Poor Invasion of Trophoblastic Cells but Normal Plaque Formation in Fibroblastic Cells despite actA Deletion in a Group of Listeria monocytogenes Strains Persisting in Some Food Processing Environments

Abstract

We determined mammalian cell invasion and virulence gene (inlA, inlB, and actA) sequences of Listeria monocytogenes strains belonging to a molecular subtype (RAPD 9) that often persists in Danish fish-processing plants. These strains invaded human placental trophoblasts less efficiently than other L. monocytogenes strains, including clinical strains, and they carry a premature stop codon in inlA. Eight of 15 strains, including the RAPD 9 and maternofetal strains, had a 105-nucleotide deletion in actA that did not affect cell-to-cell spread in mouse fibroblasts. The RAPD 9 strains may still be regarded as of low virulence with respect to human listeriosis.Listeria monocytogenes is a Gram-positive pathogenic bacterium that can cause foodborne listeriosis, which affects immunocompromised individuals, causing septicemia and meningitis, and pregnant women, causing preterm delivery, miscarriage, or stillbirth. It is a ubiquitous environmental bacterium, and it is therefore continuously introduced to food-processing plants, where some molecular subtypes are able to persist despite thorough cleaning and disinfection procedures (1,27,32,42). Such persistent strains are likely to contaminate the food products and may be the cause of foodborne infections (30).We have shown that specific molecular subtypes of L. monocytogenes can persist for years in the seafood-processing environment (45), and strains representing a particularly prevalent, persistent molecular subtype, RAPD type 9 ([RAPD 9] random amplified polymorphic DNA), had a lower virulence potential than clinical strains in simple eukaryotic models (12). However, in a more complex biological model using oral dosing of pregnant guinea pigs, the tested RAPD 9 strain (strain La111) surprisingly infected the placentas and fetuses just as efficiently as a clinical strain (13). We therefore hypothesized that this specific subtype may have an altered (enhanced) ability to invade placental cells (e.g., trophoblasts) or an enhanced ability to spread intracellularly. (29), the United States (25), and Japan (9), and these mutations lead to attenuation in the invasion of intestinal epithelial cells (25,28,33), but it is not known if invasion into trophoblasts is affected. ActA is important for cell-to-cell spread (5) and is involved in invasion of epithelial cells (39), and ActA-mediated cell-to-cell spread plays a major role in crossing the fetoplacental barrier in both a guinea pig and a mouse model (3,22).The purpose of this study was to determine if the high level of prevalence of a RAPD 9 strain in guinea pig fetuses after oral dosing could be explained by increased invasion into and spread between trophoblastic and fibroblastic cells, respectively. Subsequently, we sequenced selected virulence genes to determine if strain variations in cell invasion and spread could be explained by differences in sequences.Strains, culture conditions, and characterization. Fifteen L. monocytogenes strains representing different origins, RAPD types, serotypes, and lineages were used (Tabl...

Characterization of specific alleles in InlA and PrfA of Listeria monocytogenes isolated from foods in Osaka, Japan and their ability to invade Caco-2 cells

“…Isolates with inlA PMSCs exhibited 147 times lower average invasion efficiency than isolates with full-length InlA (P b 0.01; t test), while isolates with a 3-codon deletion of InlA and with a stop-codon deletion of PrfA exhibited invasion ability comparable to isolates with full-length InlA and PrfA (P N 0.05) after growth at 30°C. Ferreira et al (2011) and Roche et al (2012) characterized isolates with the 3-codon deletion as low-virulence strains; however, isolates with this mutation exhibited invasive activity equivalent to or surpassing isolates with full-length InlA in previous reports (Handa-Miya et al, 2007;Kovacevic et al, 2013), and this mutation has also been found in a clinical isolate from materno-fetal fluid in Denmark (Holch et al, 2010). It seems likely that both high-virulence isolates and low-virulence isolates are present among the isolates with the 3-codon deletion of InlA.…”

“…This mutation has been reported in isolates of serotype 4b from RTE seafood samples in Japan (Handa-Miya et al, 2007); in an isolate of serogroup D, including serotypes 4b, 4d, and 4e, from a meat facility in Portugal (Ferreira et al, 2011); in isolates of serotype 4b from food products in France (Roche et al, 2012); and in isolates of serotype 4b and 1/2a from foods and food-processing environments in Canada (Kovacevic et al, 2013). Furthermore, this mutation has been found in a clinical isolate of serogroup 4 including 4a, 4ab, 4b, 4c 4d, and 4e, from materno-fetal fluid in Denmark (Holch et al, 2010). It is therefore considered that serotype 4b/4e isolates with the 3-codon deletion of InlA are relatively common and easy to identify.…”

Genome Sequencing Identifies Two Nearly Unchanged Strains of Persistent Listeria monocytogenes Isolated at Two Different Fish Processing Plants Sampled 6 Years Apart

“…The differences between the two strains likely derive from missing data in the La111 assembly. Differences in the number of predicted proteins and predicted tRNAs were observed when using (16), and phenotypic behavior (13,16,17). However, a whole-genome comparison of these two strains had not yet been attempted.…”

“…Although some SNPs may derive from sequencing errors, true SNPs may be either silent or nonsynonymous, in which case they may change the function of the transcribed protein or result in a truncated protein. An example of the latter possibility entailed the listerial surface protein InlA, where a nucleotide substitution from C to T results in a stop codon and production of a truncated protein (16,42,43). In the present study, SNP analyses assessing only nonsynonymous changes were carried out after mapping raw reads of the queried strain against a reference strain (Fig.…”

“…In simple eukaryotic cell models and simple animal models (Caenorhabditis elegans and Drosophila melanogaster), the highly persistent strains were less invasive than human clinical strains (13,(16)(17)(18). Surprisingly, in a more complex biological model (using oral dosing of pregnant guinea pigs), the strains infected placentas and fetuses just as efficiently as the clinical strains (18).…”