ABSTRACTPrevious studies showed that a considerable proportion ofListeria monocytogenesisolates obtained from foods carry a premature stop codon (PMSC) mutation ininlAthat leads to production of a truncated and secreted InlA. To further elucidate the role these mutations play in virulence ofL. monocytogenes, we created isogenic mutants, including (i) natural isolates where aninlAPMSC was reverted to a wild-typeinlAallele (without a PMSC) and (ii) natural isolates where a PMSC mutation was introduced into a wild-typeinlAallele; isogenic mutant sets were constructed to represent two distinctinlAPMSC mutations. Phenotypical and transcriptional analysis data showed thatinlAPMSC mutations do not have a polar effect on the downstreaminlB. Isogenic and natural strains carrying aninlAPMSC showed significantly reduced invasion efficiencies in Caco-2 and HepG2 cell lines as well as reduced virulence in oral guinea pig infections. Guinea pigs were also orally infected with a natural strain carrying the most commoninlAPMSC mutation (vaccinated group), followed by challenge with a fully virulentL. monocytogenesstrain 15 days postvaccination to probe potentially immunizing effects of exposure toL. monocytogeneswithinlAPMSC mutations. Vaccinated guinea pigs showed reduced bacterial loads in internal organs and improved weight gain postchallenge, indicating reduced severity of infections in guinea pigs exposed to natural strains withinlAPMSC mutations. Our data support that (i)inlAPMSC mutations are causally associated with attenuated virulence in mammalian hosts and (ii) naturally occurring virulence-attenuatedL. monocytogenesstrains commonly found in food confer protective immunity.