Changes in global gene expression of Vibrio parahaemolyticus induced by cold- and heat-stress

Background Vibrio (V.) parahaemolyticus causes seafood-borne gastro-intestinal bacterial infections in humans worldwide. It is widely found in marine environments and is isolated frequently from seawater, estuarine waters, sediments and raw or insufficiently cooked seafood. Throughout the food chain, V. parahaemolyticus encounters different temperature conditions that might alter metabolism and pathogenicity of the bacterium. In this study, we performed gene expression profiling of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C to describe the cold and heat shock response. Methods Gene expression profiles of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C were investigated via microarray. Gene expression values and RT-qPCR experiments were compared by plotting the log2 values. Moreover, volcano plots of microarray data were calculated to visualize the distribution of differentially expressed genes at individual temperatures and to assess hybridization qualities and comparability of data. Finally, enriched terms were searched in annotations as well as functional- related gene categories using the Database for Annotation, Visualization and Integrated Discovery. Results Analysis of 37 °C normalised transcriptomics data resulted in differential expression of 19 genes at 20 °C, 193 genes at 4 °C, 625 genes at 42 °C and 638 genes at 15 °C. Thus, the largest number of significantly expressed genes was observed at 15 and 42 °C with 13.3 and 13 %, respectively. Genes of many functional categories were highly regulated even at lower temperatures. Virulence associated genes (tdh1, tdh2, toxR, toxS, vopC, T6SS-1, T6SS-2) remained mostly unaffected by heat or cold stress. Conclusion Along with folding and temperature shock depending systems, an overall temperature-dependent regulation of expression could be shown. Particularly the energy metabolism was affected by changed temperatures. Whole-genome gene expression studies of food related pathogens such as V. parahaemolyticus reveal how these pathogens react to stress impacts to predict its behaviour under conditions like storage and transport

Population analysis of Vibrio parahaemolyticus originating from different geographical regions demonstrates a high genetic diversity

Vibrio parahaemolyticus is frequently isolated from environmental and seafood samples and associated with gastroenteritis outbreakes in American, European, Asian and African countries. To distinguish between different lineages of V. parahaemolyticus various genotyping techniques have been used, incl. multilocus sequence typing (MLST). Even though some studies have already applied MLST analysis to characterize V. parahaemolyticus strain sets, these studies have been restricted to specific geographical areas (e.g. U.S. coast, Thailand and Peru), have focused exclusively on pandemic or non-pandemic pathogenic isolates or have been based on a limited strain number. To generate a global picture of V. parahaemolyticus genotype distribution, a collection of 130 environmental and seafood related V. parahaemolyticus isolates of different geographical origins (Sri Lanka, Ecuador, North Sea and Baltic Sea as well as German retail) was subjected to MLST analysis after modification of gyrB and recA PCRs. The V. parahaemolyticus population was composed of 82 unique Sequence Types (STs), of which 68 (82.9%) were new to the pubMLST database. After translating the in-frame nucleotide sequences into amino acid sequences, less diversity was detectable: a total of 31 different peptide Sequence Types (pSTs) with 19 (61.3%) new pSTs were generated from the analyzed isolates. Most STs did not show a global dissemination, but some were supra-regionally distributed and clusters of STs were dependent on geographical origin. On peptide level no general clustering of strains from specific geographical regions was observed, thereby the most common pSTs were found on all continents (Asia, South America and Europe) and rare pSTs were restricted to distinct countries or even geographical regions. One lineage of pSTs associated only with strains from North and Baltic Sea strains was identified. Our study reveals a high genetic diversity in the analyzed V. parahaemolyticus strain set as well as for geographical strain subsets, with a high proportion of newly discovered alleles and STs. Differences between the subsets were identified. Our data support the postulated population structure of V. parahaemolyticus which follows the 'epidemic' model of clonal expansion. Application of peptide based AA-MLST allowed the identification of reliable relationships between strains

Prevalence, diversity and stress response of Vibrio parahaemolyticus

Vibrio spp. sind ubiquitär in marinen Lebensräumen verbreitet und können regelmäßig aus der Umwelt und aus Lebensmitteln (z. B. Garnelen und Krabben) isoliert werden. Der Verzehr von bzw. Kontakt zu Lebensmitteln, die Vibrio spp. enthalten, kann beim Menschen zu Erkrankungen führen. Die Spezies sind durch eine hohe genetische Diversität gekennzeichnet. So kann bei V. parahaemolyticus z. B. zwischen pandemischen Typen und umweltassoziierten Stämmen unterschieden werden. Neben der genetischen Ausstattung spielen auch Umweltbedingungen, wie z. B. Hitze oder Kälte, eine entscheidende Rolle, Resistenzen gegenüber der Prozessierung und Zubereitung von Lebensmitteln auszubilden bzw. Krankheiten auszulösen. In 28 % der im deutschen Einzelhandel erstandenen Garnelen- und Krabbenerzeugnisse konnten eine oder mehrere Vibrio spp. nachgewiesen werden. Dabei war in nahezu 50 % der Vibrio spp.-positiven Proben mindestens ein V. parahaemolyticus-Isolat vorhanden. In 45,5 % der V. parahaemolyticus-positiven Proben konnte trh und in 4,5 % tdh/trh nachgewiesen werden. Mit Hilfe der Multi-Locus-Sequenz-Typisierung (MLST) konnte eine hohe genetische Diversität für die verschiedenen untersuchten V. parahaemolyticus- Stamm-Gruppen gezeigt werden. Dabei wurde, wie für Umweltstammsammlungen typisch, ein hoher Anteil neuer Allele und Sequenztypen identifiziert. Stammcluster enthielten hauptsächlich Stämme, die vom selben Kontinent stammten. Die hohe Diversität der Sequenztypen führte zu wenig verlässlichen Verbindungen bei den Analysen auf Nukleotid-Ebene. Die Verlässlichkeit konnte erhöht werden, indem die Beziehungen auf Peptid-Ebene mit Hilfe des neu entwickelten AA-MLST-Typisierungsschemas untersucht wurden. Die dabei beobachtete Abnahme der Diversität beruht vor allem darauf, dass nur nicht- synonyme Nukleotidsubstitutionen zu einer veränderten Aminosäure führen. Auf Peptid-Ebene zeigten die Cluster keine Abhängigkeit zur geographischen Herkunft der Stämme. Vor allem die häufig auftretenden Peptid-Sequenztypen waren durch eine globale Verbreitung gekennzeichnet. Allerdings konnten auch nur lokal auftretende Peptid-Sequenztypen identifiziert werden. Die Analyse der Populationsstruktur ergab – übereinstimmend mit der Literatur –, dass diese dem epidemischen Modell der klonalen Expansion folgt und sich vor allem durch die Lebensweise von V. parahaemolyticus erklären lässt, die häufige Anpassungen auch auf genetischer Ebene nötig macht. Die Studien zur Veränderung der Genexpression bei V. parahaemolyticus ergaben, dass Temperaturstress – sowohl bei Kälte als auch Hitze – zu einer globalen Anpassung der Expression durch Repression und Induktion führt. Bei niedrigen Temperaturen wurden vor allem Gene induziert, die mit der Anpassung an atypische Umweltbedingungen assoziiert sind. Dazu zählen u. a. die klassischen Kälteschock-Proteine (CSP), Proteine zur Modifikation der DNA-Topologie, Reparatur der DNA sowie für die Biosynthese und Degradation von Membranbestandteilen. Bei Hitze überwog dagegen die Induktion von Genen, die am Metabolismus oder an Transportprozessen beteiligt sind. Zusätzlich wurden die klassischen Hitzeschock-Proteine (HSP) induziert. Generell konnte eine antagonistische Expression von CSPs und HSPs beobachtet werden. Durch Hitze wurden Gene induziert, die ggf. zu einer Kreuzprotektion gegenüber oxidativem Stress führen könnten. Dagegen könnte durch Kälte die Resistenz gegenüber Säurestress erhöht werden, da Gene der molekularen Säurestress-Antwort induziert wurden. Von den klassischen Pathogenitätsfaktoren, wie z. B. Exotoxinen und Sekretionssystemen, wurden keine umfassend durch Temperaturstress induziert. Allerdings wurde bei 42 °C eine Induktion von Genen beobachtet, die an der Adhärenz, am Schwärmen und an der Auflösung von Geweben beteiligt sind. Somit könnten die Kolonisierung und/oder Invasion von V. parahaemolyticus nach einem Hitzeschock erleichtert sein.Vibrio spp. are distributed ubiquitously in marine habitats and are frequently isolated from the environment and seafood (e. g. prawns and mussels). The consumption of and contact to Vibrio spp. containing products can lead to infections in humans. The species are characterized by a high genetic diversity. V. parahaemolyticus can be differentiated for example into pandemic and environmental strains. Besides the genetic properties, environmental conditions like heat or cold can play a decisive role to mount a resistance against processing and preparation methods of foods or the ability to cause infections. At least one Vibrio spp. was present in 28% of the purchased retail prawn and shrimp samples. Almost 50% of the Vibrio spp.-containing samples contained a minimum of one isolate of V. parahaemolyticus. In the V. parahaemolyticus-positive samples trh and in tdh/trh were detected in 45.5% and 4.5%, respectively. Multi Locus Sequence Typing (MLST) revealed a high genetic diversity of all strain groups analyzed. Thereby a high number of new alleles and sequence types was identified as expected for environmental strain collections. Clusters of strains contained mostly strains originating from the same continent. The portion of supra-regionally distributed sequence types differed among the analyzed strain collections. The high diversity of sequence types led to less reliable relationships on nucleotide level. Reliability could be increased by applying the newly developed AA-MLST typing scheme to analyze relationships on peptide level. The observed decrease in diversity is mainly due to the fact that mostly non-synonymous nucleotide substitutions result in an altered amino acid. On peptide level clusters were not related to the geographic origin of strains. Especially the common peptide sequence types were characterized by a global distribution. However peptide sequence types were identified that showed a local distribution only. The analysis of the population structure of V. parahaemolyticus revealed – in concordance to the literature – the epidemic model of clonal expansion. Since the lifestyle of V. parahaemolyticus in the marine habitat frequently requires adaptations also on genetic level. The conducted studies on the global gene expression of V. parahaemolyticus revealed a global adaptation of expression via repression and induction induced by temperature stresses – by heat as well as by cold. At low temperatures especially genes were induced that are associated to adaptation to atypical conditions, including classical cold shock proteins (CSP), proteins for modification of DNA-topology, DNA-repair as well as for biosynthesis and degradation of membrane components. At high temperatures the induction of genes that are associated to metabolism and transport predominated. Additionally the classical heat shock proteins (HSP) were induced. In general an antagonistic expression of CSPs and HSPs at low and high temperatures was observed. Genes that could lead to a cross protection against oxidative stress were induced by heat. In contrast stress from cold temperatures could improve the resistance against low pH as genes associated to the molecular acid stress response were induced. None of the classical pathogenicity markers (e.g. exotoxins and secretion systems) was induced extensively by temperature stress. However at 42°C an induction of genes was monitored that are related to adherence, swarming and degradation of tissues. Consequently colonization and/or invasion of V. parahaemolyticus could be eased after heat shock

Changes in global gene expression of Vibrio parahaemolyticus induced by cold- and heat-stress

Background Vibrio (V.) parahaemolyticus causes seafood-borne gastro-intestinal bacterial infections in humans worldwide. It is widely found in marine environments and is isolated frequently from seawater, estuarine waters, sediments and raw or insufficiently cooked seafood. Throughout the food chain, V. parahaemolyticus encounters different temperature conditions that might alter metabolism and pathogenicity of the bacterium. In this study, we performed gene expression profiling of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C to describe the cold and heat shock response. Methods Gene expression profiles of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C were investigated via microarray. Gene expression values and RT-qPCR experiments were compared by plotting the log2 values. Moreover, volcano plots of microarray data were calculated to visualize the distribution of differentially expressed genes at individual temperatures and to assess hybridization qualities and comparability of data. Finally, enriched terms were searched in annotations as well as functional-related gene categories using the Database for Annotation, Visualization and Integrated Discovery. Results Analysis of 37 °C normalised transcriptomics data resulted in differential expression of 19 genes at 20 °C, 193 genes at 4 °C, 625 genes at 42 °C and 638 genes at 15 °C. Thus, the largest number of significantly expressed genes was observed at 15 and 42 °C with 13.3 and 13 %, respectively. Genes of many functional categories were highly regulated even at lower temperatures. Virulence associated genes (tdh1, tdh2, toxR, toxS, vopC, T6SS-1, T6SS-2) remained mostly unaffected by heat or cold stress. Conclusion Along with folding and temperature shock depending systems, an overall temperature-dependent regulation of expression could be shown. Particularly the energy metabolism was affected by changed temperatures. Whole-genome gene expression studies of food related pathogens such as V. parahaemolyticus reveal how these pathogens react to stress impacts to predict its behaviour under conditions like storage and transport.BioMed Central open acces

RESEARCH ARTICLE Open Access

Changes in global gene expression of Vibrio parahaemolyticus induced by cold- and heat-stres

Additional file 2: of Changes in global gene expression of Vibrio parahaemolyticus induced by cold- and heat-stress

Differentially expressed genes with log 2 fold change >1.5 and adjusted p -value <0.05 in each condition. Additional file includes the homolog and antagonistic reacting genes. (XLSX 192 kb

Additional file 1: of Changes in global gene expression of Vibrio parahaemolyticus induced by cold- and heat-stress

DAVID categories. Additional file includes DAVID data for all temperatures. (XLSX 25 kb