4 years ago

Spontaneous virulence loss in natural populations of Listeria monocytogenes.

Vales G, Disson O, Chenal-Francisque V, Lecuit M, Scortti M, Gouin E, Moura A, Han L, Bracq-Dieye H, Vázquez-Boland JA, Leclercq A, Maury MM
Listeria monocytogenes (Lm) pathogenesis depends on its ability to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for Lm virulence. Here we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in Lm Sixty (0.1%) non-hemolytic isolates were identified among a collection of 57,820 confirmed Lm strains isolated from a variety of sources. In most cases (56/60), the non-hemolytic phenotype resulted from nonsense, missense or frameshift mutations in prfA Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function mutants belonged to phylogenetically diverse clades of Lm and most were identified among non-clinical strains (57/60). In line with the extremely low frequency of loss of virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA-/LLO- mutational events in Lm lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.Importance The hemolytic phenotype of Lm is a key identification criterion in food and clinical microbiology. Here we characterized 60 non-hemolytic Lm strains, identified by screening a vast collection of natural Lm isolates collected in the context of epidemiological surveillance of listeriosis. Phenotypic and genomic analyses demonstrated that the absence of hemolysis was due to loss-of-function mutations in prfA or hly, leading to strong virulence attenuation in mice. We also identified the first natural Lm strain which spontaneously lost the gshF gene, required for the PrfA-dependent transcriptional activation of hly and other virulence genes. Previous phylogenomic studies have indicated that some non-pathogenic Listeria species derive from pathogenic ones, and the virulence-attenuating mutations characterized in this study illustrate the possible early events that could have determined their emergence and evolution.

Publisher URL: https://www.ncbi.nlm.nih.gov/pubmed/28827366

DOI: PubMed:28827366

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