title: Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.
abstract: 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 actin-assembly-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-l-alanine 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.
authors.affiliation: Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: Kathrin.rychli@vetmeduni.ac.at.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Rychli
authors.affiliation: Functional Microbiology, Institute of Microbiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: Tom.grunert@vetmeduni.ac.at.
authors.forename: Tom
authors.initials: T
authors.lastname: Grunert
authors.affiliation: Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria; "Dunarea de Jos" University of Galaţi, 47 Domneasca St., 800008 Galaţi, Romania. Electronic address: luminita.pricope@ugal.ro.
authors.forename: Luminita
authors.initials: L
authors.lastname: Ciolacu
authors.affiliation: Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: Andreas.zaiser@vetmeduni.ac.at.
authors.forename: Andreas
authors.initials: A
authors.lastname: Zaiser
authors.affiliation: VetCORE facility for research, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: Ebrahim.razazzi@vetmeduni.ac.at.
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Razzazi-Fazeli
authors.affiliation: Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: Stephan.schmitz-esser@vetmeduni.ac.at.
authors.forename: Stephan
authors.initials: S
authors.lastname: Schmitz-Esser
authors.affiliation: Functional Microbiology, Institute of Microbiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: Monika.ehling-schulz@vetmeduni.ac.at.
authors.forename: Monika
authors.initials: M
authors.lastname: Ehling-Schulz
authors.affiliation: Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: Martin.Wagner@vetmeduni.ac.at.
authors.forename: Martin
authors.initials: M
authors.lastname: Wagner
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D005511:Food Handling; D005516:Food Microbiology; D059022:Food Safety; D005517:Foodborne Diseases / Q000382:microbiology; D006801:Humans; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics* / Q000254:growth & development* / Q000472:pathogenicity; D008088:Listeriosis / Q000382:microbiology / Q000473:pathology*; D008565:Membrane Proteins / Q000235:genetics; D058885:Multilocus Sequence Typing; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics; D010802:Phylogeny; D025341:Principal Component Analysis; D020543:Proteome / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013312:Stress, Physiological; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D020543:Proteome; C443120:SvpA protein, Listeria monocytogenes; C072980:actA protein, Listeria monocytogenes; D009238:N-Acetylmuramoyl-L-alanine Amidase
keywords: Alkaline stress; Exoproteome; Listeria monocytogenes; Persistence