Multiple point mutations in virulence genes explain the low virulence of Listeria monocytogenes field strains.
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.
Témoin
S
S
INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de Recherche de Tours, Nouzilly 37380, France.
Roche
S M
SM
INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de Recherche de Tours, Nouzilly 37380, France.
Grépinet
O
O
INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de Recherche de Tours, Nouzilly 37380, France.
Fardini
Y
Y
INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de Recherche de Tours, Nouzilly 37380, France.
Velge
P
P
INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de Recherche de Tours, Nouzilly 37380, France.
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002460:Cell Line; D002522:Chlorocebus aethiops; D018389:Codon, Nonsense; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004847:Epithelial Cells / Q000382:microbiology; D005347:Fibroblasts / Q000382:microbiology; D005816:Genetic Complementation Test; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology*; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D013154:Spleen / Q000382:microbiology; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics*
D001426:Bacterial Proteins; D018389:Codon, Nonsense; D004269:DNA, Bacterial; D008565:Membrane Proteins; D037521:Virulence Factors; C095768:inlB protein, Listeria monocytogenes; C069761:internalin protein, Bacteria; D054801:Phosphoinositide Phospholipase C