Browsing by Person "Schmidt, Herbert"

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Broad time‐dependent transcriptional activity of metabolic genes of E. coli O104:H4 strain C227/11Φcu in a soil microenvironment at low temperature
(2023) Detert, Katharina; Währer, Jonathan; Nieselt, Kay; Schmidt, Herbert
In the current study, metabolic genes and networks that influence the persistence of pathogenic Escherichia coli O104:H4 strain C227/11Φcu in agricultural soil microenvironments at low temperature were investigated. The strain was incubated in alluvial loam (AL) and total RNA was prepared from samples at time point 0, and after 1 and 4 weeks. Differential transcriptomic analysis was performed by RNA sequencing analysis and values obtained at weeks 1 and 4 were compared to those of time point 0. We found differential expression of more than 1500 genes for either time point comparison. The two lists of differentially expressed genes were then subjected to gene set enrichment of Gene Ontology terms. In total, 17 GO gene sets and 3 Pfam domains were found to be enriched after 1 week. After 4 weeks, 17 GO gene sets and 7 Pfam domains were statistically enriched. Especially stress response genes and genes of the primary metabolism were particularly affected at both time points. Genes and gene sets for uptake of carbohydrates, amino acids were strongly upregulated, indicating adjustment to a low nutrient environment. The results of this transcriptome analysis show that persistence of C227/11Φcu in soils is associated with a complex interplay of metabolic networks.
Characterization of function and regulation of the subtilase cytotoxin and Shiga toxin of pathogenic Escherichia coli
(2021) Heinisch, Laura; Schmidt, Herbert
Food-borne diseases caused by enterohemorrhagic Escherichia coli (EHEC) constitute a great threat to human health worldwide. Pathogenicity of EHEC strongly depends on the ability to produce virulence factors such as amongst others bacterial toxins. One of these toxins are the so-called Shiga toxins (Stx), which is why EHEC are assigned to the group of Shiga toxin-producing Escherichia coli (STEC). Stx belong to the family of AB5 protein toxins consisting of two subunits. One of them, the StxA-subunit causes depurination of the 28S rRNA in eukaryotic ribosomes by exhibiting N-glycosidase activity subsequently leading to inhibition of the protein biosynthesis followed by apoptosis of the host cell. The second one is the homopentameric B-subunit, which mediates binding to the host cell surface via the receptor glycolipid globotriaosylceramide (Gb3). Besides Stx, the subtilase cytotoxin (SubAB) has been described in STEC in recent years. SubAB, also assigned to the family of AB5 toxins, generates its cytotoxic activity via cleavage of the endoplasmic chaperone binding immunoglobulin protein (BiP) by its A-subunit. This cleavage leads to an unfolded protein response, resulting in apoptosis of the host cell. The B-subunit forms a ring-like homopentameric structure which is responsible for the binding to the receptor N-glycolylneuraminic acid (Neu5Gc) and other O-glycans. Although the mode of cytotoxicity of AB5 toxins have been studied extensively, some mechanisms remain unsolved. The scope of this thesis was to analyze further the mode of action of AB5 toxins and the gene regulation of stx and subAB. Both publications included in this thesis combine the characterization of the cytotoxic activity of AB5 toxins, the regulation of their genes, their subunits, and the combination of subunits of Stx and SubAB. In the first publication the regulation of gene expression of AB5 toxins was investigated in more detail. In this study, the gene expression of subAB1 was analyzed with a luciferase reporter gene assay and by quantitative real-time polymerase chain reaction. To unravel the regulatory mechanisms, both the laboratory E. coli strain DH5α and the STEC O113:H21 strain TS18/08 were used. Expression of subAB1 and promoter activity was studied using standard cultivation methods. Moreover, this work shed light on the impact of the global regulatory proteins host factor of bacteriophage Qβ (Hfq) and histone-like nucleoid structuring protein (H-NS) on subAB1 gene expression. Therefore, isogenic deletion mutants of hfq and hns gene were generated in the respective strains. Afterwards, plasmid-based complementation was conducted to verify that the observed effects were due to the deletion. Analysis of subAB1 promoter activity revealed impact of both Hfq and H-NS during different growth phases in both strains. In addition, the influence of both regulatory proteins on the expression toxin genes in STEC strain TS18/08 was investigated. This study did not only focus on the expression of stx2a and subAB1, but also the gene expression of the gene of the cytolethal distending toxin V (cdtV) was analyzed. Interestingly, all three toxin genes studied were upregulated in the deletion mutants of Δhfq and Δhns. Those results demonstrate the impact of global regulatory proteins on AB5 toxin gene expression and show that all three toxin genes investigated are integrated into the same regulatory network. In the second publication, the mode of action of AB5 toxins on the example of Stx2a was analyzed in more detail. The paradigm of AB5 toxin was known as the receptor binding B-subunit which mediates uptake of the enzymatic A-subunit and the subsequent cytotoxic activity. Previous studies have questioned this paradigm by showing cytotoxic effects of the SubA-subunit in absence of its corresponding B-subunit. This work analyzed whether this cytotoxic effect of the A-subunit is not only true for SubAB, but also for Stx. Thus, seperate recombinant expression of StxA2a subunits and subsequent His tag-based purification was performed. Both StxA2a-His and StxB2a-His were analyzed on cytotoxicity separately or in combination with the other subunit. Strikingly, cytotoxic effects of the StxA2a-His was observed in the absence of its corresponding B-subunit cell-type independently on HeLa, Vero B4, and HCT-116 cells. Studies on the B-subunit revealed no cytotoxicity on all cell lines. Additionally, combinations of different A- and B-subunits of Stx2a and SubAB1 proteins were analyzed. The hybrid combination showed that the cytotoxic effect of StxA2a-His on HeLa and HCT-116 cells could be reduced in the presence of the SubB1-His. Contrary, the cytotoxic effects of SubA1- His were unaltered in combination with StxB2a-His. Those results give the assumption that the Stx2aA-subunit binds to a target cell receptor blocked by SubB1-His. Additional experiments on the binding capacity of the Stx2a-subunits to Gb3 revealed that while StxB2a-His was able to bind to the receptor, no binding of the recombinant A-subunit was observed. The results indicate a cytotoxic effect of StxA2a on different cell types in absence of its corresponding B-subunit, which is designated as “single-A” effect in this work. The role of this effect in STEC pathogenicity, the uptake mechanism and subsequent transport inside the host cells of StxA-subunit need to be further analyzed in the future.
Entwicklung und Validierung schneller und selektiver Verfahren zum Nachweis von Salmonella enterica, Cronobacter spp. und Bacillus cereus in Milcherzeugnissen
(2014) Zimmermann, Jennifer; Schmidt, Herbert
The presence of pathogens is a serious problem in the food industry and contaminations of food with Bacillus cereus, Cronobacter spp. and Salmonella enterica are responsible for a large number of diseases worldwide. Milk products like milk, whey or cream powder are widely used in industry as an ingredient in other foods. Therefore it requires a fast and reliable identification of pathogenic microorganisms. The official methods according to § 64 LFGB or ISO/TS 22964 apply a common scheme of pre-enrichment, selective enrichment, detection and confirmation and take between three and six days. The aim of this work was the development and validation of a real-time PCR based method, which identifies the existence of the three pathogens in dairy products within 24 hours. The identification of B. cereus, Cronobacter spp. and S. enterica with the developed TaqMan real-time PCR was performed using specific genetic characteristics and an internal amplification control to eliminate false negative results. For B. cereus, the groEL gene, which codes for a heat shock protein, was selected as target. For the detection of Cronobacter spp. the ompA gene and for S. enterica the invA gene was chosen. Both genes are responsible for the invasion of the pathogens in the human epithelial cells. The adaptation of the method to the food matrix and an optimization of the enrichment time were affected by an artificial contamination of various dry dairy products. It was possible to detect 105 cfu/g C. sakazakii and S. Enteritidis cells with an initial concentration of 100 cfu/g in reconstituted powdered infant formula after enrichment of six hours. To simulate a natural contamination, powdered infant formula was contaminated with desiccated C. sakazakii cells in various concentrations and analyzed with the developed real-time PCR method. It was possible to detect an inoculum concentration of 0.01 CFU/g dry stressed C. sakazakii cells at low aw values (0.22). The new TaqMan real-time PCR is fast, reliable and specific for the clearly detection of the three major pathogenic microorganisms in milk products and was carried out within 24 hours.
Escherichia coli O157:H7 und seine Adaption an verschiedene Wachstumsbedingungen in vitro : Untersuchung des intrazellulären Proteoms und Kohlenhydratmetabolismus
(2014) Polzin, Sabrina; Schmidt, Herbert
The aim of this study was to investigate the linkage of metabolism and pathogenicity of EHEC bacteria under certain environmental conditions in vitro. During the investigations the influence of environmental conditions could be shown on the protein as well as on the gene expression level. In experiments using the culturel media SIEM and SCEM compared to TSB as a reference, differential expression of proteins and genes were shown. For example, decreasing motility and the formation of biofilm-like substances were observed. Furthermore, differences in metabolism and expression of chaperons and stress response systems were detected. After culturing EHEC bacteria in experimental media, increased glucose and pyruvate metabolism, increased amino acid biosynthesis, as well as increased chaperon levels were found, in contrast to decreased expression levels of stress associated proteins. The experimental procedure also affected the presence of pathogenicity factors, like EspP and EspB. Notably, the environmental conditions induced the glutamic acid resistance, providing the EHEC bacteria an unhindered passage through the human gastrointestinal tract, especially the acidic environments like the stomach. Additionally, the sialic acid esterase Z1466 along with shiga toxin, was strongly expressed. This could provide an advantage over the intestinal micro flora, in respect of growth. Effects onto glucose metabolism and amino acid biosynthesis were investigated by isotopoloque profiling experiments, prepared thankworthy by Dr. rer. nat. Wolfgang Eisenreich (Munich). To conclude, stress response and expression of pathogenicity factors could be induced in vitro in the cultural media elected, and without the need of a human host. The experiment also advices the complex regulation of different processes in bacterial cells as response to external factors.
Funktionelle Charakterisierung von 9-O-Acetylesterasen enterohämorrhagischer Escherichia coli
(2019) Feuerbaum, Stefanie; Schmidt, Herbert
Enterohemorrhagic Escherichia coli (EHEC) colonize the human colon and compete against commensal E. coli for limited nutrition. Colonic goblet cells produce glycoproteins called mucin, which are part of the mucosal barrier. This barrier is free of bacterial penetration and is important for the protection of the epithelium. The main glycoprotein in the large intestine is mucin 2 (MUC2), which is heavily O-glycosylated with terminal glycan-bounded sialic acids. These aminosugars naturally exists with up to four O-acetylgroups at position C4, C7, C8 and C9. The grade of O-acetylation acts as a protection for enzymatic degradation by glycosidases. Commensal E. coli and the pathogenic strain O157:H7 EDL933 encodes the single chromosomal 9-O-acetylesterase NanS, which is important for the metabolismus of sialic acids by de-O-acetylation of Neu5,9Ac2 to the basic structure Neu5Ac and Acetate. Both can be used as an energy source. Pathogenic E. coli O157:H7 EDL933 and O104:H4 C227-11Φcu encode further several prophage-encoded 9-O-acetylesterases (NanS-p). Recent studies demonstrated that NanS-p producing EHEC bacteria reveal a higher replication rate in Neu5,9Ac2 containing medium compared with commensal E. coli. This could be an advantage during colonization in human large intestine, where Neu5,9Ac2 is the most common sialic acid in mucins. The aim of this study was to further characterize the enzymatic function of prophage-encoded 9-O-acetylesterases in carbohydrate metabolism and during infection in vitro. To analyze the NanS-p mediated cleavage of mucin-derived O-acetylneuraminic acids, HPTLC and nanoESI MS analyses were performed. The results revealed, that recombinant expressed NanS-p cleave-off acetyl residues from up to tri-O-acetylated Neu5Ac and Neu5Gc. While NanS-p were able to de-O-acetylate glycan-bounded sialic acids at positions C7, C8 and C9, the tested enzymes were not able to hydrolyze the acetyl ester from position C4. The lower specificity of the NanS-p leads to higher availability of mucin-derived substrates for sialidases of commensal bacteria B. thetaiotaomicron. Further analyses reveal a NanS-p dependent mucin degradation in cell culture assays. Mutant strains EDL933ΔnanSΔnanS-p1-nanS-p7 and EDL933ΔnanSΔnanS-p1-nanS-p5 were not able to degrade the mucinlayer, while the wildtype strain O157:H7 EDL933 could disrupt the mucosal barrier of LS 180 cells. The NanS-p dependent adherence of O157:H7 EDL933 and O104:H4 C227-11Φcu to epithelial cells HT-29 was demonstrated by performing adherence assays. The deletion of nanS-p genes revealed less adhered bacteria compared to wildtype strains. Further performed swimming assays could show the impact of NanS-p on motility. The wildtype strains O157:H7 EDL933 and O104:H4 C227-11Φcu have shown less motility compared to nanS-p deletion mutants. Taken together, NanS-p show an important role during infection and could contribute to the preferred colonization of the large intestine due to their impact of the motility, disruption of the mucosal barrier and mediation of adherence of EHEC to the epithelium.
Isolierung universell einsetzbarer und mikrobiologisch stabiler Sauerteigstarterkulturen durch spontane Fermentationen mit Amaranth.
(2009) Sterr, Yasemin Arzu; Schmidt, Herbert
Spontaneous fermented sourdoughs prepared from five amaranth flours were screened for the presence of lactic acid bacteria (LAB) that predominate the autochthonous microbiota and thus may be suitable as starter cultures. The doughs were fermented with daily backslopping on the laboratory scale for 10 days with a dough yield of 200 at 30°C. Every 24 hours, the pH-values and total titratable acidity degrees were determined and samples were analyzed for the presence of LAB and yeasts by cultural methods. The identity of the isolates was traced during the fermentation with RAPD-PCR and two different primers, and the strains were identified by sequence analysis of the 16S rDNA genes. The strains Lactobacillus plantarum RTa12, L. sakei RTa14, and Pediococcus pentosaceus RTa11 were selected and applied as starters in further laboratory fermentations. All strains were predominant in repeated experiments, both, as single strains and in combination. During the first 24 h, L. plantarum RTa12 and P. pentosaceus RTa11 grew quite similar. The pH-value dropped earlier with P. pentosaceus RTa11, while both strains gave the same pH-values after 10 h of fermentation. In the challenge test with the autochthonous mikrobiota both strains overgrew the other LAB of the spontaneous fermented dough within the first eight hours, and were therefore considered dominant over the resident microbiota. Influences of the incubation temperature on the fermentation characteristics were mainly assessed in the viable cell counts, the pH-values and the titratable acidity degrees at 25°C. The pH-values for both strains were at high incubation temperatures (30 and 35°C) during the fermentation lower than at 25°C, respectively. However, after 24 h of fermentation both strains reached a pH-value of approximately 4.0 after 24 h. Further sugar, organic acid, mannitol and ethanol profiles of fermented doughs were determined with HPLC. Mainly analyzed metabolites in the doughs were glucose, sucrose, lactate, and acetate. To compare the potential starter cultures with commercial available startercultures, fermentations with two industrial startercultures were performed for 24 h at 30°C and a dough yield of 200. Both strains were able to compete with the commercial available starter cultures concerning viable cell counts, total titratable acidity and pH-values. Because of the dominance of both strains in sourdough fermentations with amaranth, the ability for acidification in a short time, the capacity to compete with the autochthonous mikrobiota, the robustness against lesser effects of the environment, e. g. variation of the temperature, and at least because of the ability to compete with commercial available startercultures, thus, the characterized strains L. plantarum RTa12 and P. pentosaceus RTa11 are interesting candidates as starter cultures for amaranth sourdoughs.
Mikrobiologische und biochemische Analyse der Fermentationseigenschaften von Lactobacillus paralimentarius AL28 und Lactobacillus plantarum AL30 in Sauerteigen aus Pseudozerealien
(2011) Vogel, Antje; Schmidt, Herbert
Pseudocereals are absent of gluten and therefore are important for people having a gluten-intolerance. Today no commercial starter cultures are available for sourdough fermentations with pseudocereals. This PHD-Thesis shows results of the characterisation of L. paralimentarius AL28 and L. plantarum AL30 concerning an application in pseudocereal sourdoughs. The fermentation properties of the strains, applied as single strains and in combination, were assessed in laboratory scale fermentations with amaranth and buckwheat. The fermentation studies were performed with a dough yield of 200 and over a period of two to ten days at 30°C with daily refreshment step. The investigated strains acidified the sourdoughs fast within the first two propagation steps as single strains as well as in combination (approximately pH 4). In amaranth higher total titratable acidity (TTA) -values (TTA between 25 and 30) were measured than in buckwheat (TTA of 20). 16S rDNA / 28S rDNA-PCR sequencing and RAPD-PCR were applied to determine the bacterial and eucaryotic species affiliation, respectively, and to trace specific strains during the fermentation process. The analysed strains competed against the autochthonous microbiota with the result of suppression the majority of yeasts and moulds as well as strains of their own species within the first 12 h of sourdough fermentation. They also suppressed an autochthonous microbiota grown up to 10^8 cfu/g sourdough. Single strains as well as the combination of both strains dominated the microflora in all tested flours / fermentation batches. Both strains displayed reproducible results concerning their over-all fermentation characteristics. L. paralimentarius AL28 and L. plantarum AL30, respectively, dominated the LAB viable counts in all flours after 10 days of fermentation as single strains (>/= 68 % and >/= 98 % of LAB, respectively) as well as in combination. In the latter case strain L. plantarum AL30 was especially competitive in buckwheat (AL28:AL30 = 1:1), more than in amaranth (AL28:AL30 = 4:1). The strains were characterised by their short lag-phase of
Molekularbiologische und physiologische Untersuchungen zur Bedeutung phagenkodierter Sialinsäureesterasen von enterohämorrhagischen Escherichia coli (EHEC)
(2019) Saile, Nadja; Schmidt, Herbert
Enterohemorrhagic Escherichia coli (EHEC) are responsible for severe disease in humans such as hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. The main virulence factors are Shiga toxins (Stx) and a type-III-secretion system. For colonization of the colon, they have to compete with the intestinal microbiota for limiting substrates such as 5-N-acetyl neuraminic acid (Neu5Ac). For the catabolism of Neu5Ac, E. coli possess the genes nanA, nanT, nanE, nanK, nagA, and nagB. Many sialic acids are terminally bound to mucins of mucus in the colon. E. coli is not able to use bound Neu5Ac, because it cannot express sialidases for cleavage of Neu5Ac from mucin. However, sialic acids can be cleaved by sialidases from anaerobic bacteria in the colon and are then available. Many E. coli strains encode the nanCMS operon. NanC is a porin protein, NanM is a mutarotase and NanS is an O-acetyl esterase. NanS converts 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2) and Neu5,8Ac2 to Neu5Ac and offers a substrate niche. There are multiple prophage-located nanS-homologous open reading frames in the chromosomes of EHEC, that are designated as nanS-p (p = phage) in this study. The question of this thesis was, why EHEC possess multiple nanS-p genes, if their corresponding proteins are used for energy production and for a competition advantage of EHEC. Furthermore, the influence of the sialidase BTSA of Bacteroides thetaiotaomicron on the NanS-p dependent utilization of mucin should be investigated. The strains used in this study were the pathogenic E. coli O157:H7 strain EDL933 as well as the pathogenic O104:H4 strains LB226692 and C227-11phicu (variant of C227-11 cured in its stx-prophage), and the apathogenic E. coli strains AMC 198 (nanS+, nanS-p-) and C600deltananS (nanS-, nanS-p-). The chromosome sequences of EDL933 and LB226692 were analyzed in silico. NanS-p2 and NanS-p4 of EDL933 were expressed recombinantly and the pH- and temperature-optimum as well as potential substrates were investigated. nanS/nanS-p deletion mutants of EDL933 and C227-11phicu were generated and cultivated with Neu5,9Ac2 or mucin. The courses of the growth curves were analyzed by turbidity measurement or determination of the viable cell counts. In co-cultivation experiments the medium was supplemented with the AMC 198 strain and/or BTSA and/or NanS-p. EDL933 possess the chromosomal nanS and seven nanS-p genes, while LB226692 has no nanS, but five nanS-p genes. It was confirmed, that all nanS-p genes are located in the late regulated gene region of the prophages. The putative NanS-p proteins include the domain 303, which is known for its esterase activity in NanS, and the domain of unknown function 1737. The analyzed recombinant NanS-p proteins de-O-acetylated the substrates Neu5,9Ac2 and mucin of bovine submaxillary gland and showed a temperature- and pH-optimum of 40-50 °C and 7-9, respectively. A gene-dose effect was identified because the generation times of the deletion mutant strains increased with the number of deleted nanS-p genes in the chromosomes. After extracellular supplementation with NanS-p, the mutant strains regained the original growth kinetic of the wildtype strain. The mono-deletion of nanS in EDL933 (EDL933deltananS) caused no chances in the growth kinetic of the strain with Neu5,9Ac2. Therefore, NanS is not necessary for growth of EDL933 on Neu5,9Ac2. C600deltananS could not catabolize Neu5,9Ac2, as expected. The original course of the growth curve of a C600 cultivation was recuperated by C600deltananS if NanS-p was added to the medium. In a co-cultivation the viable cell count of C227-11phicu increased, while AMC 198 increased hardly. Only after deletion of all nanS-p genes in C227-11phicu, the viable cell count of AMC 198 increased. In a mucin-containing medium with BTSA as supplement EDL933 and EDL933deltananS could grow in contrast to EDL933deltananSdeltananS-p1a-p7. This shows, that the combination of BTSA and NanS-p supports the growth of EDL933 in a mucin-containing environment. The results of this work showed for the first time the importance of prophage-encoded O-acetyl esterases for EHEC bacteria and are an excellent basis for further work that contribute to a profound scientific understanding of the sialic acid catabolism in EHEC and other pathogenic E. coli. They identify sialic acids as substrates of a potential nutrient niche in the gut and establish a potential location for therapeutic approaches.
Molekulare Interaktionen von Milchsäurebakterien mit enterohämorrhagischen Escherichia coli und humanen Darmepithelzellen
(2011) Stöber, Helen; Schmidt, Herbert
The interactions of 19 benign strains of lactic acid bacteria, bifidobacteria and staphylococci with five enterohemorrhagic Escherichia coli (EHEC) strains of different serotypes and virulence gene spectrum were investigated using a HT29 cell culture infection model. As a parameter for the infection the secretion of Interleukin 8 (IL-8) of the infected cells was analyzed by ELISA. None of the used benign strains induced an IL-8 secretion, whereas the infection with the EHEC strains leads ? independent of their virulence profile - to high amounts of IL-8. In coinfection assays with the pathogen EDL933 (O157:H7) and different test strains the secretion of IL-8 of the cultured cells was decreased by a few strains. With 12 of 19 tested strains, a weak reduction < 30 % of IL-8 secretion of HT29 cells after coinfection with EHEC O157:H7 strain EDL933 was observed. Six strains reduced the IL-8 secretion up to 60 % and the strain B. breve DSMZ 20083 decreased the IL-8 production about 73 %. Coinfection assays with different strains of one species (B. adolescentis DSMZ 20083 and DSMZ 20086 as well as L. johnsonii BFE 633 and DSMZ 10533) showed the strain specificity of the observed anti-inflammatory effect, due to different capabilities of IL-8 reduction. In further coinfection assays with different EHEC strains of the serotypes O103:H2, O26:H-, 0157:H- and O113:H21 different abilities of the benign strains to influence the infection with the different pathogen strains were noted. Therefore the protective anti-inflammatory effect is strain specific for the tested benign bacteria and also depends on the application of EHEC strains with different sero- and virulence types. Further investigations indicated the imperative of living bacteria for the observed protective effect; neither culture supernatant nor inactivated bacteria showed an effect on the IL-8 secretion of the EDL933 infected HT29 cells. The analysis of the cell culture supernatants 6 h after infection with different bacteria detected the production of lactic and acetic acid. The application of these acids in infection assays with EDL933 did not lead to an reduced IL-8 secretion of the infected cells. Therefore the production of organic acids did not explain the protective effect. The induction of IL-8 could not be traced back to the influence of a single virulence factor. Four PMK5 strains with deletions in different virulence genes induced similar IL-8 secretions in comparison to cells infected with the wild-type strain. Coinfection assays with the mutants and S. pasteuri LTH 5211 showed also similar IL-8 reductions than coinfection assays with the wild-type strain. It is to suppose that the anti-inflammatory effects of the benign bacteria do not influence a single virulence factor of the tested EHEC strains. As a second parameter the activation of the transcription factor ?Nuclear Factor kappa B? (NF-κB) of coinfected HT29 cells was monitored using a reporter-genassay. In comparison to the single EHEC-infection, the NF-κB activation was reduced by all tested lactic acid bacteria, bifidobacteria and S. pasteuri LTH 5211 in coinfection trials significantly. No strain-specificity and no pathogen-specificity could be observed. Interestingly, stimulation of the HT29 cells with benign bacteria led to inhibition of NF-κB activity, the measured values were less than the values of the negative control PBS. A gene expression analysis of toll-like receptors (TLRs), recognizing bacteria on cell surfaces and initiating the immune response, showed no regulation for TLR2. Infection with EDL933 led to down regulation of TLR4 and to up regulation of TLR9. Stimulation with L. rhamnosus GG, L. johnsonii DSMZ 10533 or L. fermentum DSMZ 20052 led neither to regulation of TLR4 nor TLR9. The benign bacteria did not influence the EHEC-induced TLR4 regulation in coinfection trials; in contrast the regulation of TLR9 was reduced significantly. The model described here is useful for screening basic effects of protective bacteria that are able to counteract EHEC-mediated effects on human cells and to study the molecular interaction between bacteria as well as between bacteria and human cultured cells.
Molekulargenetische Untersuchungen zur Expression des Typ III Effektors NleA 4795 von Shiga Toxin-produzierenden Escherichia coli
(2010) Schwidder, Maike; Schmidt, Herbert
Shiga toxin-producing E. coli (STEC) are the causative agents of foodborne infections in many countries and can lead to severe diseases like hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. The bacteria colonize the human intestine where they normally cause the formation of characteristic ?attaching and effacing?-lesions. Essential for this effect is a pathogenicity island, termed as ?locus of enterocyte effacement? (LEE), that encodes the components of a type III secretion system and several effector proteins, which are translocated directly into the host cells by the TTSS machinery. In addition to the LEE-encoded effectors a large number of effector proteins have been identified which are encoded outside of the pathogenicity island. Among these is the ?non-LEE encoded effector A? (NleA), which is encoded on cryptic or inducible prophages and is widely distributed among pathogenic E. coli strains. In the present study, the expression and regulation of the nleA-variant nleA4795 of E. coli O84:H4 strain 4795/97 was investigated, which is located on the Shiga toxin-converting bacteriophage BP-4795. Therefore, different environmental conditions as well as certain regulatorproteins were tested on their influence on nleA4795-expression using a luciferase-reportersystem and the quantitative real-time PCR. Among the analyzed environmental factors, certain concentrations of NaCl and KCl were identified to activate nleA4795-expression, indicating an osmotic-based influence. The suggested induction of nleA4795 in preconditioned medium due to quorum sensing could not be confirmed, since none of the so far known autoinducers showed a positive influence on the expression. The increased expression of nleA4795 could be associated with a reduced amount of nutrients in subsequent investigations and therefore demonstrated a relation between nleA4795-expression and bacterial stress-response-systems. Furthermore, a possible correlation of nleA4795-expression with the induction of phage BP 4795 and Shiga toxin-expression was analyzed. Different from the expression of Shiga toxin, induction-experiments with norfloxacin showed no activation, but a strong repression of nleA4795-expression. Analysis of the regulatory level demonstrated that the expression of nleA4795 depends on the three LEE-encoded regulators Ler, GrlA und GrlR as well as on the Pch-regulators, which are encoded outside of the LEE. The non-LEE encoded regulator EtrA showed no influence on the expression of nleA4795. In addition, the regulator proteins Ler, GrlA and PchA were tested for direct binding to the nleA4795-promoterregion. Regulators GrlA and PchA showed no specific binding and were therefore classified as indirect regulators of nleA4795-expression. In contrast, regulator Ler showed a specific binding to different areas of the nleA4795-promoter region and thereby confirmed the integration of nleA4795 in the Ler-mediated circuit of LEE-regulation.
The prophage and us - Shiga toxin phages revisited
(2023) Schmidt, Herbert; Muniesa, Maite
Rapid acidification and off-flavor reduction of pea protein by fermentation with lactic acid bacteria and yeasts
(2024) Zipori, Dor; Hollmann, Jana; Rigling, Marina; Zhang, Yanyan; Weiss, Agnes; Schmidt, Herbert
Pea protein is widely used as an alternative protein source in plant-based products. In the current study, we fermented pea protein to reduce off-flavor compounds, such as hexanal, and to produce a suitable fermentate for further processing. Laboratory fermentations using 5% (w/v) pea protein suspension were carried out using four selected lactic acid bacteria (LAB) strains, investigating their growth and acidification capabilities in pea protein. Rapid acidification of pea protein was achieved with Lactococcus lactis subsp. lactis strain LTH 7123. Next, this strain was co-inoculated together with either the yeasts Kluyveromyces lactis LTH 7165, Yarrowia lipolytica LTH 6056, or Kluyveromyces marxianus LTH 6039. Fermentation products of the mixed starter cultures and of the single strains were further analyzed by gas chromatography coupled with mass spectrometry to quantify selected volatile flavor compounds. Fermentation with L. lactis LTH 7123 led to an increase in compounds associated with the “beany” off-flavors of peas, including hexanal. However, significant reduction in those compounds was achieved after fermentation with Y. lipolytica LTH 6056 with or without L. lactis LTH 7123. Thus, fermentation using co-cultures of LAB and yeasts strains could prove to be a valuable method for enhancing quality attributes of pea protein-based products.
A robust one-step recombineering system for enterohemorrhagic escherichia coli
(2022) Peng, Lang; Dumevi, Rexford Mawunyo; Chitto, Marco; Haarmann, Nadja; Berger, Petya; Koudelka, Gerald; Schmidt, Herbert; Mellmann, Alexander; Dobrindt, Ulrich; Berger, Michael
Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3–4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.
Untersuchungen zur spezifischen Genexpression von enterohämorrhagischen Escherichia coli (EHEC) in der Lebensmittelmatrix
(2012) Kroj, Andrea; Schmidt, Herbert
Ground beef as a high risk food is known to be an cause of human infection with Shiga toxin-producing E. coli (STEC). The pathogens infect humans by the ingestion of undercooked ground meat and cause severe diseases like hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. E. coli O157:H7 strain EDL933 as a representative of enterohemorrhagic E. coli (EHEC), a subgroup of STEC, was analysed for in vivo induced genes in ground beef with the help of the in vivo expression technology. It could be demonstrated that the promoter selection vector pKK232-8, which contains a promoterless chloramphenicol resistance gene, is not a suitable vector for a study of gene expression in this matrix. The detection of in vivo expressed genes using the alcohol-soluble and bacteriostatic antibiotic was not possible. Therefore, the promoter selection vector pAK-1 was developed. The new vector system was based on a water-soluble and bactericidal kanamycin resistance gene for selection. In the present study, the vector was established and used for analysis of the gene expression in ground meat. 20 in vivo induced genes that were expressed during growth in ground meat under elevated temperature conditions at 42°C could be detected. Eight genes were associated with energy and nucleotide metabolism, macromolecule synthesis, transport and stress response of the cell. The major part of 12 genes was attributed to a putative or unknown function. Predominantly, identified genes could not be associated with virulence or stress response of the cell. The results of this study, using the in vivo expression technology, showed that genes which are expressed under specific conditions in ground meat could be detected with the help of the chosen method. A first insight into the gene expression of strain EDL933 in ground beef could be acquired. During further investigations a comparison of the fitness of 23 E. coli strains belonging to serogroups O26, O103 and O157 was realized. The isolates originating from foods, patients with HUS and animals were compared in ground beef. The determined differences showed strain-specificity and temperature-specificty. The fitness of the strains varied dependent on the chosen temperatures at 15, 20 and 37 degrees. The analysis of the strains based on ten virulence factors showed that the observed differences could not be attributed to the presence or the number of virulence genes. A correlation between the fitness and the production of a bacteriocin could not be found.
Uptake of enterohemorrhagic Escherichia coli into the roots of lettuce plants
(2020) Eißenberger, Kristina; Schmidt, Herbert
Within the last 10 years, the annual numbers of human infections with enterohemorrhagic Escherichia coli (EHEC) in Germany increased by a factor of 2.4. The peak was reached during the large German outbreak in 2011. Intriguingly, the source of the outbreak was supposedly traced back to organic fenugreek sprouts. Moreover, the number of EHEC outbreaks traced back to plant-based foods, e.g. fresh produce, increased also in the United States. This trend poses a serious threat to public health as fresh produce is mostly consumed raw. Also, these observations gave rise to investigate the interactions of plants and human pathogens in more detail especially as fresh produce may be contaminated directly on the field. In the present thesis, the capability of different EHEC strains and an enteroaggregative/enterohemorrhagic E. coli (EAEC/EHEC) strain, to adhere to and to internalize into the roots of different lettuce plants was investigated. These studies conducted within the scope of this dissertation focused on different aspects of the mentioned processes, such as different bacterial strains, the bacterial genetic equipment, and different environmental conditions, such as plant variety, soil type used for plant growth, and the soil microbiota. To mimic the natural conditions as close as possible, plants were grown from unsterile plant seed in unsterile soil under greenhouse conditions. In the first publication, the overall ability of EHEC O157:H7 strain Sakai to adhere to and internalize into the roots of Valerianella locusta, also known as lamb’s lettuce, grown in diluvial sand soil was described. It was demonstrated that EHEC O157:H7 strain Sakai is indeed able to attach to and internalized into the lettuce roots under the conditions tested. Moreover, this paper shed light on potentially important intrinsic bacterial factors, i.e. genes/proteins, which are putatively involved in adherence and/or internalization. Therefore, deletion mutants lacking hcpA and/or iha, were also investigated regarding adherence to and internalization into the lamb’s lettuce roots. Both genes, coding for the major subunit of the hemorrhagic coli pilus HcpA and the adhesin Iha, respectively, are supposed to be associated with adherence and therefore called “adherence factors”. However, deletion mutants lacking one or both of these genes did not show significant differences in root attachment compared to the wild-type strain. Regarding internalization, deletion of either of these genes resulted in significantly lower numbers of internalized bacteria clearly indicating that both of these genes – or the proteins encoded by these genes – play an important role during invasion of EHEC O157:H7 strain Sakai into the roots of lamb’s lettuce. Interestingly, deletion of both genes did not result in further reduction of internalization compared to single deletion mutants. Hence, hcpA and iha encode rather internalization factors than adherence factors. Moreover, internalization does not solely depend on these two factors. The second paper focused on the influence of lettuce varieties and soil type on adherence and internalization of E. coli O104:H4 strain C227/11φcu. In this study, the lettuce varieties Valerianella locusta and Lactuca sativa, also known as lamb’s lettuce and lettuce, respectively, were both grown in two different soil types, diluvial sand (DS) and alluvial loam (AL), to address the impact of plant host and environment on bacterial attachment and invasion into lettuce roots. To approach the latter aspect in more detail, the composition of the soil microbial community was analyzed in parallel by partial 16S rRNA gene sequencing. Adherence to the roots was positively influenced by the soil type as the number of adherent E. coli O104:H4 strain C227/11φcu bacteria significantly rose by a factor of three to four when the plants were grown in DS compared to AL. However, when grown in the same type of soil, no statistically significant differences in attachment were detected between the distinct lettuce varieties. On the other hand, internalization significantly differed predominantly between the two types of lettuce. Internalization into the roots of L. sativa compared V. locusta was found to be increased by a factor of 12 upon growth in DS, and by a factor of 108 when the plants were grown in AL. Moreover, internalization into the roots of L. sativa was five-times higher in AL than in DS. Consequently, the lettuce variety significantly influences to ability of E. coli O104:H4 strain C227/11φcu to internalize into the lettuce roots, while the soil type affected bacterial invasion only at the roots of L. sativa under the conditions tested. Moreover, by microbiota analysis, the inoculated strain was found within the soil microbiota, and this analysis demonstrated that soil type, lettuce variety, and the combination of both result in large differences in the composition of the soil microbiota.
Uptake of pathogenic Escherichia coli into crop plants
(2023) Detert, Katharina Margarete; Schmidt, Herbert
Contaminations of fresh produce by enterohemorrhagic Escherichia coli (EHEC) have been an increasing source of human disease outbreaks in the past few decades. In particular, the large disease outbreak in 2011 in Germany has left many questions about the survival of EHEC in soil and the colonization of plants unanswered. Agricultural soil as a reservoir for EHEC strains represents an important contamination source for crop plants. The use of cattle manure for soil fertilization or contaminated irrigation water can result in the introduction of pathogens in fields. To prevent the transfer of pathogens into the food chain, the German fertilizer ordinance disallows the use of organic fertilizer 12 weeks before harvesting the crop plants. However, it is expected that EHEC survives for longer and therefore continue to pose a risk for crop plant contamination. In this study, the overall survival ability of the pathogenic E. coli O104:H4 strain C227/11Φcu, which represents a stx2a-negative derivative of the 2011 outbreak strain C227/11, in agricultural soil was investigated. Thereby, different environmental conditions, soil types and genetic factors of the bacterial strain were identified as influencing factors. Furthermore, the ability of E. coli O104:H4 strain C227/11Φcu to colonize lamb’s lettuce via the root system was investigated to demonstrate that contamination of soil can result in crop plant contamination. In the first publication, the survival of E. coli O104:H4 strain C227/11Φcu in soil microenvironments containing either diluvial sand or alluvial loam was investigated. Two different temperatures (4°C and 22°C) were used and the samples were incubated for more than 12 weeks. The study aimed to analyze whether cattle manure addition prolongs EHEC survival in the soil microenvironments. In the last step, the survival studies were performed using ΔrpoS and ΔfliC deletion mutant strains of C227/11Φcu. The results demonstrated that E. coli O104:H4 strain C227/11Φcu survived for at least 12 weeks in the soil microenvironment model. The survival rate was influenced by the soil type and the temperature. In more detail, the incubation at lower temperature prolonged the survival rate and pathogens were detected up to 20 weeks after inoculation. The application of contaminated cattle manure increased the survival ability at 22°C. Sigma factor RpoS was recognized as an important factor for soil survival. The rpoS deletion mutants showed significant reduction of the survival period while FliC did not influence the overall survival ability in these experiments. To investigate the influence of further genetic factors of the bacterial strain in more detail and thereby characterize all transcription activities, transcriptome analysis was performed in the second publication. Since the strain showed the best survival in alluvial loam at 4°C, this condition was used for the next experiments. To decrease the amount of competing soil microbiota, the soil samples were autoclaved prior inoculation. After inoculation and after 1 and 4 weeks, samples were taken for RNA isolation. The differential expression analysis was performed using the sample from time point 0 as control. The analysis revealed that stress response genes and genes of the primary metabolism were upregulated after 1 and 4 weeks. In addition, genes and gene sets for the uptake of various carbohydrates or amino acids were upregulated, indicating adaption to an environment with low nutrient availability. Moreover, the results of the second publication demonstrated that persistence of C227/11Φcu in soil is related with a complex interface of metabolic networks. The third paper focused on the colonization of lamb’s lettuce by E. coli O104:H4 strain C227/11Φcu via the root system. Surface-sterilized seeds were cultivated on Murashige-Skoog agar or in autoclaved agricultural soil and migration into the edible portions of the plants was analyzed after 2, 4 and 8 weeks. The results of this publication demonstrated that migration into the edible parts occurred when the surrounding agar or soil was contaminated. This highlighted the threat of plant contamination with pathogenic E. coli on the field as a result of soil contamination.
Vergleichende Transkriptomanalyse und funktionelle Untersuchungen von enterohämorrhagischen Escherichia coli nach Kultivierung in Pflanzenmedium
(2020) Bufe, Thorsten; Schmidt, Herbert
Enterohemorrhagic Escherichia coli (EHEC) are human pathogens which are able to cause severe gastrointestinal diseases in humans. The gastrointestinal tract of cattle is considered as the main reservoir for EHEC and contaminated raw meat represents the primary source of infection. Yet there have been increasing reports over the last few decades of EHEC infections that were linked to the consumption of raw vegetables. Today it is generally accepted that EHEC bacteria are able to use plants as their secondary hosts, thus favouring the transmission to humans. To improve the understanding of this pathogen-plant interaction fundamental knowledge about the pathogens’ molecular adaptions towards plant material is urgently required. In the cope of this study the adaption of different EHEC strains towards components of the plant was examined. Therefore O157:H7 strain Sakai, O104:H4 strain C227-11phicu and O157:H strain 3072/96 were chosen as surrogates. In growth experiments performed with an artificial lettuce medium it could be shown that components of the lettuce were sufficient for the proliferation of the three strains. RNA-sequencing was performed to study the differential gene expression of the three strains after the growth in lettuce medium compared to the growth in M9 minimal medium. In order to compare genes according to standardized gene denotations, the differential gene expression analysis was performed on the basis of a shared genome including the genomes of the three pathogenic strains as well as the genome of Escherichia coli strain K-12 substrain MG1655. Analogous to the successful growth in presence of components of the plant an upregulation of genes involved in carbohydrate and peptide metabolism throughout all three strains was observed. Especially genes involved in the catabolism of lactose (lacZ), ribose (rbsAC) and xylose (xylF) were found to be uniformly upregulated. The greatest differences among the strains accounted for the regulation of motility and chemotaxis genes. O104:H4 strain C227-11phicu showed a strong upregulation of all three classes of the flagellar hierarchy (class I, II and III) in presence of plant derived compounds. These included genes involved in the establishment of the basal body hook structure (fli, flg), the synthesis of the flagellar filament (fliC), and the chemotaxis-system (che, tap, tar). In contrast, O157:H7 strain Sakai only featured upregulation of class I and class II genes. According to the transcriptional data both of these strains also showed increased swimming and swarming behaviour on motility plates in presence of lettuce extract. Solely O157:H- strain 3072/96, which is non-motile due to a deletion in the flhC gene, showed an upregulation of virulence factors encoded on the LEE pathogenicity island, including genes involved in the establishment of the T3SS (esc) and T3SS secreted effectors (esp). Interestingly, it was shown for O157:H- strain 3072/96 to have a powerful capacity to form biofilms in M9 minimal medium. Furthermore it was proven that the complementation of an intact flhC gene restored motility in O157:H- strain 3072/96. In this regard it could be shown that the deletion in flhC was not the mere reason for the augmented biofilm formation capacity. In addition to the biofilm formation, the strains’ potential to adhere to HT-29 cells was examined. Here a significantly increased adherence potential for O157:H- strain 3072/96 with respect to the motile strains could be observed, the lowest adherence potential was determined for O157:H7 strain Sakai. The results presented in this study clearly indicate that the different EHEC strains are capable to adapt towards the nutrient availability provided by their plantal host. It can be assumed that flagella and the chemotaxis system play a fundamental role in the finding and exploitation of the plant. Furthermore curli structures might play a crucial role in the initial adherence and the subsequent establishment of a biofilm on plant tissues. Presumably, besides the typical plant associated outbreak strain O157:H7 strain Sakai, there are further strains capable of utilizing their genetic repertoire in order to adapt towards the atypical environmental conditions within this niche. The findings of this study suggest that the strains, besides sharing multiple coinciding mechanisms, are able to adapt in a strain specific manner and use different strategies in coping with plants as their secondary hosts.