the IFN--dependent production of O₂^– or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The sodC and sodB sodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the sodC mutant was restored in ifn-^–/–, inos^–/–, and phox^–/– mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.

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* Corresponding author. Mailing address: Center for Immunology and Microbial Disease, MC 151, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Phone for Chandra Shekhar Bakshi: (518) 262-6263. Fax: (518) 262-6161. E-mail: bakshis{at}mail.amc.edu. Phone for J. Andres Melendez: (518) 262-8791. Fax: (518) 262-6161. E-mail: melenda{at}mail.amc.edu

Published ahead of print on 14 August 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

^# A.A.M. and M.M. contributed equally to this work.

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Journal of Bacteriology, October 2009, p. 6447-6456, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00534-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.