Genotypic and phenotypic characterization of hemolysin-producing Vibrio parahaemolyticus strains

Vibrio parahaemolyticus ist weltweit der bedeutendste Erreger von Lebensmittelinfektionen durch Verzehr von rohen oder ungenügend erhitzten Meeresfrüchten und Fisch. Dabei können Symptome einer akuten Gastroenteritis, wie Erbrechen, Durchfall oder abdominale Krämpfe auftreten. Als wichtige Pathogenitätsfaktoren gelten die Hämolysine TDH (thermostable direct hemolysin) und TRH (TDH-related hemolysin) sowie das T3SS2 (Typ 3 Sekretionssystem 2). In deutschen Küstengewässern treten tdh positive Stämme nur sehr selten auf, während trh positive Isolate regelmäßig detektiert werden. In dieser Arbeit wurde eine Vielzahl an V. parahaemolyticus Stämmen verschiedenen geographischen Ursprungs auf Virulenzgene mittels PCR untersucht und über verschiedene phänotypische Tests, wie z.B. hämolytische Aktivität, Sekretion extrazellulärer Enzyme, Resistenz gegenüber humanem Serum und Biofilmbildung, welche als potentiell pathogene Eigenschaften gelten, charakterisiert. Zur genaueren Untersuchung und Charakterisierung der Hämolysine TDH und TRH wurde deren Synthese in einem zellfreien System etabliert. Um den Einfluss des Signalpeptids und verschiedener Protein-Tags auf die Funktionalität zu untersuchen, wurden acht TDH-Konstrukte mittels zellfreier Proteinsynthese hergestellt und anschließend die hämolytische Aktivität getestet. TDH-Konstrukte mit Signalpeptid, also Voräluferproteine, wiesen fast keine Löslichkeit in wässriger Lösung auf und waren nicht hämolytisch, während TDH-Konstrukte ohne Signalpeptid, also reife Proteine, löslich waren und eine eindeutige hämolytische Aktivität zeigten. Die Protein- Tag-Kombinationen zeigten eine geringe bis starke Hemmung der Proteinfunktionalität. Als zentraler Aspekt dieser Arbeit wurde eine Auswahl an einheimischen trh positiven V. parahaemolyticus Stämmen mit trh positiven Stämmen aus anderen Regionen der Welt bezüglich ihres pathogenen Potentials und des Verwandtschaftsgrads miteinander verglichen. Die kodierende Sequenz der trh-Gene zeigte eine Clusterung in drei Varianten: trh1, trh2 und ψtrh. Letzteres ist vermutlich ein Pseudogen. Alle deutschen Stämme, welche aus Muscheln, Meerwasser und Patienten isoliert wurden, trugen die trh2-Genvariante. Die Multilokus-Sequenz-Analyse zeigte eine nahe Verwandtschaft zwischen deutschen und norwegischen Isolaten, welche somit wahrscheinlich Teil der autochthonen marinen Mikroflora in Nordeuropa sind. Bei Stämmen, welche das Pseudogen ψtrh im Genom trugen, konnte das vopC-Gen, welches für einen Effektor des T3SS2 kodiert, nicht nachgewiesen werden. Alle anderen trh positiven Stämme dagegen besaßen das vopC-Gen. Die Transkription der trh-Varianten sowie des vopC-Gens wurde unter verschiedenen Wachstumsbedingungen untersucht. Dabei wurde der Einfluss von Galle und Harnstoff untersucht, welche u.a. in den menschlichen Gastrointestinaltrakt ausgeschieden werden. Die trh1-Transkription konnte mithilfe von Gallenextrakt induziert werden, während keine Induktion der trh2-Genexpression beobachtet wurde. Die vopC-Transkription konnte in trh2 positiven Stämmen durch Harnstoff induziert werden. Die meisten trh1-tragenden Stämme waren gegenüber Schaferythrozyten hämolytisch, während alle trh2 positiven Stämme keine hämolytische Aktivität zeigten. Auch das zellfrei hergestellte TRH1-Protein war stark hämolytisch, während zellfrei synthetisierte TRH2-Proteine keine Aktivität zeigten. Die Ergebnisse dieser Arbeit zeigen eine hohe Diversität unter trh positiven V. parahaemolyticus Stämmen und stellen die Funktionalität der TRH2-Proteine sowie die Rolle des ψtrh-Gens als Pathogenitätsfaktor in Frage. Für eine Bewertung des pathogenen Potentials von V. parahaemolyticus Stämmen wäre eine Differenzierung zwischen den trh-Varianten sowie die Detektion von Komponenten des T3SS2 wie VopC eine sinnvolle Maßnahme in der Diagnostik und könnte zu einer verbesserten Risikobewertung beitragen.Vibrio parahaemolyticus is the major pathogen associated with the consumption of raw or undercooked seafood and causing acute gastroenteritis. The hemolysins TDH (thermostable direct hemolysin) and TRH (TDH-related hemolysin) as well as T3SS2 (type 3 secretion system 2) are considered to be major pathogenicity factors. While tdh positive strains are rarely detected in coastal waters of Germany, trh harbouring strains are found regularly. In this work V. parahaemolyticus strains of different geographic origin were analyzed for pathogenicity factors using PCR. Selected phenotypic traits which might contribute to pathogenicity, like hemolytic activity, secretion of extracellular enzymes, serum resistance and biofilm formation were tested in various assays. The cell-free synthesis of hemolysins TDH and TRH was established to analyze and compare the hemolysins. Eight TDH constructs were synthesized in a cell-free system to investigate the influence of signal peptide and protein tags on protein functionality. Precursor proteins with signal peptide and mature proteins were tested by hemolysis assays showing that precursor proteins were not soluble and therefore not active while mature proteins were soluble and revealed hemolytic activity. Inhibition of protein functionality by protein tags varied between tag types and tag combinations. Major part of this work was the characterization of the pathogenic potential of trh habouring V. parahaemolyticus strains from Germany which were compared to trh positive strains from other geographical regions. The complete trh sequences revealed a clustering into three different types: trh1 and trh2 genes and a pseudogene ψtrh. All German isolates possessed alleles of the trh2 gene. Multilocus sequence typing analysis indicated a close relationship of German and Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene ψtrh were negative for T3SS2 effector vopC while all other trh positive strains exhibited vopC gene. Transcription of trh and vopC genes was analyzed under different growth conditions to examine the influence of bile and urea as both substances are present in the human intestinal tract. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all. This work reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene ψtrh as a pathogenicity factor are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2 components like vopC would improve V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and clinical diagnostics

Molecular characterization of regulatory polymorphisms in the promoter region of the STAT6 gene in a Gabonese population

Parasites remain competent invaders of host immunity. Their invasion strategies have proven to impact immunorelevant genes leading to diversity among gene families. We focussed on signal transducer and activator of transcription (STAT6) factor that plays a fundamental role in signal transduction and activation of transcription. Recent studies have highlighted the role of STAT6 variants in control of infection levels. We identified and investigated regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of the STAT6 gene in a group of Gabonese individuals exposed to a variety of parasitic infections. Three promoter variants were identified in 40 individual subjects. We further validated these promoter variants for their allelic gene expression using transient transfection assays. One promoter variant, rs3024944 (G/C), revealed an altered expression of the marker gene. The identification of function-altering SNPs in the promoter may facilitate studying parasite susceptibility in association studies

Characterization of trh2 harbouring Vibrio parahaemolyticus strains isolated in Germany.

Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany.Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2β effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all.Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2β components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and clinical diagnostics

Primers and probes used for qRT-PCR.

<p>Primers and probes used for qRT-PCR.</p

Polymerase chain reaction (PCR) primers, targets and amplification sizes used for genotyping.

<p>Polymerase chain reaction (PCR) primers, targets and amplification sizes used for genotyping.</p

Hemolytic activity of cells and supernatants on human and sheep blood.

<p>Bacteria were cultivated in LB medium with and without 0.04% bile bovine. Cultures [A] and [C] and filtered supernatants [B] and [D] were added to a suspension of each blood type and incubated for four hours. Red blood cell lysis was determined at OD₅₄₀. Control strains were VN-0022 (<i>tdh/trh</i> negative) and ATCC439996 (<i>tdh</i> positive, <i>trh</i> negative). All experiments were performed twice.</p

List of <i>V</i>. <i>parahaemolyticus</i> strains used in this study.

<p>¹⁾ Travel associated, isolated in Germany after return</p><p>²⁾ Environmental German strains were from seawater.</p><p>³⁾ Strains isolated from German mussel primary productions</p><p>⁴⁾ Strains isolated in Germany from imported seafood</p><p>AWI: Alfred Wegener Institute, Helgoland, Germany;</p><p>BfR: Federal Institute for Risk Assessment, Berlin, Germany;</p><p>CEFAS: Centre for Environment, Fisheries and Aquaculture Science, Weymouth, United Kingdom;</p><p>IFH: Institute of Food Hygiene, Department of Veterinary Medicine, Freie Universität Berlin, Germany;</p><p>LAGuS: State Office for Health and Social Affairs, Rostock, Germany;</p><p>LAVES: Lower Saxony State Office for Consumer Protection and Food Safety, Cuxhaven, Germany</p><p>List of <i>V</i>. <i>parahaemolyticus</i> strains used in this study.</p

Primers used in various combinations for amplification of complete <i>trh</i> gene.

<p>Annealing temperature was 54°C, amplicon sizes ranged from 600–700 bp.</p><p>Primers used in various combinations for amplification of complete <i>trh</i> gene.</p