Identification of new genes involved in the virulence of Listeria monocytogenes by signature-tagged transposon mutagenesis

Listeria monocytogenes is a gram-positive, facultative intracellular pathog en that can cause severe food born infections in humans and animals. We hav e adapted signature tagged transposon mutagenesis to L. monocytogenes to id entify new genes involved in virulence in the murine model of infection. We used transposon Tn1545 carried on the integrative vector pAT113. Forty-eig ht tagged transposons were constructed and used to generate banks of L. mon ocytogenes mutants. Pools of 48 mutants were assembled, taking one mutant f rom each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants teste d, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to prev iously unknown genes, The properties of four loci involving putative cell w all components were further studied in vitro and in vivo. The data suggeste d that these components are involved in bacterial invasion and multiplicati on in the brain.