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Infection and Immunity, September 2005, p. 5789-5798, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5789-5798.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Pathogenicity and Immunogenicity of a Vaccine Strain of Listeria monocytogenes That Relies on a Suicide Plasmid To Supply an Essential Gene Product

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received 4 February 2005/ Returned for modification 31 March 2005/ Accepted 29 April 2005

Listeria monocytogenes is a bacterial pathogen that elicits a strong cellular immune response and thus has potential use as a vaccine vector. An attenuated strain, L. monocytogenes dal dat, produced by deletion of two genes (dal and dat) used for D-alanine synthesis, induces cytotoxic T lymphocytes and protective immunity in mice following infection in the presence of D-alanine. In order to obviate the dependence of L. monocytogenes dal dat on supplemental D-alanine yet retain its attenuation and immunogenicity, we explored mechanisms to allow transient endogenous synthesis of the amino acid. Here, we report on a derivative strain, L. monocytogenes dal dat/pRRR, that expresses a dal gene and synthesizes D-alanine under highly selective conditions. We constructed the suicide plasmid pRRR carrying a dal gene surrounded by two res1 sites and a resolvase gene, tnpR, which acts at the res1 sites. The resolvase gene is regulated by a promoter activated upon exposure to host cell cytosol. L. monocytogenes dal dat/pRRR was thus able to grow in liquid culture and to infect host cells without D-alanine supplementation. However, after infection of these cells, resolvase-mediated excision of the dal gene resulted in strong down-regulation of racemase expression. As a result, this system allowed only transient growth of L. monocytogenes dal dat/pRRR in infected cells and survival in animals for only 2 to 3 days. Nevertheless, mice immunized with L. monocytogenes dal dat/pRRR generated listeriolysin O-specific effector and memory CD8⁺ T cells and were protected against lethal challenge by wild-type Listeria. This vector may be an attractive vaccine candidate for the induction of protective cellular immune responses.

Editor: J. T. Barbieri

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