2. Universidad Cardenal Herrera-CEU

Background: Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis, an infectious disease in animals and people, with pigs acting as asymptomatic reservoirs. In August 2019 an outbreak associated with the consumption of pork meat caused 222 human cases of listeriosis in Spain. Determining the diversity as well as the virulence potential of strains from pigs is important to public health. Methods: The behaviour of 23 L monocytogenes strains recovered from pig tonsils, meat and skin was compared by studying (1) internalin A, internalin B, listeriolysin O, actin assembly-inducing protein and PrfA expression levels, and (2) their invasion and intracellular growth in eukaryotic cells. Results: Marked differences were found in the expression of the selected virulence factors and the invasion and intracellular replication phenotypes of L monocytogenes strains. Strains obtained from meat samples and belonging to serotype 1/2a did not have internalin A anchored to the peptidoglycan. Some strains expressed higher levels of the studied virulence factors and invaded and replicated intracellularly more efficiently than an epidemic L monocytogenes reference strain (F2365). Conclusion: This study demonstrates the presence of virulent L monocytogenes strains with virulent potential in pigs, with valuable implications in veterinary medicine and food safety.

Most human listeriosis outbreaks are caused by Listeria monocytogenes evolutionary lineage I strains which possess four exotoxins: a phosphatidylinositol-specific phospholipase C (PlcA), a broad-range phospholipase C (PlcB), listeriolysin O (LLO) and listeriolysin S (LLS). The simultaneous contribution of these molecules to virulence has never been explored. Here, the importance of these four exotoxins of an epidemic lineage I L. monocytogenes strain (F2365) in virulence was assessed in chicken embryos infected in the allantoic cavity. We show that LLS does not play a role in virulence while LLO is required to infect and kill chicken embryos both in wild type transcriptional regulator of virulence PrfA ( PrfAWT) and constitutively active PrfA (PrfA*) backgrounds. We demonstrate that PlcA, a toxin previously considered as a minor virulence factor, played a major role in virulence in a PrfA* background. Interestingly, GFP transcriptional fusions show that the plcA promoter is less active than the hly promoter in vitro, explaining why the contribution of PlcA to virulence could be observed more importantly in a PrfA* background. Together, our results suggest that PlcA might play a more important role in the infectious lifecycle of L. monocytogenes than previously thought, explaining why all the strains of L. monocytogenes have conserved an intact copy of plcA in their genomes.

Objectives: To investigate the contribution to virulence of the surface protein internalin B (InlB) in the Listeria monocytogenes lineage I strain F2365, which caused a deadly listeriosis outbreak in California in 1985. Methods: The F2365 strain displays a point mutation that hampers expression of InlB. We rescued the expression of InlB in the L. monocytogenes lineage I strain F2365 by introducing a point mutation in the codon 34 (TAA to CAA). We investigated its importance for bacterial virulence using in vitro cell infection systems and a murine intravenous infection model. Results: In HeLa and JEG-3 cells, the F2365 InlBþ strain expressing InlB was z9-fold and z1.5-fold more invasive than F2365, respectively. In livers and spleens of infected mice at 72 hours after infection, bacterial counts for F2365 InlBþ were significantly higher compared to the F2365 strain (z1 log more), and histopathologic assessment showed that the F2365 strain displayed a reduced number of necrotic foci compared to the F2365 InlBþ strain (Mann-Whitney test). Conclusions: InlB plays a critical role during infection of nonpregnant animals by a L. monocytogenes strain from lineage I. A spontaneous mutation in InlB could have prevented more severe human morbidity and mortality during the 1985 California listeriosis outbreak.