Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes

Abstract

The pathogenesis of <i>Listeria monocytogenes</i> 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 (<i>hly</i>) requires the transcriptional activator PrfA, and both <i>hly</i> and <i>prfA</i> genes are essential for <i>L. monocytogenes</i> virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in <i>L. monocytogenes</i> Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed <i>L. monocytogenes</i> 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 <i>prfA</i> Five strains carried <i>hly</i> mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both <i>hly</i> and <i>gshF</i> (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 <i>L. monocytogenes</i>, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that <i>prfA</i> and <i>hly</i> are under purifying selection. Although occurring at a low frequency, PrfA^-/LLO^- mutational events in <i>L. monocytogenes</i> lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.