Spontaneous virulence loss in natural populations of Listeria monocytogenes

Abstract
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-haemolytic 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.