Correia, A.

Ferreira, Vânia

Silva, M. Ferreira da

Teixeira, Paula

English

Silva, Ferreira da M.

Ferreira, V.

Correia, A

Repositório Institucional da Universidade Católica Portuguesa

Detec%on	   of	   premature	   stop	   codons	   leading	  to	   truncated	   Internalin	  A	  among	   food	  and	  clinical	  strains	  of	  Listeria	  monocytogenes	  Ferreira	  da	  Silva,	  M.	  1,	  2,	  Ferreira,	  V.	  1,	  Correia,	  A.2,	  Teixeira,	  P.1	  1	  CBQF	  –	  Centro	  de	  Biotecnologia	  e	  Química	  Fina,	  Laboratório	  Associado,	  Escola	  Superior	  de	  Biotecnologia,	  Universidade	  Católica	  Portuguesa/Porto,	  Porto,	  Portugal	  	  2	  Department	  of	  Biology,	  University	  of	  Aveiro,	  3810-­‐193	  Aveiro,	  Portugal	  Magalhães	  (SFRH/BD/71704/2010)	  IntroductionMethodsConclusions      Listeria monocytogenes is a major intracellular bacterial foodborne pathogen capable of causing listeriosis, a severe invasive disease.  The elderly,  immunocompromised,  pregnant  women and neonates  are  risk  groups  for  listeriosis  [1].  In  developed countries, the globalization of the food industry and the increased consumption of ready-to-eat, particularly chilled foods, has increased listeriosis outbreaks in the last 30 years [2]. Listeriosis was the most frequent cause of death due to the consumption of contaminated food in Europe (2008-2011) [3], and the third foodborne infection concerning illness cost and quality life losses in the USA [4]; mortality is one of the highest among bacterial infections (> 30%) [3]. Although an active surveillance program for listeriosis doesn’t exist in Portugal, our research group demonstrated that listeriosis occurs at levels similar to those encountered in other countries [5, 6]. 	      Although 13 serotypes have been identified in L. monocytogenes, 95% of the strains isolated from foods and human clinical cases belong to serotypes 1/2a, 1/2b, 1/2c, and 4b [7]. Strains of L. monocytogenes can be also distinguished into four genetic lineages (I to IV). The majority of human listeriosis outbreaks have been linked to lineage I serotype 4b isolates, even though some outbreaks have been caused by lineage I serotype 1/2b and lineage II serotype 1/2a isolates [8].  The majority of studies involving clinical isolates characterization have been performed by France and USA research groups, and point out that lineage I isolates are overrepresented among isolates from human listeriosis cases (predominantly serotype 4b), while lineage II isolates appear to be overrepresented among food isolates [9]. As overrepresentation of lineage I strains among human listeriosis cases cannot be attributed to more frequent exposure to these strains it has been hypothesized that lineage I isolates have a higher pathogenicity in comparison to isolates of lineage II. 	      Mutations leading to a PMSC in inlA  are responsible for attenuated mammalian virulence. In this study, as previously observed by other authors, PMSC mutations in inlA were more common in food isolates (4 /11) and underrepresented in clinical isolates (1/11), despite the relatively low number of strains analyzed. Previous molecular epidemiology studies reported that a significant proportion of L. monocytogenes strains isolated from food products carry PMSC mutations in inlA, indicating that there is a significant subpopulation of L. monocytogenes strains in the food supply with a limited ability to cause human disease. Taken together, in addition to the significance of these findings for food safety and public health, these data contribute to our understanding of the ecology and evolution of L. monocytogenes. Future efforts to elucidate the selective advantages associated with  the  production  of  a  truncated  and  secreted  InlA protein  may  provide  a  better  understanding  of  the  function  of  L. monocytogenes internalins in different hosts and nonhost environments.	      After the ingestion of contaminated food, L. monocytogenes has the ability to induce its own entry into mammalian cells such  as  macrophages,  epithelial  cells  and  endothelial  cells  of  the  gastrointestinal  tract  [10].  Internalin  A protein  (InlA) encoded by inlA has a key role in L. monocytogenes infection mechanism. InlA facilitates uptake of L. monocytogenes  by host cells expressing certain isoforms of E-cadherin, making the interaction between InlA and E-cadherin a critical first step for crossing the intestinal barrier during the initial stages of infection, in a process called internalization. A previous study characterized a set of paired isogenic mutants with and without an inlA premature stop codons (PMSC) using an intragastric guinea  pig  infection  model  demonstrate  that  inlA PMSC  mutations  appear  to  be  causally  associated  with  attenuated mammalian virulence [11]. L. monocytogenes isolates that carry a PMSC in inlA produce a truncated form of InlA that is secreted rather  than anchored to the bacterial  cell  wall  and demonstrate attenuated invasion of  Caco-2 human intestinal epithelial cells. [12, 13]. A number of studies suggests that lineages differ in their virulence characteristics; lineage II in particular including a considerable proportion of virulence-attenuated isolates (>30%) due to premature stop codons in inlA, while serotypes 4b and 1/2b isolates typically harbor a full length intact InlA [8, 9]. 	      While there is substantial information on L. monocytogenes genetic diversity and epidemic potential, additional studies may reveal additional diversity and contribute to understanding on how L. monocytogenes subtypes differ in their virulence potential. This study aimed to characterize 22 L. monocytogenes strains, collected in Portugal, from human clinical cases (n=11) and different food products (n=11) in terms of invasion efficiency in Caco-2 cell line and detection of virulence-attenuated mutations in inlA gene. 	Prevalence of PMSC in L. monocytogenes strains	Table 2. Characterization of L. monocytogenes strains with PMSC.	Acknowledgements We	  thank	  Dr.	  Didier	  Cabanes	  for	  the	  help	  with	  the	   invasion	  assays	   in	  Caco-­‐2	  cell	   line	  at	  the	  Molecular	  Microbiology	  Research	  Group,	   Ins%tute	  for	  Molecular	  and	  Cell	  Biology.	  Financial	  support	  by	  Pest-­‐OE/EQB/LA0016/2011	  -­‐	  FCT.	  Financial	  support	  for	  author	  Vânia	  Ferreira	  was	  provided	  by	  postdoctoral	  fellowship	  SFRH/BPD/72617/2010	  FCT.	  	  This	  work	  was	  supported	  by	  Na%onal	  Funds	  from	  FCT	  –	  Fundação	  para	  a	  Ciência	  e	  a	  Tecnologia	  through	  project	  PEst-­‐OE/EQB/LA0016/2011.	   L. monocytogenes strains	inoculum (~107 cells/ml) 	Infection	60 min, 37 ºC, 7% CO2	Gentamicin 20% action	90 min, 37 ºC, 7% CO2	Triton 0,2% (v/v)	10 min, RT	~104 Caco-2 cells/ml 	1.  Serial dilutions in PBS	2.  Spread in BHI agar plates	3.  Incubation 37 ºC, 24h	4.  Enumeration (CFU/ml)	Strains	 Origin	 Geno-serogroup	Mutation type b	Predicted truncated InlA (aa)	Invasion efficiency (%)	1216	 Food	 1/2b	 6	 492	 0.9	1761	 Clinical	 1/2a	 6	 492	 9.5	1305	 Food	 1/2c	 11	 685	 1.4	747	 Food	 1/2b	 11	 685	 54.3	842	 Food	 1/2c	 12	 576	 1.4	Invasion efficiency in L. monocytogenes strains with PMSC mutations in inlA show	reduced invasion efficiency in Caco-2 	Figure 1. Caco-2 average invasion efficiency for selected L. monocytogenes strains, normalized to that of the control strain FSL F8-146 (full length inlA strain).	Results       Table 2 shows the 3 different mutation types leading to PMSC detected in five L. monocytogenes strains recovered from food and clinical sources, belonging to different geno-serogroups. The remaining seventeen strains tested did not show PMSC in inlA.	      The invasion efficiency in Caco-2 cell of the strains with PMSC were evaluated and compared with the positive control (FSL F8-146), 100% efficient.  	Strain Code	 Origin	 Sample	Geno-Serogroupa	747	 Food 	 Cheese 	 IIb 	842	 Food 	 Plant environment	 IIc 	854	 Food 	 Lettuce salad 	 IVb 	903	 Food 	 Roast chicken 	 IVb 	925/3 	 Food 	 Goat cheese 	 IIb 	930/1 	 Food 	 Goat milk 	 IIb 	994/1 	 Food 	 Goat milk 	 IIa 	1044	 Food 	 Meal 	 IVb 	1216	 Food 	 Raw chicken 	 IIb 	1305	 Food 	 Sausage 	 IIc 	2652	 Food 	 Cheese 	 IVb 	1001	 Clinical 	 Blood 	 IVb 	1547	 Clinical 	 Blood 	 IIb 	1761	 Clinical 	 CSF 	 IIb 	1891	 Clinical 	 CSF 	 IIb 	2065	 Clinical 	 Blood 	 IIb 	2074	 Clinical 	 Blood 	 IVb 	2086	 Clinical 	 Blood 	 IIa 	2092	 Clinical 	 Blood 	 IVb 	2103	 Clinical 	 Blood 	 IIa 	2562	 Clinical 	 Blood 	 IVb 	2666	 Clinical 	 Blood 	 IVb 	      1. Listeria monocytogenes strains		       For this study a set of 22 bacterial isolates from food and clinical  origin collected in Portugal  between were selected from  the  L.  monocytogenes  culture  collection  of  Escola Superior de Biotecnologia. 		      Table 1. Description of L. monocytogenes strains	a Detection of serotype-specific marker genes was performed using a multiplex-PCR assay previously reported by Doumith et al. [13]. This  assay  differentiates  isolates  in  five  major  subtypes,  which each  represents  more  than  one  serotype,  including  (i)  geno-seroproup IIa (which includes serotypes 1/2a and 3a);  (ii)  geno-serogroup IIb (serotypes 1/2b, 3b, and 7); (iii) genoserogroup IIc (serotypes 1/2c and 3c ); and (iv) genoserogroup IVb (serotypes 4b, 4d, and 4e).	      2. Caco-2 invasion assays		      For  invasion assays in the human intestinal epithelial caco-2 cell line a laboratory control strain presenting a full-length  InlA (FSL F8-146)  and  a  strain  presenting  an  inlA PMSC (FSL F8-144) (previoully reported by Ferreira et al. [15])  were included as controls  in each invasion assay.  At least three independent invasion assays were performed for each  isolate.  Results  were  reported  as  percent  invasion efficiency  (i.e.,  bacterial  numbers  recovered/bacterial numbers inoculated), normalized to the values obtained for the positive control strain (set as 100%). 		Intracellular L. monocytogenes cells were collected	      3. inlA sequencing		     L.  monocytogenes  strains  were  screened  for  the presence  of  PMSCs  in  inlA,  which  encodes  a  protein critical  for  invasion  of  Caco-2  cells.  Primer  sequences, PCR conditions, and sequencing strategies have previously been detailed [16]. 	DNA extraction	1. Grow of 1 colony on BHI agar, at 37 ºC, overnight	2. Centrifugation (10.000 g, 10 min) of 250 µL of bacterial cells	3. Pellet resuspention  in 95 µL of 1xPCR buffer and 4 µL of 50 mg/mL lysozyme solution	4. Incubation at RT, 15 min	5. Addiction of 1 µL of 20 mg/mL proteinase K, 58 ºC, 60 min	6. Enzymes heat inactivation , 95 ºC, 10 min	PCR amplification of inlA											Four pairs of primers:	•  inlA proF/inlA proR, 	•  inlA F1/inlA S1R	•  inlA F2/inlA S2R 	•  inlA seqF/inlA R		KAPA HiFi DNA Polymerase	PCR product Purification - EzWay PCR Clean-Up Kit 		DNA Sequencing - Macrogen Inc., Netherlands		Sequence analysis - Geneious 4.8.2 program; Mega 5.05. 	b	  Seventeen mutations leading to PMSC in inlA have been detected between 1998 and 2010. In this study mutations have been classified according to categorization proposed by Van Stelten et al. [18]. 	        From the 22 L. monocytogenes strains characterized only five presented PMSC in inlA, namely one clinical isolate  (geno-serogroup IIa,  lineage II),  and four food isolates (two genoserogroup IIb isolates,  lineage I,  and two geno-serogroup IIc, lineage II). These mutation types have been previously described in isolates from France, Portugal and USA [12, 15, 17]. 	      Phenotypically, it was possible to relate the presence of PMSCs with invasion efficiencies below that of the positive control FSL F8-146, that shows a full-length InlA. 		0.0	  20.0	  40.0	  60.0	  80.0	  100.0	  1216	   FSL	  F8-­‐144	  842	   1305	   1761	   747	   FSL	  F8-­‐146	  Invasion	  Efficiency	  (%)	  Strain	  code	  1. Swaminathan, B. and P. Gerner-Smidt, 2007. Microbes Infect. 9: 1236–1243; 2. Cartwright, E. L. et al., 2013. Emerg. Infect. Dis. 19: 1-9; 3. EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control), 2013. EFSA J. 11:3129:-250; 4. Hoffmann, et al., 2012. J. Food Protect. 75: 1292-1302; 5. Almeida, G. et al., 2006. BMC Infect. Dis. 6:152-156; 6. Almeida, G., et al., 2010. Eur. J. Clin. Microbiol. Infect. Dis. 29:1219-1227; 7. McLauchlin, J., et al., 2004. Int. J. Food Microbiol. 92:15–33; 8. Orsi, R. H., et al., 2006. Int. J. Med. Microbiol. 301:79–96; 9. Nightingale, K. K., et al., 2006. J. Clin. Microbiol. 44:3742–3751; 10. Cossart, P. 2002. Int. J. Med. Microbiol. 291: 401-409; 11. Nightingale, K.K., et al., 2008. Appl. Environ. Microbiol. 74: 6570–6583; 12. Olier, M., et al., 2003. Infect. Immun. 71:1217–1224; 13. Orsi, R. H., et al., 2007. Microbiol. 153:2666–2678; 14. Doumith, M., et al., 2004. J. Clin. Microbiol. 42:3819-3822; 15. Ferreira, V., et al., 2011. Appl. Environ. Microbiol. 77:2701-2715; 16. Nightingale, K. K., et al., 2005. Appl. Environ. Microbiol. 71:8764-8772; 17. Van Stelten, A., et al., 2010. Appl. Environ. Microbiol. 2010: 2783-2790.	References

Detection of premature stop codons leading to truncated internalin a among food and clinical strains of Listeria monocytogenes

 1                   PORTUGUESE CONGRESS OF  MICROBIOLOGY AND BIOTECHNOLOGY Micro Biotec‘13 6th – 8th December | Aveiro Portugal Abstracts Book   431   Microbial Pathogenesis P338/F09 DETECTION OF PREMATURE STOP CODONS LEADING TO TRUNCATED INTERNALIN A AMONG FOOD AND CLINICAL STRAINS OF LISTERIA MONOCYTOGENES   M. Ferreira da Silva1; V.Ferreira2; A. Correia1; P.Teixeira2  1Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal 2CBQF – Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Porto, Portugal  Listeria monocytogenes is a food-borne pathogen capable of causing listeriosis, a severe invasive disease and responsible for sporadic outbreaks. Internalin A protein (InlA) encoded by inlA has a key role in the infection mechanism, being necessary for L. monocytogenes to cross the intestinal barrier and to establish a systemic infection. Studies on inlA in L. monocytogenes have shown that mutations leading to premature stop codons (PMSC) occur naturally and are associated with attenuated virulence of this pathogen. The aim of this study was to evaluate the prevalence of PMSCs in inlA of eleven food and eleven clinical strains of L. monocytogenes from Portugal. The characterization was accomplished genetically by inlA sequencing to find mutations carrying PMSC leading to production of a truncated and secreted InlA. The PMSC were detected in three strains recovered from different food products (namely, from raw chicken, non-fermented sausage, and cheese), one strain recovered from a food-processing environment sample, and in one strain from a human clinical case. These strains belonged to different geno-serogroups, including: 1/2a-3a, 1/2b-3b-7 and 1/2c-3c. The mutations found lead to a predicted truncated InlA with 492, 576 and 685 amino acids, have been previously reported by other authors. The remaining seventeen strains tested did not show PMSC in inlA. Invasion efficiency of strains with PMSC in the human colorectal adenocarcinoma epithelial cell line (Caco-2 cells) was further evaluated. The results show a lower invasiveness efficiency of strains with PMSC in inlA compared to the positive control strain. InlA protein is necessary to cross the intestinal barrier, thus being a critical virulence factor promoting L. monocytogenes infection. When inlA shows PMSC leading to a truncated form of InlA, the isolate has reduced invasion efficiency. Understanding the importance of attenuated-virulence strains with PMSC in inlA, occurring mainly in foods, could be explored to prevent risk of human disease. Despite the important results obtained by Caco-2 invasion assays, it is necessary to study other in vitro and animal models to understand the pathophysiology of listeriosis in vivo.    

Detection of premature stop codons leading to truncated internalin a among food and clinical strains of listeria monocytogenes

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Detection of premature stop codons leading to truncated internalin a among food and clinical strains of listeria monocytogenes

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