Spontaneous Loss of Virulence in Natural Populations of Listeria Monocytogenes

The pathogenesis of depends on the ability of this bacterium 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 () requires the transcriptional activator PrfA, and both and genes are essential for virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in Five strains carried mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both and (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 (PrfA/LLO) mutants belonged to phylogenetically diverse clades of , and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that and are under purifying selection. Although occurring at a low frequency, PrfA/LLO mutational events in lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.