inlAPremature Stop Codons Are Common amongListeria monocytogenesIsolates from Foods and Yield Virulence-Attenuated Strains That Confer Protection against Fully Virulent Strains

Abstract

Previous studies showed that a considerable proportion of Listeria monocytogenes isolates obtained from foods carry a premature stop codon (PMSC) mutation in inlA that leads to production of a truncated and secreted InlA. To further elucidate the role these mutations play in virulence of L. monocytogenes, we created isogenic mutants, including (i) natural isolates where an inlA PMSC was reverted to a wild-type inlA allele (without a PMSC) and (ii) natural isolates where a PMSC mutation was introduced into a wild-type inlA allele; isogenic mutant sets were constructed to represent two distinct inlA PMSC mutations. Phenotypical and transcriptional analysis data showed that inlA PMSC mutations do not have a polar effect on the downstream inlB. Isogenic and natural strains carrying an inlA PMSC 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 common inlA PMSC mutation (vaccinated group), followed by challenge with a fully virulent L. monocytogenes strain 15 days postvaccination to probe potentially immunizing effects of exposure to L. monocytogenes with inlA PMSC 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 with inlA PMSC mutations. Our data support that (i) inlA PMSC mutations are causally associated with attenuated virulence in mammalian hosts and (ii) naturally occurring virulence-attenuated L. monocytogenes strains commonly found in food confer protective immunity.

Listeria monocytogenes Associated with New Zealand Seafood Production and Clinical Cases: Unique Sequence Types, Truncated InlA, and Attenuated Invasiveness

“…Similar to our finding that isolates from human cases can harbor a PMSC, isolates with PMSCs have previously been associated with food-related outbreaks of listeriosis (e.g., the 1985 cheese outbreak in the United States and the 1988 pâté outbreak in the United Kingdom [40,45]). These outbreak strains showed attenuated invasion abilities when tested in Caco-2 cell lines, leading to the suggestion that their capacity to cause listeriosis may have been due to inlA-independent properties (e.g., better survival and growth in food and food-associated environments).…”

“…Nonsense mutations in inlA are reported to be at least partially responsible for an attenuated invasion phenotype (7,24,(39)(40)(41). These mutations may have accumulated as a result of selection for environmentally adapted ecotypes with reduced virulence, which do not require a fulllength InlA protein for ecological success (22).…”

“…Nightingale et al (40) suggested that mutations in inlA are related to the genotype of L. monocytogenes (i.e., ribotype, ST, or pulsotype) rather than to the source of isolation (i.e., they are more likely to be due to founder effects and not causally linked to invasiveness). Orsi et al (6) also suggest that infrequent reporting of the PMSCs in listeriosis-related strains may have resulted in the overrepresentation of 4b isolates in clinical samples in comparison to 1/2a strains (harboring inlA-PMSC), while the PMSC may have resulted in positive selection for strains with a truncated InlA protein during environmental adaptation.…”

“Epidemic Clones” of Listeria monocytogenes Are Widespread and Ancient Clonal Groups

“…Finally, strain CLIP 11308 had a 1-nt deletion at position 5 in gene inlA that resulted in a predicted stop codon at amino acid position 9. Functional, nontruncated InlA has been associated with the clinical origin of L. monocytogenes isolates (42), and premature stop codons in inlA leading to the secretion of a nonfunctional truncated protein have been associated with reduced pathogenicity (9,(41)(42)(43). Patterns of recombination, selection, and phylogenetic relationships.…”

Invasiveness of Listeria monocytogenes strains isolated from animals in Poland

“…A number of mutations in inlA gene, leading to premature stop codons (PMSCs), have been observed in L. monocytogenes. Such mutations result in production of truncated InlA, contributing to attenuated invasion efficiency of the bacteria (Nightingale et al 2005, Nightingale et al 2008). However, recent study by Roche et al (2012) reports on low virulent L. monocytogenes strains expressing full length InlA.…”