RESUMEN
The low-virulence Listeria monocytogenes strains have been previously assigned to 4 phenotypic groups. This study aimed to characterize the A23 strain, which exhibits a pulsed-field gel electrophoresis profile specific to low-virulence strains. This strain has the same causal mutations as the group III strains and a supplementary mutation in the mpl gene, leading to the absence of internalin A expression and the presence of inactive internalin B, phosphatidyl-inositol phospholipase C, and phosphatidylcholine phospholipase C. Despite these mutations in major virulence genes, the A23 strain formed plaques in cell monolayers and contaminated 100% of inoculated mice, suggesting that it evolved from group III strains by acquiring new virulence genes.
Asunto(s)
Eliminación de Gen , Listeria monocytogenes/patogenicidad , Factores de Virulencia/genética , Animales , Proteínas Bacterianas/genética , Línea Celular , Humanos , Listeriosis/microbiología , Proteínas de la Membrana/genética , Ratones , Fosfoinositido Fosfolipasa C/genética , Fosfolipasas de Tipo C/genética , VirulenciaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Mutación Puntual , Factores de Virulencia/genética , Sustitución de Aminoácidos/genética , Animales , Línea Celular , Chlorocebus aethiops , Codón sin Sentido , Recuento de Colonia Microbiana , ADN Bacteriano/química , ADN Bacteriano/genética , Células Epiteliales/microbiología , Fibroblastos/microbiología , Prueba de Complementación Genética , Humanos , Listeria monocytogenes/aislamiento & purificación , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Fosfoinositido Fosfolipasa C/genética , Fosfoinositido Fosfolipasa C/metabolismo , Análisis de Secuencia de ADN , Bazo/microbiología , VirulenciaRESUMEN
The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDDeltaprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix alphaH. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix-turn-helix (HTH) motif.
Asunto(s)
ADN Bacteriano/metabolismo , Listeria monocytogenes/patogenicidad , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Sustitución de Aminoácidos , Animales , Cristalografía por Rayos X , Dimerización , Secuencias Hélice-Giro-Hélice , Factores de Hemolisina/genética , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/metabolismo , Modelos Moleculares , Mutación , Factores de Terminación de Péptidos/química , Conformación Proteica , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , VirulenciaRESUMEN
Several models have shown that virulence varies from one strain of Listeria monocytogenes to another, but little is known about the cause of low virulence. Twenty-six field L. monocytogenes strains were shown to be of low virulence in a plaque-forming assay and in a subcutaneous inoculation test in mice. Using the results of cell infection assays and phospholipase activities, the low-virulence strains were assigned to one of four groups by cluster analysis and then virulence-related genes were sequenced. Group I included 11 strains that did not enter cells and had no phospholipase activity. These strains exhibited a mutated PrfA; eight strains had a single amino acid substitution, PrfAK220T, and the other three had a truncated PrfA, PrfADelta174-237. These genetic modifications could explain the low virulence of group I strains, since mutated PrfA proteins were inactive. Group II and III strains entered cells but did not form plaques. Group II strains had low phosphatidylcholine phospholipase C activity, whereas group III strains had low phosphatidylinositol phospholipase C activity. Several substitutions were observed for five out of six group III strains in the plcA gene and for one out of three group II strains in the plcB gene. Group IV strains poorly colonized spleens of mice and were practically indistinguishable from fully virulent strains on the basis of the above-mentioned in vitro criteria. These results demonstrate a relationship between the phenotypic classification and the genotypic modifications for at least group I and III strains and suggest a common evolution of these strains within a group.