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Listeria monocytogenes (Lm) is a Gram-positive intracellular pathogen that can elicit strong cellular immunity. An attenuated strain (Lmdd) with deletions in two genes (dal and dat) required for d-alanine synthesis and viability has been shown to induce long-lived protective systemic and mucosal immune responses in mice when administered in the presence of the required amino acid. To bypass the necessity for exogenous d-alanine without compromising the safety of the original strain, the defect of Lmdd was complemented with a heterologous Bacillus subtilis dal gene, and the effects of truncating the upstream region of the gene on its transcription efficiency and of modifying its protein product with an ssrA tag at the 3'-terminus were examined. The strains with 551 bp and 80 bp upstream regions showed high levels of transcription and grew without d-alanine. The strains with the shortest upstream regions, 48 bp and 18 bp, showed greatly decreased levels of transcription and failed to grow in the absence of d-alanine. Addition of an ssrA tag to the longer genes resulted in a somewhat altered growth pattern in media and a reduced plaque size on L2 fibroblasts. These bacteria contained low levels of racemase protein and reduced free pools of d-alanine. One of the strains tested further, Lmdd/pA80S, was rapidly cleared from the spleens of infected mice but nevertheless induced a strong immune response that protected mice against challenge by wild-type L. monocytogenes. These bacteria can thus induce immune responses in mice comparable to the original Lmdd strain, but without the need for exogenous d-alanine, and may have use as a live vaccine vector against infectious diseases and cancers.

A truncated Bacillus subtilis dal gene with a 3′ ssrA gene tag regulates the growth and virulence of racemase-deficient Listeria monocytogenes