Genetic markers linked to and/or responsible for Listeria monocytogenes virulence attenuation Martin Wiedmann Department of Food Science Cornell

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1 Genetic markers linked to and/or responsible for Listeria monocytogenes virulence attenuation Martin Wiedmann Department of Food Science Cornell University, Ithaca, NY Phone:

2 Take home messages L. monocytogenes represents a diverse set of strains, including some that are virulence attenuated A large proportion of L. monocytogenes found in foods may show mutations in the gene inla, which is essential for invasion of human intestinal epithelial cells Epidemiological, animal, and tissue culture data suggest significantly reduced infectivity of inla PMSC mutants Some studies suggest some remaining ability to cause pregnancy associated infections in mice and guinea pigs Virulence attenuating mutations in other genes (e.g., prfa) are rare To date findings have largely been ignored in risk assessments and regulatory policy

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4 L. monocytogenes major virulence genes inla inlb

5 Molecular characterization of human, animal, and food isolates Lineage Human isolates (n=507) Food isolates (n=502) Animal isolates (n=126) Lineage I 54.4% 37.3% 40% Lineage II 42.6% 62.4% 52% Lineages III & IV 2.4% 0.4% 8% Lineage I predominantly represents serotypes 1/2b and 4b, lineage II predominantly represents serotypes 1/2a and 1/2c

6 1) Ribotype Number of isolates P-value 1) Comments Food Human DUP-1030A 8 8 NS DUP-1030B 0 10 ** not found in food DUP-1038B **** DUP-1039A ** DUP-1039B ** DUP-1039C NS DUP-1042A NS DUP-1042B **** DUP-1042C 14 0 *** multiple food types, not in humans DUP-1043A * DUP-1044A ** DUP-1044B 1 19 *** rarely found in food DUP-1044E 10 0 ** blue cheese only DUP-1045B NS DUP-1052A * DUP-1053A * DUP-1062A **** rarely found in humans DUP-1062D 28 1 **** rarely found in humans rare * Ribotypes with 1-4 isolates uncommon NS Ribotypes with 5-8 isolates Total **** Overall analysis of ribotype vs. origin P-values refer to comparison of origin between ribotype specified in that row vs. all other ribotypes where NS = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P <

7 Human virulence attenuation of ribotype DUP-1062A Isolates with ribotype DUP-1062A carry a premature stop codon in inla, which leads to reduced invasion of human epithelial cells Wildtype inla (745 aa) MA DUP-1062A inla (631 aa) L. mono L. mono Full-Length InlA E- Cadherin Truncated & Secreted InlA Host Cell Host Cell

8 inla premature stop codons in other strains Van Stelten et al. AEM. 2010

9 Human intestinal epithelial cell invasion by L. monocytogenes with and without inla premature stop codons Nightingale et al. AEM. 2008

10 Invasion into human intestinal epithelial cells of seafood, seafood-processing environmental, and human clinical L. monocytogenes with and without inla premature stop codons ST1, ST2 and ST5 isolates encode full-length inla inla PMSC type 3 isolates Cruz et al. AEM. 2014

11 Average dose of Lm in foods by inla subgroups Chen et al., Appl. Environ. Microbiol.

12 Dose response for guinea pig and human listeriosis varies between Lm strains with/without inla SNPs Dose-response (DR) modeling in oral guinea pig model for VA and fully-virulent strains Shift in DR curves (1.3 Log 10 shift) between strains Lm shows notable difference in ID 50 required for systemic infection DR models used to generate r values r = probability of a single cell causing illness Mean log(10) r values were: Retail level -8.1 for fully virulent Lm for VA Lm with inla PMSC Consumer level for fully virulent Lm for VA Lm with inla PMSC Van Stelten et al., 2011 and Chen et al., Appl. Environ. Microbiol.

13 Estimated 3.4 log lower infectious dose for inla PMSC strains 45% of food isolates from diverse sources in the US carry inla PMSC

14 Studies on prevalence of inla PMSC strains in different countries France: inla premature stop codon strains represent 35% of food isolates and 4% of human clinical isolates (Jacquet et al JID 189) Spain: 3 out of 11 food isolates from raw retail poultry meat had a different inla PMSC type (López et al JFP) New Zealand: inla premature stop codon strains represent 16/31 seafood-processing environmental isolates, 3/15 seafood isolates and 2/6 seafood-associated human clinical isolates (Cruz et al AEM) Canada: inla PMSC found in 35% of 54 isolates from foods and food-processing environments from fish- (33% of isolates) and meat-processing facilities (60% of isolates) in Vancouver (Kovacevic et al AEM) China: 13 out of 27 sequenced isolates from raw meat samples harbored inla copies with PMSC (Shen et al., Food Microbiol.)

15 inla PMSC strains are capable to cross the placental barrier and infect mice and guinea pig fetuses 2 food-processing plant isolates with inla PMSC type 6 were used for pregnant guinea pig and pregnant mice oral infection Previous studies showed that these isolates were impaired in their ability to invade human intestinal epithelial (CaCo-2) cells (Jensen et al., 2007, JFP) and human throphoblastic (JAR) cells (Holch et al., 2010, AEM) InlA has been shown to not be necessary for crossing the fetoplacental barrier in the guinea pig (Bakardjiev et al., 2004, Infect. Immun.) and mice animal models (Lecuit et al., 1999, EMBO J.) although it is required in the gerbil model (Disson et al., 2008, Nature) and for invasion of human trophoblastic cell line (Lecuit et al., 2004, PNAS; Bakadjiev et al., 2004, Infect. Immun.)

16 inla PMSC 1 inla PMSC 2 inla full-length inla PMSC 1 inla full-length Fetoplacental invasion by inla PMSC strains This difference may be due to a higher ability to cross the intestinal barrier by the strain expressing full-length InlA EGD-inlA m No statistical analysis due to small sample size Guinea-pigs Mice Holch et al., J. Med. Microbiol.

17 prfa mutations linked to virulence attenuation Appear to be much less common than inla mutations that reduce virulence and infectivity Roche et al., 2005 (AEM) found 2 different prfa mutations linked to attenuated virulence attenuation in France An A-to-C transition leading to a Lysine-to-Threonine amino acid change (30% of low-virulence strains) A 7 nt insertion leading to a premature stop codon (12% of low-virulence strains) Lindback et al., 2011 (AEM) showed that the insertion can be reverted during high dose infection in mice López et al., 2013 (JFP) found an isolate with this same insertion in a study in the US Rupp et al., 2015 (Vet Microbiol) observed a 3 -end nt deletion resulting in a premature stop codon in prfa (truncated PrfA) associated with virulence attenuation in a bovine brain-slice culture

18 Take home messages L. monocytogenes represents a diverse set of strains, including some that are virulence attenuated A large proportion of L. monocytogenes found in foods may show mutations in the gene inla, which is essential for invasion of human intestinal epithelial cells Epidemiological, animal, and tissue culture data suggest significantly reduced infectivity of inla PMSC mutants Some studies suggest some remaining ability to cause pregnancy associated infections in mice and guinea pigs Virulence attenuating mutations in other genes (e.g., prfa) are rare To date findings have largely been ignored in risk assessments and regulatory policy