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Multiple point mutations in virulence genes explain the low virulence of Listeria monocytogenes field strains

INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de Recherche de Tours, Nouzilly 37380, France

Correspondence
S. M. Roche
sylvie.roche{at}tours.inra.fr

In order to understand the causes of the low virulence of Listeria monocytogenes field strains, five low-virulence strains were analysed. These five strains showed changes in relation to invasion, phosphatidyl-inositol phospholipase C (PI-PLC) activity, plaque formation and in vivo virulence. Molecular analyses revealed the same mutations in the plcA, inlA and inlB genes in all five strains. The Thr262Ala substitution in the PI-PLC protein was responsible for the absence of PI-PLC activity. This residue, conserved in certain L. monocytogenes species, is located at the outer rim of the active site pocket and could impair the cleavage activity of the enzyme. The low invasion rate of these strains was due to a nonsense codon leading to a lack of InlA protein synthesis, and to an Ala117Thr substitution in the leucine-rich repeat of InlB, which altered the interaction with the Met receptor. Single trans complementation with the inlA_EGDe, inlB_EGDe or plcA_EGDe genes restored the capacity of low-virulence strains either to enter epithelial and fibroblastic cells or to express PI-PLC activity. Complementation by allelic exchange of the plcA_EGDe gene on the chromosome and trans complementation with either the inlA_EGDe or the inlB_EGDe gene restored the ability to form plaques, but only partly restored the in vivo virulence, suggesting that there were other gene mutation(s) with consequences that could mainly be observed in vivo. These results indicate that the low virulence of L. monocytogenes strains can be explained by point mutations in a number of virulence genes; these could therefore be important for detecting low-virulence strains. Moreover, the fact that all the strains had the same substitutions suggests that they have a common evolutionary pathway.

The GenBank accession numbers for the plcA, inlA and inlB nucleotide sequences were: AY367414, EF990797 and EF990802 for strain BO43; AY367410, EF990793 and EF990798 for strain CNL895807; AY367411, EF990796 and EF990801 for strain CNL895795; AY367412, EF990794 and EF990799 for strain 416; and AY367413, EF990795 and EF990800 for strain 417.

Three supplementary figures, showing immunofluorescence labelling of actin tails, nucleotide and amino acid substitutions observed in plcA, inlA and inlB genes of the Group III strains, and a schematic representation of the crystal structure of PI-PLC, and also a supplementary table, listing the PCR primers used, are available with the online version of this paper.

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