Karin Hummel

Species-specific identification and differentiation of Arcobacter, Helicobacter and Campylobacter by full-spectral matrix-associated laser desorption/ionization time of flight mass spectrometry analysis.

Rapid and reliable identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, has become increasingly important, since many of them are now recognized as human and/or animal pathogens. Matrix-associated laser desorption/ionization-time of flight (MALDI-TOF) MS has been shown to be a rapid and sensitive method for characterization of micro-organisms. In this study, we therefore established a reference database of selected Arcobacter, Helicobacter and Campylobacter species for MALDI-TOF MS identification. Besides the species with significance as food-borne pathogens - Arcobacter butzleri, Helicobacter pullorum, Campylobacter jejuni and Campylobacter coli - several other members of these genera were included in the reference library to determine the species specificity of the designed MALDI Biotyper reference database library. Strains that made up the reference database library were grown on Columbia agar, and yielded reproducible and unique mass spectra profiles, which were compared with the Bruker Biotyper database, version 2. The database was used to identify 144 clinical isolates using whole spectral profiles. Furthermore, reproducibility of MALDI-TOF MS results was evaluated with respect to age and/or storage of bacteria and different growth media. It was found that correct identification could be obtained even if the bacteria were stored at room temperature or at 4 degrees C up to 9 days before being tested. In addition, bacteria were correctly identified when grown on Campylosel agar; however, they were not when grown on modified charcoal cefoperazone deoxycholate agar. These results indicate that MALDI-TOF MS fingerprinting is a fast and reliable method for the identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, with applications in clinical diagnostics.

A Comparative Analysis of Industrial Escherichia coli K–12 and B Strains in High-Glucose Batch Cultivations on Process-, Transcriptome- and Proteome Level

Escherichia coli K–12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (EttanTM DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K–12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation.

Proteomic analysis of porcine saliva.

Saliva contains a number of proteins that may be useful as biomarkers of health and disease and can be easily obtained from large numbers of animals in a non-invasive, stress-free way. The objective of this study was to explore the protein composition of porcine saliva from 10 specific pathogen free pigs using first one-dimensional SDS-PAGE and then two-dimensional electrophoresis and mass spectrometry. A reference proteome pattern for porcine saliva was established with the identification of 13 different, mainly saliva-specific, proteins. These reference data will facilitate the investigation of salivary proteins potentially altered in disease and could serve as novel diagnostic biomarkers.

Cor a 14, the allergenic 2S albumin from hazelnut, is highly thermostable and resistant to gastrointestinal digestion

Scope Allergens from nuts frequently induce severe allergic reactions in sensitive individuals. The aim of this study was to elucidate the physicochemical characteristics of natural Cor a 14, the 2S albumin from hazelnut. Methods and results Cor a 14 was purified from raw hazelnuts using a combination of precipitation and chromatographic techniques. The protein was analyzed using gel electrophoresis, MS, and far‐UV circular dichroism (CD) analyses. The immunoglobulin E (IgE) binding of native, heat‐treated, and in vitro digested Cor a 14 was studied. We identified two different Cor a 14 isoforms and showed microclipping at the C‐terminus. CD spectra at room temperature showed the typical characteristics of 2S albumins, and temperatures of more than 80°C were required to start unfolding of Cor a 14 demonstrating its high stability to heat treatment. In vitro digestion experiments revealed that Cor a 14 is resistant to proteolytic degradation. Native and heat‐treated protein was recognized by sera from hazelnut allergic patients. However, denaturation of the allergen led to significantly reduced IgE binding. Conclusion We identified two different isoforms of Cor a 14 displaying high stability under heating and gastric and duodenal conditions. Data from IgE‐binding experiments revealed the existence of both, linear and conformational epitopes.

Downregulation of Cellular Protective Factors of Rumen Epithelium in Goats Fed High Energy Diet

Energy-rich diets can challenge metabolic and protective functions of the rumen epithelial cells, but the underlying factors are unclear. This study sought to evaluate proteomic changes of the rumen epithelium in goats fed a low, medium, or high energy diet. Expression of protein changes were compared by two-dimensional differential gel electrophoresis followed by protein identification with matrix assisted laser desorption ionisation tandem time-of-flight mass spectrometry. Of about 2,000 spots commonly detected in all gels, 64 spots were significantly regulated, which were traced back to 24 unique proteins. Interestingly, the expression profiles of several chaperone proteins with important cellular protective functions such as heat shock cognate 71 kDa protein, peroxiredoxin-6, serpin H1, protein disulfide-isomerase, and selenium-binding protein were collectively downregulated in response to high dietary energy supply. Similar regulation patterns were obtained for some other proteins involved in transport or metabolic functions. In contrast, metabolic enzymes like retinal dehydrogenase 1 and ATP synthase subunit beta, mitochondrial precursor were upregulated in response to high energy diet. Lower expressions of chaperone proteins in the rumen epithelial cells in response to high energy supply may suggest that these cells were less protected against the potentially harmful rumen toxic compounds, which might have consequences for rumen and systemic health. Our findings also suggest that energy-rich diets and the resulting acidotic insult may render rumen epithelial cells more vulnerable to cellular damage by attenuating their cell defense system, hence facilitating the impairment of rumen barrier function, typically observed in energy-rich fed ruminants.

Development of Dictyostelium discoideum is associated with alteration of fucosylated N-glycan structures

The social amoeba Dictyostelium discoideum has become established as a simple model for the examination of cell-cell interactions, and early studies suggested that shifts in glycosylation profiles take place during its life cycle. In the present study, we have applied HPLC and mass spectrometric methods to show that the major N-glycans in axenic cultures of the AX3 strain are oligomannosidic forms, most of which carry core fucose and/or intersecting and bisecting N-acetylglucosamine residues, including the major structure with the composition Man8GlcNAc4Fuc1. The postulated alpha1,3-linkage of the core fucose correlates with the cross-reactivity of Dictyostelium glycoproteins with a horseradish peroxidase antiserum; a corresponding core alpha1,3-fucosyltransferase activity capable of modifying oligomannosidic N-glycans was detected in axenic Dictyostelium extracts. The presence of fucose on the N-glycans and the reactivity to the antiserum, but not the fucosyltransferase activity, are abolished in the fucose-deficient HL250 strain. In later stages of development, N-glycans at the mound and culmination stages show a reduction in both the size and the degree of modification by intersecting/bisecting residues compared with mid-exponential phase cultures, consistent with the hypothesis that glycosidase and glycosyltransferase expression levels are altered during the slime mould life cycle.

Growth promotion in pigs by oxytetracycline coincides with down regulation of serum inflammatory parameters and of hibernation-associated protein HP-27.

The growth promoting effect of supplementing animal feed with antibiotics like tetracycline has traditionally been attributed to their antibiotic character. However, more evidence has been accumulated on their direct anti‐inflammatory effect during the last two decades. Here we used a pig model to explore the systemic molecular effect of feed supplementation with sub therapeutic levels of oxytetracycline (OTC) by analysis of serum proteome changes. Results showed that OTC promoted growth, coinciding with a significant down regulation of different serum proteins related to inflammation, oxidation and lipid metabolism, confirming the anti‐inflammatory mechanism of OTC. Interestingly, apart from the classic acute phase reactants also down regulation was seen of a hibernation associated plasma protein (HP‐27), which is to our knowledge the first description in pigs. Although the exact function in non‐hibernators is unclear, down regulation of HP‐27 could be consistent with increased appetite, which is possibly linked to the anti‐inflammatory action of OTC. Given that pigs are good models for human medicine due to their genetic and physiologic resemblance, the present results might also be used for rational intervention in human diseases in which inflammation plays an important role such as obesity, type 2 diabetes and cardiovascular diseases.

Glutathione S-transferase affects permethrin detoxification in the brown dog tick, Rhipicephalus sanguineus

Control of ticks on dogs is often done by application of repellents that contain permethrin as the active ingredient. In this research, we studied the role of a glutathione S-transferase (GST) gene in detoxification of permethrin by ticks using a gene silencing method RNA interference (RNAi). The brown dog tick, Rhipicephalus sanguineus, used in these studies, has a notable host preference for dogs, but also infests other mammals. In this research, R. sanguineus females were injected with gst double-stranded RNA (dsRNA) to effect gene silencing by RNAi and then exposed to sublethal doses of permethrin. Sixty hours after injection, the females were allowed to feed on sheep. The female ticks subjected to RNAi proved to be more susceptible to permethrin than the untreated controls. The effect of gene silencing was most notable in the highest dose group (50.3 ppm) in which all ticks died, while in the corresponding controls that were not subjected to RNAi this dose was not lethal. The acaricide treatment of the ticks resulted in a change in tick attachment behavior. Acaricide-treated ticks attached in a scattered pattern in contrast to the control ticks that attached and fed tightly clustered together. The time required for repletion for both the injected and non-injected females exposed to the higher permethrin level was shorter than that observed in the lower-dose groups and unexposed controls, and this more rapid attachment and feeding would likely favor more rapid transmission of pathogens. However, engorgement and egg mass weights were not significantly different among the experimental groups. This research demonstrated that the silencing of the gst gene increased the ticks susceptibility to permethrin. Overall, these results have contributed to our understanding of the detoxification mechanism of ticks and provide new considerations for the formulation of treatment strategies.

Shotgun proteomic analysis of Yersinia ruckeri strains under normal and iron-limited conditions

Yersinia ruckeri is the causative agent of enteric redmouth disease of fish that causes significant economic losses, particularly in salmonids. Bacterial pathogens differentially express proteins in the host during the infection process, and under certain environmental conditions. Iron is an essential nutrient for many cellular processes and is involved in host sensing and virulence regulation in many bacteria. Little is known about proteomics expression of Y. ruckeri in response to iron-limited conditions. Here, we present whole cell protein identification and quantification for two motile and two non-motile strains of Y. ruckeri cultured in vitro under iron-sufficient and iron-limited conditions, using a shotgun proteomic approach. Label-free, gel-free quantification was performed using a nanoLC-ESI and high resolution mass spectrometry. SWATH technology was used to distinguish between different strains and their responses to iron limitation. Sixty-one differentially expressed proteins were identified in four Y. ruckeri strains. These proteins were involved in processes including iron ion capture and transport, and enzymatic metabolism. The proteins were confirmed to be differentially expressed at the transcriptional level using quantitative real time PCR. Our study provides the first detailed proteome analysis of Y. ruckeri strains, which contributes to our understanding of virulence mechanisms of Y. ruckeri, and informs development of novel control methods for enteric redmouth disease.

In between — Proteomics of dog biological fluids

Dogs are relevant to biomedical research in connection both to veterinary medicine for their role as pets and to basic investigations for their use as animal models in pathology, pharmacology and toxicology studies. Proteomic analysis of biological fluids is less advanced for dogs than for other animal species but a wealth of information has already been gathered, which we summarize in this review. As a remarkable feature, we also assemble here for due reference a number of 2-DE serum/plasma or urine patterns in health and disease; some of them correspond to unpublished data from the University of Veterinary Medicine Vienna.