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Infection and Immunity, October 2004, p. 5622-5629, Vol. 72, No. 10
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.10.5622-5629.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Growth, Virulence, and Immunogenicity of Listeria monocytogenes aro Mutants

Lehrstuhl für Mikrobiologie am Biozentrum der Universität Würzburg,¹ Lehrstuhl für Lebensmittelchemie der Universität Würzburg, Am Hubland,³ Institut für Medizinische Strahlenkunde und Zellforschung der Universität Würzburg, Würzburg,⁴ Institut für Medizinische Mikrobiologie und Hygiene, Mannheim, Germany²

Received 2 June 2004/ Returned for modification 22 June 2004/ Accepted 13 July 2004

Mutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA. aroA, aroB, and in particular aroE mutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of the aro mutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K₂, suggesting that the aro mutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of the aro mutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and all aro mutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 10⁴-fold for the aroA, aroB, and aroA/B mutants and >10⁵-fold for the aroE mutant compared to the parent strain. Nevertheless, mice preimmunized with aro mutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 x 10⁶ aroA, aroB, and aroA/B mutant bacteria were sufficient to obtain a protective T-cell response, while 5 x 10⁸ aroE or aroA/E mutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating that Listeria strains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.

Editor: J. D. Clements

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