Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains

International Journal of Food Microbiology 218 (2016) 17–26

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains Kathrin Rychli a,⁎, Tom Grunert b, Luminita Ciolacu a,c, Andreas Zaiser a, Ebrahim Razzazi-Fazeli d, Stephan Schmitz-Esser a, Monika Ehling-Schulz b, Martin Wagner a a

Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria Functional Microbiology, Institute of Microbiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria “Dunarea de Jos” University of Galaţi, 47 Domneasca St., 800008 Galaţi, Romania d VetCORE facility for research, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria b c

a r t i c l e

i n f o

Article history: Received 3 March 2015 Received in revised form 4 November 2015 Accepted 8 November 2015 Available online 10 November 2015 Keywords: Listeria monocytogenes Persistence Exoproteome Alkaline stress

a b s t r a c t The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actinassembly-inducing protein ActA and the surface virulence associated protein SvpA. Furthermore proteins involved in cell wall modification, such as the lipoteichonic acid primase LtaP and the N-acetylmuramoyl-Lalanine amidase (Lmo2591) are more abundant in EGDe than in the persistent strains and could indirectly contribute to virulence. In conclusion this study provides information about a set of proteins that could potentially support survival of L. monocytogenes in abiotic niches in food processing environments. Based on these data, a more detailed analysis of the role of the identified proteins under stresses mimicking conditions in food producing environment is essential for further elucidate the mechanism of the phenomenon of persistence of L. monocytogenes. © 2015 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author at: Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. E-mail addresses: [email protected] (K. Rychli), [email protected] (T. Grunert), [email protected] (L. Ciolacu), [email protected] (A. Zaiser), [email protected] (E. Razzazi-Fazeli), [email protected] (S. Schmitz-Esser), [email protected] (M. Ehling-Schulz), [email protected] (M. Wagner).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.11.002 0168-1605/© 2015 Elsevier B.V. All rights reserved.

Foodborne pathogens are able to survive for prolonged period of time in certain habitats. For example the facultative intracellular pathogen Listeria monocytogenes can persist in the food processing environment for months and even years (Ferreira et al., 2014; Larsen et al., 2014). L. monocytogenes is responsible for listeriosis, a rare but severe disease in humans and animals (Vazquez-Boland et al., 2001). The ability of L. monocytogenes to survive, adapt and grow under extreme environmental conditions (e.g. at low temperatures or in a wide pH range)

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allows L. monocytogenes to colonize the food processing environment, to cross-contaminate food products and to ultimately infect humans (Soni et al., 2011). There is evidence that certain strains are persistent, defined as the frequently recovery of genetically indistinguishable strains, whereas other subtypes are recovered only sporadically (Ferreira et al., 2014). Persistent strains have been linked to listeriosis outbreaks, for example the source of a multistate outbreak in the United States in 2000 has been traced back to an isolate also responsible for a single listeriosis case in 1988 (Olsen et al., 2005; Orsi et al., 2008). These data indicate that the outbreak strain persisted in the same plant at least 12 years underlying the public health importance of persistence. Various factors have been investigated for their role in persistence of L. monocytogenes including biofilm formation and stress and disinfectant resistance, with contradicting results (Carpentier and Cerf, 2011; Ferreira et al., 2014; Larsen et al., 2014). Hence our understanding of the mechanisms and the genetic determinants of persistence is still fragmented. Recently it has been reported that the presence of quaternary ammonium compounds, often used for disinfection, resulted in increased expression of genes involved in carbohydrate uptake (Casey et al., 2014; Fox et al., 2011b). Genome analysis of persistent L. monocytogenes strains revealed that certain conserved prophage regions and plasmids might provide important adaptation for survival in food producing environments (Holch et al., 2013; Orsi et al., 2008; Schmitz-Esser et al., 2015). Proteome studies using persistent L. monocytogenes have not been performed to date. The aim of this study was to compare the extracellular proteome of three representative persistent L. monocytogenes strains and the reference strain EGDe to identify proteins linked to persistence. The exoproteome is highly dynamic, reflects the adjustment to environmental conditions and plays an important role in virulence of L. monocytogenes (Cabrita et al., 2014; Dumas et al., 2008). Additionally we characterized the stress survival under acidic and alkaline conditions and the in vitro virulence potential of these strains. 2. Materials and methods 2.1. Bacterial growth conditions L. monocytogenes strains were incubated for 8 h at 37 °C in brain heart broth infusion (BHI, Merck), centrifuged 10 min at room temperature at 3220 × g, resuspended in 25 ml chemically defined medium (CM) consisting of RPMI-1640 (Life Technologies), 1% L-glutamine and 0.08 g/l ferric citrate and incubated overnight at 37 °C with shaking (120 rpm). Cultures were adjusted to OD 0.2 in 100 ml CM and incubated at 37 °C with shaking at 120 rpm. Bacterial cultures were harvested at early stationary phase by centrifugation (10 min, 3220 ×g, RT; Fig. S1). Each strain was cultured three times independently. 2.2. Protein preparation Supernatants were filtered using a 0.22 μm sterile filter (Millipore) and proteins were precipitated with cold trichloroacetic acid/acetone (final concentration 10%) supplemented with sodium deoxycholate (final concentration 0.2%) at 4 °C for 24 h. Precipitated proteins were collected by centrifugation (40 min, 48,400 ×g, 4 °C), washed 3 times with acetone, dried and dissolved in 30 mM Tris buffer, pH 8.5 containing 7 M urea, 2 M thiourea, and 4 M CHAPS. Protein samples were adjusted to pH 8.5, aliquoted and stored at −80 °C. 2.3. 2D differential gel electrophoresis Differential gel electrophoresis (DIGE) labelling and 2D separation was performed as previously described (Grunert et al., 2011; Radwan et al., 2008). Briefly, 25 μg of protein per sample (measured by using Bradford reagent) were labelled with CyDye™ Fluor minimal dyes (GE Healthcare Life Sciences). An internal standard consisting of all samples

was labelled with Cy2. Proteins were separated using 18 cm IPG Dry strips with linear pH gradient of pH 4–7 using an Ettan IPGphor3 isoelectric focusing unit (GE Healthcare) under the following conditions: 150 V 2 h, 300 V 2 h, 600 V 1 h, a gradient to 8000 V 0.5 h, 8000 V for 4 h, and 500 V for 4 h. The second dimension was carried out in an Ettan DALTsix electrophoresis system (GE, Healthcare). 2.4. Image acquisition and analysis Gels were scanned with a Typhoon 9400 scanner and image analysis was performed using the DeCyder™ software version 7.0 (GE Healthcare). The differential in-gel analysis (DIA) and the biological variation analysis (BVA) modules were used to perform spotcodetection, spot quantification by normalization and ratio calculation, gel-to-gel matching of spots and quantitative comparison of protein expression across multiple gels. The abundance of protein spots, presented as log10 standardized abundance, was calculated as log10 abundance of Cy3- or Cy5-labeled spot over log10 abundance of Cy2-labeled standard spot. Differentially abundant spots between the L. monocytogenes persistent strains and strain EGDe were selected according to volume ratio with a cut-off value of ≥ 2 and Student's test p b 0.05. Only protein spots present in greater than 80% of the images (8/9) were included in further analyses. Spot matching and quality of proteins of interest were manually checked to avoid false positives. Additionally principal component analysis (PCA) including all spots (one-way ANOVA, p b 0.05) was used to investigate the relation between the four strains and to test the reproducibility of the three independent experiments. 2.5. Protein identification Semi-preparative 2D gels (n = 3) with a protein load of 150 μg were used to recover enough material for MS analysis. Protein spots were detected by silver nitrate staining, manually excised, pooled and destained as previously described (Radwan et al., 2008). Digestion was carried out for 20 min at 4 °C by swelling the gel pieces in 50 mM ammonium bicarbonate containing 5 mM CaCl2 and 12.5 μg/μl trypsin (Promega). Excessive trypsin solution was removed and the gel piece was incubated for 8 h at 37 °C in 30 μl 50 mM ammonium bicarbonate. The supernatant was collected and the peptides were extracted three times with 50% acetonitrile (ACN), 5% trifluoroacetic acid (TFA). Supernatants were pooled, dried in a vacuum centrifuge, dissolved in 0.1% TFA and desalted using μZipTips C18 (Millipore). Proteins were identified either on a Matrix Assisted Laser Desorption Ionization Tandem Time-of-Flight (MALDI-TOF/TOF) mass spectrometer (Ultraflex II, Bruker Daltonics, Germany) or on a Nano HPLC-MS/ MS system coupled to an IonTrap (HCT Esquire, Bruker Daltonics). For MALDI-TOF/TOF analysis peptides were spotted onto an AnchorChip MALDI target plate pre-spotted with α-cyano-4-hydroxycinnamic acid (PAC target, Bruker Daltonics). Spectra processing and peak annotation was carried out using FlexAnalysis and Biotools (Bruker Daltonics). Data was searched at the National Center for Biotechnology Information (NCBI) homepage, limiting the taxonomy to Listeria using following parameters: MS tolerance 100 ppm; MS/MS tolerance 1 Da; one missed cleavage allowed; fixed modifications: carbamidomethylation of cysteins and variable modifications: oxidation of methionine. Identifications were considered statistically significant with p b 0.05. For nano-HPLC-MS sample pre-concentration and desalting was performed with a 1 mm pre-column cartridge (Dionex, Netherlands), separation on a 15 cm PepMap C18 column (Ultimate RSLC system, Dionex) with an injection volume of 1 μl (full loop) and a flow rate of 300 nl/min. Mobile phase A consisted of ultrapure water with 0.1% formic acid and B consisted of 80% ACN with 0.1% formic acid. A gradient from 4% B to 55% B within 30 min was followed by a washing step with 90% B. Data were directly analyzed by data-dependent Auto-MSn with following settings: Drying gas flow 10 l/min, drying gas temperature

K. Rychli et al. / International Journal of Food Microbiology 218 (2016) 17–26

150 °C, V cap typically 1500 V, capillary exit offset 139 V, trap drive 80 V with averages of 5. ICC was on; maximum accumulation time 150 ms, smart target 200,000, and MS scan range is 100–1500 m/z. Automatic MS/MS acquisition was done in peptide scan mode with MS(n) averages of 2, precursor ions of 3, active exclusion on (after 1 spectrum for 0.2 min). Single charged ions were excluded and the MS/MS scan range was 100–1800 m/z. 2.6. Bioinformatic analysis Proteins were further characterized using the databases Uniprot (http://www.uniprot.org/) and NCBI (www.ncbi.nlm.nih.gov). The subcellular protein localization was predicted by using the CBS prediction server (http://www.cbs.dtu.dk/services) for signal peptides (SignalP 4.0), LOCtree3 (https://rostlab.org/services/loctree3/) and the TOPCONES platform (http://topcones.net)(Tsirigos et al., 2015) for signal peptides and transmembrane domains. 2.7. Phosphoprotein staining Phosphoprotein staining of the semi-preparative 2D gels (150 μg protein load) was performed using Pro-Q Diamond Phosphoprotein Gel stain (Invitrogen) according to the manufacturer's instructions. PeppermintStick™ Phosphoprotein Molecular Weight Standard (Invitrogen) was used as a positive control. Gels were scanned with a Typhoon 9400 scanner. Subsequently proteins were visualized by silver staining. 2.8. Phylogenetic analyses by multilocus sequence typing L. monocytogenes multilocus sequence typing (MLST) is based on seven housekeeping genes: abcZ (ABC transporter), bglA (beta glucosidase), cat (catalase), dapE (succinyl diaminopimelate desuccinylase), dat (D-amino acid aminotransferase), ldh (L-lactate dehydrogenase), lhkA (histidine kinase) (Salcedo et al., 2003). We have retrieved fulllength MLST genes of 22 L. monocytogenes strains of different serotypes from GenBank. For each strain, the MLST genes were concatenated and aligned using Muscle implemented in MEGA6 using the Tamura-Nei evolutionary model and the maximum likelihood treeing method (Tamura et al., 2013). Gapless alignment was checked manually. Classification of the STs into clonal complexes was performed according to Cantinelli et al. (2013). 2.9. In vitro virulence assay Invasion efficiency of strain EGDe, 4423, 6179 and R479a were determined using human epithelial colon carcinoma Caco2; % of intracellular bacteria were determined in macrophage like THP1 cells according to Ciolacu et al. (2015). Briefly, bacteria were grown to stationary phase in CM as described in section 2.1. Cells were infected for 1 h at 37 °C with bacteria at a multiplicity of infection of 25. Cells were washed twice and incubated for 45 min with the appropriate media supplemented with 100 μg/ml gentamycin. Cells were washed and lysed with 1 ml Triton x-100. The number of intracellular bacteria was determined by plating serial dilutions on tryptic soya agar plates. Invasion efficiency (%) for Caco2 and % of intracellular bacteria for THP1 were calculated as the number of intracellular bacteria divided by the number of inoculated bacteria multiplied by 100. Each experiment was performed at least 4 times independently and mean value and SD have been calculated including all values (%).

19

°C. Colony forming units (CFU) were determined by serial plating on tryptic soya agar plates in triplicate. Survival is represented as log10 reduction. Each experiment was performed four times. 2.11. mRNA isolation and quantitative RT-PCR To investigate the expression of the lmo2637, namA, fhs and qoxA genes, one colony of L. monocytogenes EGDe and 6179 was grown overnight in 16 ml BHI broth at 37 °C with shaking (125 rpm). The bacterial culture was adjusted to an optical density (OD600) of 0.2 in a final volume of 30 ml CM. Cells were grown at 37 °C and 10 °C with shaking (125 rpm) to early stationary phase (7–8 h at 37 °C, 48–52 h at 10 °C). Cells were exposed to alkaline stress (in 15 ml CM adjusted to pH 9.5 with 1 M NaOH) for 0.5 h at the appropriate temperature (37 °C or 10 °C). Cells were harvested by centrifugation (3220 ×g, 10 min, at the appropriate temperature), resuspended in 0.4 ml RNAlater Solution (Life Technologies) and stored at 4 °C until RNA isolation. Experiments were performed in two biological independent replicates and repeated twice. RNA isolation was performed using the GeneJet RNA purification kit (Thermo Scientific) performed according to the manufacturer's instructions. Cell pellets were resuspended in lysis buffer and disrupted using beadbeating in Lysing Matrix A tubes with a FastPrep FP120 instrument (both MP Biomedicals) with the following parameters: three times 45 s at speed 5.5 at 4 °C. The remaining DNA was digested using the Turbo DNA-free Kit (Life Technologies) according to manufacturer's instructions. The absence of DNA was confirmed by performing a PCR targeting the 16S rRNA gene using BiomixRed (Bioline, Netherlands). (PCR conditions: 1 × 94 °C 5 min; 30 × 94 °C 30 s, 48 °C 30 s, 72 °C 1 min; 1 × 72 °C 5 min; 4 °C hold). RNA amount was quantified using Qubit RNA Kit and Qubit® 2.0 Fluorometer (Life technologies, USA). 50 ng RNA were used for cDNA synthesis with random primers using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) according to the protocol of the manufacturer. Primers targeting the L. monocytogenes 16S rRNA, lmo2637, namA, fhs and qoxA gene were designed using the online tool Primer3 (v. 0.4.0) (Table S1). BrilliantIII Ultra Fast SYBR QPCR Master Mix with Low ROX and the cycler Mx3000P™Stratagene® (both Agilent Technologies, USA) were used for qRT-PCR. Cycling conditions for all five genes were: 95 °C for 3 min, 40 cycles of the three steps: 95 °C for 15 s and 60 °C for 20 s and 72 °C for 30 s. A subsequent dissociation curve (95 °C for 1 min, 55–95 °C, 0.1 °C per s) was performed. A dilution series of genomic DNA from L. monocytogenes 6179 (1 to 10− 6 ng/μl) was used as an internal amplification control and for calculation of the primer efficiency (Table S1). Data were analyzed using Mx300P MxPro software (Stratagene). Each sample was measured in duplicates. Relative quantification was performed using the comparative Ct method. Values, given as x-fold of EGDe control at 37 °C or 10 °C, were normalized using 16S rRNA. Mean values ± SD of two biological replicates performed in duplicate and measured in duplicate were calculated. 2.12. Statistical analysis Microsoft Excel® 2007 and SPSS.22.0 were used for statistical analysis. We calculated the mean value and SD of invasion efficiency (%), % of intracellular bacteria and survival (log10 reduction) from four biological replicates, performed in triplicate. Values were compared statistically using ANOVA Post-Hoc test LSD. p-Values b 0.05 were considered to be significant. 3. Results and discussion

2.10. Survival under stress conditions

3.1. Bacterial strains and strain variability

Bacterial strains in stationary growth phase were incubated in CM adjusted to pH 2.5 (with HCl), and to pH 11 (with NaOH) for 2 h at 37

The main aim of this study was to obtain first insights in the protein expression of persistent strains analysing the extracellular proteome,

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which is highly dynamic. Since different studies showed that protein expression correlates with the serotypes we used four 1/2a L. monocytogenes strains to reduce the intraspecies variability (Dumas et al., 2008; Gormon and Phan-Thanh, 1995): strain EGDe (sequence type (ST)35) as a reference strain (Murray et al., 1926) and three persistent strains: 4423 (ST121), 6179 (ST121) and R479a (ST8, Table 1). ST121 and ST8 strains are often found in food and food processing plants (Haase et al., 2014; Hein et al., 2011; Martin et al., 2014; Wang et al., 2012) and several strains of ST121 strains have already been linked to persistence (Holch et al., 2013). We analyzed the exoproteome of L. monocytogenes strains grown to early stationary phase in a chemically defined medium (Fig. S1). There is evidence that bacteria face mild stress in early stationary phase due to nutrient limitation; underlined by the finding that the stress-responsive alternative sigma factor σB is induced during entry of L. monocytogenes in the stationary phase (Becker et al., 1998; Utratna et al., 2014). The proteins were analyzed using a pH gradient of 4 to 7, because the majority of the extracellular proteins have their isoelectric points in this pH range; however other than listeriolysin O (LLO) few extracellular proteins of L. monocytogenes have a high isoelectric point (pI) (Desvaux et al., 2010; Trost et al., 2005). In total we detected 1949 spots, of which 652 were present in all images. Principal component analysis (PCA) including all differentially expressed protein spots (n = 151, p ≤ 0.05) was used to investigate the distinctiveness of the strains (Fig. 1A). The biplot, displaying the first and the second components (accounting for 49 and 21% of variance), shows that the exoproteome of the strain EGDe (ST35) is distinct compared to the persistent strains. Additionally R479a (ST8) shows a high variance to the other strains, whereas strain 4423 and 6179 (both ST121) cluster together. Exoproteome profiling has already been successfully employed for correlating genotypic and phenotypic traits of L. monocytogenes (Cabrita et al., 2013), but also for other bacteria like Bacillus cereus (Ehling-Schulz et al., 2005). Phylogenetic analyses based on seven MLST genes revealed similar grouping of the four strains (Fig. 1B). Recently Schmitz-Esser et al. showed that the genomes of ST121 L. monocytogenes strains are highly similar to each other and show a remarkable high degree of conservation among their prophages and plasmids (Schmitz-Esser et al., 2015). Our results suggest that the ST of L. monocytogenes strains might not only correlate with virulence potential and variants of virulence factors like internalin A or LLO as we recently described (Ciolacu et al., 2015), but also with the exoproteome. 3.2. Differentially abundant proteins Analysis of the differences in spot intensity between strain EGDe and the three persistent strains, using a differential tolerance of ≥ two-fold (p b 0.05), resulted in 54 differentially expressed proteins, of those 24 spots were selected for protein identification (Fig. 2). The selection was based on a combination of the following criteria: expression pattern in the 2D DIGE analysis (extent of difference between strain EGDe and the persister strains), spot quantity (spot density in silver stained gels supplying sufficient amounts for MS analysis) and spot quality (shape and compactness of spots, nonoverlap with others) (Radwan et al., 2008). We were able to identify 22 spots (92%) corresponding to only 16 proteins as multiple protein species were detected due to posttranslational

modification or proteolytic processing (Schlüter et al., 2009) (Fig. 2, Table 2, Table S2). Of these 16 proteins only seven harbour a classical signal peptide or transmembrane domains. This is in line with several studies showing that almost half of the extracellular proteins of L. monocytogenes have no recognizable signal sequence for secretion (Desvaux et al., 2010; Schaumburg et al., 2004; Trost et al., 2005). Comparing the exoproteome of the persistent L. monocytogenes strains to strain EGDe revealed that the expression of five different proteins was significantly increased in the persistent strains, whereas 10 proteins were less abundant. 3.2.1. Stress-related proteins Four proteins suggested to be linked to stress response were upregulated in the persistent strains: The membrane anchored lipoprotein Lmo2637 (function unknown), reported to be upregulated in glucose starved planctonic cells and under alkaline stress conditions (Giotis et al., 2008; Giotis et al., 2010; Helloin et al., 2003); the AA3-600 quinol oxidase QoxA, a σB dependent protein, which is involved in the respiratory chain (Chaturongakul et al., 2011); the formate tetrahydrofolate ligase (FTHFS, lmo1877), involved in the one-carbon metabolism, playing a role in stress response e.g. hypoxic stress in Escherichia coli (Sah et al., 2015) or acidic stress in Streptococcus mutans (Crowley et al., 1997); and the NADPH dehydrogenase NamA, known to be increased in alkaline stress conditions in L. monocytogenes (Giotis et al., 2010). In Bacillus subtilis the homologous protein of NamA (YqiM, 62% amino acid identity), is upregulated under oxidative stress conditions and plays a role in detoxification of reactive oxygen species (Fitzpatrick et al., 2003). Two spots were identified as catalase. Catalase is a major factor involved in the oxidative stress response and has five phosphorylation sites in L. monocytogenes (Misra et al., 2011). One spot was found to be significantly more abundant in the exoproteome of the persistent strains, while the spot with a higher pI was less abundant compared to EGDe. Additionally we identified the spot located between these two significantly different abundant catalase spots as catalase. This result might indicate that in the persistent strains the catalase has a higher phosphorylation state compared to that found in EGDe. Phosphorylation of the catalase in at least two of the spots was confirmed using phosphostaining (Fig. S2). The abundance of protein in the spot with the lowest pI was too low for detection of phosphorylation using this method. While this is an interesting observation, the effect of the phosphorylation status on the function of catalase is unknown. 3.2.2. Virulence-related proteins Three proteins linked to virulence were significantly increased in the exoproteome of EGDe compared to the persistent strains: the membrane protein actin-assembly-inducing protein (ActA), responsible for actin-based motility of intracellular bacteria (Freitag et al., 2009), promoting efficient entry into non-phagoyctic cells (Garcia-del Portillo et al., 2011) but also critical for bacterial aggregation and biofilm formation (Travier et al., 2013); the iron dependent protein surface virulence associated protein (SvpA), involved in bacterial escape from the phagosome of macrophages (Borezee et al., 2001) and colonization of L. monocytogenes in the liver, spleen and intestines of mice (Newton et al., 2005) and the translation elongation factor Tu (EF-Tu). Although EF-Tu has no predictable secretion signal peptide, there is evidence that EF-Tu is also secreted and present at the bacterial the cell wall (Lenz et al., 2003; Schaumburg et al., 2004). Besides its role in protein

Table 1 Strains used in this study. Name

Source

Year of isolation

Country

Time of persistence

MLST-type

Genome accession numbers

Reference

EGDe 4423 6179 R479a

Animal tissue Cheese smear water Cheese Smoked salmon

1924 2004 2000 1996

USA Austria Ireland Denmark

n.a. 1997–2004 2000–2008 1996–1999

ST35 ST121 ST121 ST8

NC_003210 NZ_CBXR000000000.1 NZ_HG813249 NZ_HG813250 NZ_HG813247 NZ_HG813248

Murray et al. (1926) Stessl et al. (2014) Fox et al. (2011a) Vogel et al. (2001)

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Fig. 1. Principal component and phylogenetic analysis Principal component analysis (PCA) including all differentially abundant protein spots in the exoproteome EGDe, 4423, 6179 and R479a (one-way ANOVA, p ≤ 0.05, panel A). The first component (PC1) accounted for 49% and PC2 for 21% of the variance, respectively. Each point refers to one replicate of the experimental group. Maximium likelihood phylogenetic tree of L. monocytogenes strains based on MLST loci (panel B). L. monocytogenes sequence types are indicated. „ST“ denotes sequence types, „CC“ denotes clonal complexes. The tree is based on concatenated full-length MLST gene sequences and was calculated with MEGA 6 (Tamura et al., 2013) using the Tamura-Nei model. Bootstrap values (500× resampling) are indicated at the respective nodes. The bar represents the number of substitutions per site. The analysis involved 22 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 9039 positions in the final dataset.

synthesis, it has recently been reported that EF-Tu has additional biological functions potentially influencing the infection process. For example Archambaud et al. showed that EF-Tu is a substrate for Stp, a serine threonine phosphatase controlling bacterial survival in the infected host (Archambaud et al., 2005). Two proteins involved in cell wall modification are more highly expressed in EGDe: the lipoteichonic acid (LTA) primase LtaP, required for LTA synthesis (Campeotto et al., 2014; Webb et al., 2009) and the Nacetylmuramoyl-L-alanine amidase (Lmo2591), both might indirectly be involved in virulence. LTA is essential for binding of the virulence factor internalin B to the bacterial surface (Jonquieres et al., 1999). Additionally it has been shown that lack of incorporation of D-alanines into LTA resulted in attenuated virulence of L. monocytogenes in the mouse infection model (Abachin et al., 2002). So far function of the Nacetylmuramoyl-L-alanine amidase has not been investigated; however other amidase (peptidoglycan hydrolases) such as the autolysin amidase Ami (lmo2558) and Auto (lmo1076) are known to play a prominent role in virulence (Asano et al., 2012; Asano et al., 2011; Bublitz et al., 2009). Additionally, we detected higher expression of two proteins involved in glycerol metabolism, essential for intracellular growth of L. monocytogenes in the host cell (Gillmaier et al., 2012; Grubmuller et al., 2014) in the exoproteome of EGDe: the dihydroxyacetone kinase subunit DhaK and the succinate semialdehyde dehydrogenase GabD, both more highly expressed if glycerol is used as a carbon source (Joseph et al., 2008). There are indications that GabD contributes also to acidic tolerance of L. monocytogenes (Abram et al., 2008).

We detected also one spot that could be assigned to listeriolysin O (LLO), which was more highly expressed in the persistent strains. LLO can be present at least in seven protein spots corresponding to different protein species with a pI range from 6.6 to 7.2 (Dumas et al., 2009). However, the pH range used in this study (pH 4–7) was not optimal for adequate separation of all the isoforms of LLO. 3.2.3. Other proteins Furthermore the translation elongation factor Ts (EF-Ts), which is essential in protein synthesis, is more highly expressed in strain EGDe, but the extracellular function of EF-Ts is so far unknown. Furthermore the phage coat protein Lmo2296 was only present in the exoproteome of EGDe. Genome analysis (data not shown) revealed that the persistent strains R479a, 4423 and 6179 lack the comK prophage (A118-like prophage) of which lmo2296 is part. The exact function of this prophage is unknown, but recently is has been suggested that excision of the A118-like prophage, induced during intracellular growth, is necessary for phagosomal escape of L. monocytogenes in macrophages (Rabinovich et al., 2012). Other prophages and phage remnants integrated into comK in many L. monocytogenes strains and have been suggested to play a role in the adaptation to the food processing environment (Verghese et al., 2011). 3.3. In vitro virulence The expression pattern of proteins linked to virulence suggests a different virulence potential of the four strains used in this study. This conclusion was confirmed by cell culture assays.

Fig. 2. Exoproteome of L. monocytogenes strains Representative 2D-gels of the exoproteome of L. monocytogenes reference strain EGDe and the persistent strains 4423, 6179 and R479a labelled with CyDyes (green: cy3, red: cy5). Numbered spots indicate identified proteins.

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Table 2 Differentially abundant proteins in the exoproteome of persistent L. monocytogenes strains (4423, 6179 and R479a) compared to EGDe. Accession number Gen Bank/SwissProt

1a 1b 1c 2a 2b 3a

ActA (Actin- assembly-inducing protein) ActA ActA SvpA (surface virulence associated protein) SvpA Catalase

NC_003210.1 P33379

3b

Catalase

2.2

3c

Catalase

−6.8

4

FTHFS formate tetrahydrofolate ligase

5b 6

GabD succinate semialdehyde dehydrogenase GabD LLO listeriolysin O precursor

7a

N-acetylmuramoyl-L-alanine amidase

7b

N-acetylmuramoyl-L-alanine amidase

8

LtaP-Lipoteichonic acid primase

9

Dihydroxyacetone kinase subunit DhaK

10

EF-Ts Translation elongation factor Ts

11

Phage coat protein

12

NamA NADPH dehydrogenase

13

EF-Tu translation elongation factor Tu

14

QoxA AA3-600 quinol oxidase, subunit II

15

Lmo2637

16

Carbonic anhydrase

5a

Locus_ tag

pI theoret.

Mr(kDa) Predicted subcellular theoret. location

Function

actA

lmo0204

5.01

70.36

Cell membrane

Virulence

NC_003210.1 Q9KGV9

lmo2185

lmo2185

6.5

63.3

Cell wall anchored

Virulence associated

NP_466307.1 Q8Y3P10

kat

lmo2785

5.4

55.8

Cytoplasm, extracellular

Detoxification

NP_465401 Q8Y624 NP_464439 Q4EVI3 NP_463733 P13128 NP_466114 Q8Y464

Gene

Average ratiob −10.1 −12.6 −8.4 −3.4 −2.9 2.2

0.002 0.004 0.012 0.011 0.023 0.032 0.055 (n.s.) 0.0013

fhs

lmo1877

5.8

45.6

Cytoplasm

One-carbon metabolism

gabD

lmo0913

5.5

53.1

Cytoplasm

Metabolism

−2.0

0.029 0.0028 0.0061 0.0093

8.1

0.000027

hly

lmo0202

6.6

58.2

Extracellular

Virulence

−2.7 3.4

lmo2591

lmo2591

6.11

57.0

Cell surface

Cell wall modification

−2.7

lmo0664

lmo0664

5.4

69.3

Cell surface

Cell wall

−3.8

0.0006

lmo2695

lmo2695

4.7

34.8

Cytoplasm

Metabolism

−3.6

0.0041

tsf

lmo1657

4.8

26.1

Cytoplasm

Elongation factor

−3.3

0.001

lmo2296

lmo2296

5.2

35.6

−6.0

b0.0001

namA

lmo2471

6.0

37.2

Cytoplasm

Detoxification

tuf

lmo2653

4.8

43.4

Cytoplasm

Elongation factor

−2.2

qoxA

lmo0013

6.4

41.6

Membrane

Respiratory chain

49.8

lmo2637

lmo2637

5

27.3

Membrane

FMN binding

lmo0811

lmo0811

4.9

34.8

Cytoplasm

Metabolism

−59.6 NP_464191 Q8Y989 NP_466217 Q8Y3Y4 NP_465182 Q8Y6M7 NP_465820 Q8Y4Y3 NP_465994 Q4ES05 NP_466175 Q8Y422 NP_463546 Q8YAV0 NP_466160 Q8Y436 NP_464338 Q8Y8T3

p-Value

3.7

3.0 −4.0

b0.0001

0.0008 0.028 b0.0001 0.0035 0.036

pI: isoelectric point, Mr.: molecular weight, and n.s.: not significant. a Spotnumber according Fig. 2; a, b, c represent different spots (protein species) of the same protein. b Differentially abundant spots between persistent strains and strain EGDe were selected according to volume ratio with a cut-off value of ≥2 and Student's test p b 0.05. Positive ratio values indicate a higher abundance in the persister strains. Negative ratios indicate a higher abundance in strain EGDe.

K. Rychli et al. / International Journal of Food Microbiology 218 (2016) 17–26

Spot-numbera Protein name

K. Rychli et al. / International Journal of Food Microbiology 218 (2016) 17–26

Strain EGDe showed the highest invasion efficiency, followed by strain R479a, whereas strain 4423 and 6179 showed strongly attenuated invasion using intestinal epithelial Caco2 cells (Fig. 3A). Recent genome analysis showed that strain 4423 and 6179 harbor a truncated internalin A (inlA) gene, which has been suggested to be primarily responsible for the attenuated invasion (Schmitz-Esser et al., 2015). Furthermore the number of bacteria internalized in macrophagelike THP1 cells was slightly higher for EGDe compared to the persister strains (Fig. 3B), potentially due to the increased expression of SvpA, which is required for bacterial escape from the phagosome of macrophages (Borezee et al., 2001). Data on the virulence potential of persistent L. monocytogenes strains are limited and contradictive (Ferreira et al., 2014). Two studies suggest that persistent L. monocytogenes strains have lower virulence than clinical strains using in vitro and in vivo infection models (Holch et al., 2010; Jensen et al., 2008a). In contrast, another study reported that one persistent strain is fully virulent, crossing the fetal placental barrier (Jensen et al., 2008b). Since a significant greater proportion of L. monocytogenes strains from ready-to-eat foods than from clinical cases carries a truncated inlA (like two of the persister strains used in this study) we can speculate that persistent strains might be on average less virulent (Van Stelten et al., 2010). There are indications that the expression of virulence factors reduces the fitness of Listeria outside the host e.g. due to mutation in the main virulence regulator PrfA (Bruno and Freitag, 2010; Vasanthakrishnan et al., 2015; Xayarath and Freitag, 2012). On the other hand, the continuous presence in the food processing environment increases dramatically the risk of food contamination resulting in higher infection risk. The fact that numerous outbreaks have been linked to persistent strains underlines the health risk (Ferreira et al., 2014).

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Fig. 4. Stress survival Log10 CFU reduction of L. monocytogenes strains EGDe, 4423, 6179 and R479a in minimal media adjusted to pH 2.5 (panel A) and pH 11 (panel B). Values represent mean values ± SD of four biological replicates performed in triplicates. Different letters indicate statistically significant differences (p b 0.05).

3.4. Stress survival We analyzed the acidic (pH 2.5) and alkaline (pH 11) stress tolerance of EGDe, and the three persistent strains. The log CFU reduction of EGDe was slightly, but significantly lower under acidic stress

conditions compared to the persistent strains indicating a slightly higher survival rate (Fig. 4A). L. monocytogenes faces strong acidic challenge in the gastrointestinal tract during the infection process; therefore survival and adaptation to an acidic environment is essential for systemic infection (Gahan and Hill, 2014). The succinate semialdehyde dehydrogenase GabD, more highly expressed in EGDe, might among other proteins, contribute to acidic tolerance leading subsequently to higher pathogenicity (Abram et al., 2008). In contrast the persistent strains showed significantly higher survival under alkaline stress conditions (Fig. 4B). L. monocytogenes strains inhabiting food processing environments are frequently exposed to sublethal alkaline stress due to the alkaline nature of most of the detergents and some of the chemical sanitizers (Soni et al., 2011; Taormina and Beuchat, 2002). Therefore alkaline tolerance could facilitate persistence of L. monocytogenes. We further identified two proteins, the lipoprotein Lmo2637 and the NADPH dehydrogenase NamA, whose expression is known to be increased under alkaline stress conditions (Giotis et al., 2010). However, the biological role of these proteins has not yet been investigated. 3.5. Expression of stress related proteins under alkaline conditions

Fig. 3. In vitro virulence Invasion efficiency (%) using intestinal epithelial Caco2 (panel A) and % of intracellular bacteria in macrophage-like THP1 cells (panel B) of L. monocytogenes strains EGDe, 4423, 6179 and R479a. Values represent mean values ± SD of four biological replicates performed in triplicates. Different letters indicate statistically significant differences (p b 0.05).

We additionally determined gene expression of lmo2637, namA, fhs and qoxA of strain EGDe and 6179 grown to early stationary phase at 37 °C and 10 °C, the common temperature in the food producing environment (Fig. 5). Transcription of all four genes was significantly higher in the persister strain compared to strain EGDe at 37 °C, which is line with our exoproteome data. In parallel expression of namA, fhs and qoxA were significantly increased at 10 °C. Regarding lmo2637 we detected only a slightly, not significantly increased expression in the persister strain compared to strain EGDe. These data support our hypothesis that at least NamA, FTHS and QoxA have a role in the survival of persistent L. monocytogenes at low temperatures. We additionally investigated gene expression of lmo2637, namA, fhs and qoxA of strain EGDe and 6179 exposed to alkaline stress at 37 °C and

24

K. Rychli et al. / International Journal of Food Microbiology 218 (2016) 17–26

Fig. 5. Expression of lmo2637, namA, fhs and qoxA in alkaline stress conditions. Gene expression of lmo2637 (panel A), namA (panel B), fhs (panel C) and qoxA (panel D) of L. monocytogenes strain EGDe and 6179 (persister) grown to early stationary growth phase at 37 °C and 10 °C (control) and exposed 30 min to alkaline stress (pH 9.5) was determined by quantitative RTPCR. Values were normalized according 16S rRNA expression levels. Values given as x-fold of EGDe control (early stationary growth phase), represent mean values ± SD of two biological replicates performed and measured in duplicate. *Different letters indicate statistically significant differences (p b 0.05).

10 °C. We revealed a complex pattern. Gene expression of lmo2637 and namA was increased under alkaline stress in both strains at 37 °C, whereas fhs and qoxA gene expression was not induced. This is in line with previous transcriptome studies (Giotis et al., 2008; Giotis et al., 2010). Expression of lmo2637 was even higher in the persistent strain compared to strain EGDe at 37 °C.

Exposure of L. monocytogenes to alkaline stress at lower temperature (10 °C) resulted in increased expression of lmo2637 in both strains. In contrast gene expression of namA was unaffected in strain EGDe and even reduced in strain 6179 after alkaline stress treatment. Expression of fhs was slightly increased in strain EGDe, but decreased in the persister strain after alkaline stress exposure at 10 °C; whereas QoxA

K. Rychli et al. / International Journal of Food Microbiology 218 (2016) 17–26

expression was unaffected in both strains. These data suggest a certain role of the lipoprotein Lmo2637 in alkaline stress response of L. monocytogenes at low temperature e.g. during sanitation in the food processing environment. More studies are essential to elucidate the role of lmo2637, namA, fhs and qoxA in stress response and persistence.

4. Conclusion The comparison of the exoproteome of persistent strains and EGDe revealed that the two persistent ST121 strains have a similar extracellular proteome. A recent study showed that the ST121 L. monocytogenes genomes are highly similar to each other and reveal a remarkably high level of conservation among prophages and plasmids (SchmitzEsser et al., 2015). The exoproteome of ST121 persisters was to some extent different from the exoproteome of the short time persistent strain R479a (ST8), but all three persistent strains shared patterns of protein expression distinct from that seen in the control strain EGDe. Since exoproteome analysis is a very demanding analytical approach we could only compare three persistent strains with one control strain in this study. Experiments including more L. monocytogenes strains of different STs are necessary to prove the hypothesis of a possible ST-specific protein expression. Analyzing different protein expression between persistent strains and EGDe showed a higher abundance of proteins that could potentially facilitate the survival and persistence of L. monocytogenes in a food processing environment such as the NADPH dehydrogenase NamA and the lipoprotein Lmo2637. This is further supported by the finding that NamA, FHTS and QoxA are more highly expressed at 10 °C, the common temperature in food producing environments. However, further studies investigating the stress response and the intra- and extracellular proteome of various environmental strains exposed to conditions mimicking the food producing environment like low nutrient conditions are essential to elucidate the mechanisms involved in the phenomena of persistence of L. monocytogenes. Understanding the factors involved in persistence will support the development of strategies to combat the survival of L. monocytogenes in the food processing environment. Besides limiting the opportunities for contamination, appropriate cleaning and sanitation procedures and the elimination of growth niches is a critical point, since these allow Listeria to grow and survive despite cleaning and sanitation (Ferreira et al., 2014; Larsen et al., 2014).

Acknowledgements Strains 6179 and R479a were kindly provided by K. Jordan, Ireland and L. Gram, Denmark, respectively. We thank I. Miller, K. Hummel, K. Nöbauer and E. M. Wagner for excellent technical help. This study was supported by the 7th Framework Programme projects PROMISE (project number 265877) and by a start-up project from the University of Veterinary Medicine Vienna, Austria. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2015.11.002. References Abachin, E., Poyart, C., Pellegrini, E., Milohanic, E., Fiedler, F., Berche, P., Trieu-Cuot, P., 2002. Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes. Mol. Microbiol. 43, 1–14. Abram, F., Starr, E., Karatzas, K.A., Matlawska-Wasowska, K., Boyd, A., Wiedmann, M., Boor, K.J., Connally, D., O'Byrne, C.P., 2008. Identification of components of the sigma B regulon in Listeria monocytogenes that contribute to acid and salt tolerance. Appl. Environ. Microbiol. 74, 6848–6858. Archambaud, C., Gouin, E., Pizarro-Cerda, J., Cossart, P., Dussurget, O., 2005. Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of Listeria monocytogenes. Mol. Microbiol. 56, 383–396.

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