Tom Grunert

Rapid and Reliable Identification of Staphylococcus aureus Capsular Serotypes by Means of Artificial Neural Network-Assisted Fourier Transform Infrared Spectroscopy

ABSTRACT Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors and represent putative targets for vaccine development. Therefore, the purpose of this study was to develop a high-throughput method to identify and discriminate the clinically important S. aureus capsular serotypes 5, 8, and NT (nontypeable). A comprehensive set of clinical isolates derived from different origins and control strains, representative for each serotype, were used to establish a CP typing system based on Fourier transform infrared (FTIR) spectroscopy and chemometric techniques. By combining FTIR spectroscopy with artificial neuronal network (ANN) analysis, a system was successfully established, allowing a rapid identification and discrimination of all three serotypes. The overall accuracy of the ANN-assisted FTIR spectroscopy CP typing system was 96.7% for the internal validation and 98.2% for the external validation. One isolate in the internal validation and one isolate in the external validation failed in the classification procedure, but none of the isolates was incorrectly classified. The present study demonstrates that ANN-assisted FTIR spectroscopy allows a rapid and reliable discrimination of S. aureus capsular serotypes. It is suitable for diagnostic as well as large-scale epidemiologic surveillance of S. aureus capsule expression and provides useful information with respect to chronicity of infection.

Separation of active and inactive forms of human antithrombin by heparin affinity chromatography

During the manufacturing of an antithrombin preparation, it is necessary to define all steps that may damage or alter the target molecule, and thus decrease the biological activity of the inhibitor in blood coagulation. Pasteurization, commonly used procedure for viral inactivation of plasma derived antithrombin concentrates, was shown to partially alter the conformation of the active native antithrombin to an inactive latent form. To study intensively the different forms of inactive antithrombin that are formed upon heat treatment, human alpha-antithrombin, human beta-antithrombin and an equimolar mixture of the two isoforms were incubated at 60 degrees C for 15 h in the presence of citrate as stabilizing agent. Using two subsequent heparin affinity chromatography steps, three different inactive fractions were separated. By comparison of the heparin binding capacities, isoelectric points and unfolding characteristics of these inactive forms, the alpha-latent and beta-latent antithrombin isoforms could be identified. It was also shown that additional inactive forms such as proteinase cleaved and/or oxidized forms of antithrombin are formed during the heat treatment process. In four commercially available antithrombin preparations, all produced by pasteurization, the amount of inactive protein varied between 0.5% and 9.5%.

Dynamics of uterine infections with Escherichia coli, Streptococcus uberis and Trueperella pyogenes in post-partum dairy cows and their association with clinical endometritis

The diversity and dynamics of the uterine microbiota of dairy cows are poorly understood although it is becoming increasingly evident that they play a crucial role in the development of metritis and endometritis. Fourier-transform infrared (FTIR) spectroscopy was used to monitor the bovine microbiota of 40 cows on the day of calving and days 3, 9, 15, and 21 after parturition, and to investigate the associations of selected species with clinical endometritis (CE). Trueperella pyogenes (43.5%), Escherichia coli (21.5%), Bacillus spp. (21.0%) and Streptococcus uberis (18.5%) were the most frequently isolated microbes. Analyses of different sampling time points revealed that the presence of S. uberis on day 3 increased the risk of subsequent T. pyogenes infection on day 9 (odds ratio [OR] = 5.1, 95% confidence interval [CI] = 1.2-22.6). T. pyogenes infection (OR = 36.0, 95% CI = 3.8-343.2) and retained fetal membranes (RFM) (OR = 12.4, 95%CI = 1.4-112.7) were significant risk factors for CE. Cows with S. uberis on day 3 tended to have greater odds of CE than S. uberis-negative cows (OR = 7.1, 95% CI = 0.9-55.6). Chemometric analysis revealed significant differences in the metabolic profile of S. uberis strains isolated from cows with different vaginal discharge scores. This is the first study showing the association of specific S. uberis subtypes with the uterine health status of post-partum dairy cows. The study demonstrates that uterine clearance is a highly dynamic process, during which time bacteria show distinct patterns of progression, and provides information about interactions between bacterial species involved in the occurrence of CE.

Tyrosine Kinase 2 Controls IL-1β Production at the Translational Level

IL-1β is an important proinflammatory cytokine with a major role in several inflammatory diseases. Expression of IL-1β is tightly regulated at the level of transcription, mRNA stability, and proteolytic processing. In this study, we report that IL-1β expression in response to LPS is also regulated at the translational level. LPS-induced IL-1β protein levels in macrophages derived from murine bone marrow are markedly increased in the absence of tyrosine kinase 2 (Tyk2). Increased IL-1β is found intra- and extracellularly, irrespective of the efficiency of IL-1β processing. We show that the absence of Tyk2 results both in higher translational rates and in enhanced association of IL-1β mRNA with polysomes. Induction and stability of IL-1β mRNA are not affected by the lack of Tyk2. We show further that the Tyk2-dependent translational inhibition is mediated by autocrine/paracrine type I IFN signaling and requires signal transducer and activator of transcription 1. Enhanced IL-1β production in Tyk2- and IFN receptor 1-deficient macrophages is also observed following Listeria monocytogenes infection. Taken together, the data describe a novel mechanism for the control of IL-1β synthesis.

Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese.

Staphylococcus aureus is one of the most important contagious mastitis pathogens in dairy cattle. Due to its zoonotic potential, control of S. aureus is not only of great economic importance in the dairy industry but also a significant public health concern. The aim of this study was to decipher the potential of bovine udder associated S. aureus as reservoir for S. aureus contamination in dairy production and processing. From 18 farms, delivering their milk to an alpine dairy plant for the production of smeared semi-hard and hard cheese. one thousand hundred seventy six one thousand hundred seventy six quarter milk (QM) samples of all cows in lactation (n = 294) and representative samples form bulk tank milk (BTM) of all farms were surveyed for coagulase positive (CPS) and coagulase negative Staphylococci (CNS). Furthermore, samples from different steps of the cheese manufacturing process were tested for CPS and CNS. As revealed by chemometric-assisted FTIR spectroscopy and molecular subtyping (spa typing and multi locus sequence typing), dairy cattle represent indeed an important, yet underreported, entrance point of S. aureus into the dairy chain. Our data clearly show that certain S. aureus subtypes are present in primary production as well as in the cheese processing at the dairy plant. However, although a considerable diversity of S. aureus subtypes was observed in QM and BTM at the farms, only certain S. aureus subtypes were able to enter and persist in the cheese manufacturing at the dairy plant and could be isolated from cheese until day 14 of ripening. Farm strains belonging to the FTIR cluster B1 and B3, which show genetic characteristics (t2953, ST8, enterotoxin profile: sea/sed/sej) of the recently described S. aureus genotype B, most successfully contaminated the cheese production at the dairy plant. Thus, our study fosters the hypothesis that genotype B S. aureus represent a specific challenge in control of S. aureus in the dairy chain that requires effective clearance strategies and hygienic measures already in primary production to avoid a potential transfer of enterotoxic strains or enterotoxins into the dairy processing and the final retail product.

The impact of tyrosine kinase 2 (Tyk2) on the proteome of murine macrophages and their response to lipopolysaccharide (LPS)

Tyrosine kinase 2 (Tyk2) belongs to the Janus kinase (Jak) family and is involved in signalling via a number of cytokines. Tyk2‐deficient mice are highly resistant to lipopolysaccharide (LPS)‐induced endotoxin shock. Macrophages are key players in the pathogenesis of endotoxin shock and, accordingly, defects in the LPS responses of Tyk2−/− macrophages have been reported. In the present study, the molecular role of Tyk2 is investigated in more detail using a proteomics approach. 2‐D DIGE was applied to compare protein patterns from wild‐type and Tyk2−/− macrophages and revealed significant differences in protein expression patterns between the genotypes before and after LPS treatment. Twenty‐one proteins deriving from 25 differentially expressed spots were identified by MALDI/ESI MS. Among them, we show for N‐myc interactor that its mRNA transcription/stability is positively influenced by Tyk2. In contrast, LPS‐induced expression of plasminogen activator 2 protein but not mRNA is strongly enhanced in the absence of Tyk2. Our data furthermore suggest an influence of Tyk2 on the subcellular distribution of elongation factor 2 and on LPS‐mediated changes in the peroxiredoxin 1 spot pattern. Thus, our results imply regulatory roles of Tyk2 at multiple levels and establish novel connections between Tyk2 and several cellular proteins.

High-resolution subtyping of Staphylococcus aureus strains by means of Fourier-transform infrared spectroscopy.

Staphylococcus aureus causes a variety of serious illnesses in humans and animals. Subtyping of S. aureus isolates plays a crucial role in epidemiological investigations. Metabolic fingerprinting by Fourier-transform infrared (FTIR) spectroscopy is commonly used to identify microbes at species as well as subspecies level. In this study, we aimed to assess the suitability of FTIR spectroscopy as a tool for S. aureus subtyping. To this end, we compared the subtyping performance of FTIR spectroscopy to other subtyping methods such as pulsed field gel electrophoresis (PFGE) and spa typing in a blinded experimental setup and investigated the ability of FTIR spectroscopy for identifying S. aureus clonal complexes (CC). A total of 70 S. aureus strains from human, animal, and food sources were selected, for which clonal complexes and a unique virulence and resistance gene pattern had been determined by DNA microarray analysis. FTIR spectral analysis resulted in high discriminatory power similar as obtained by spa typing and PFGE. High directional concordance was found between FTIR spectroscopy based subtypes and capsular polysaccharide expression detected by FTIR spectroscopy and the cap specific locus, reflecting strain specific expression of capsular polysaccharides and/or other surface glycopolymers, such as wall teichoic acid, peptidoglycane, and lipoteichoic acid. Supervised chemometrics showed only limited possibilities for differentiation of S. aureus CC by FTIR spectroscopy with the exception of CC45 and CC705. In conclusion, FTIR spectroscopy represents a valuable tool for S. aureus subtyping, which complements current molecular and proteomic strain typing.

A comparative proteome analysis links tyrosine kinase 2 (Tyk2) to the regulation of cellular glucose and lipid metabolism in response to poly(I:C)

Tyrosine kinase 2 (Tyk2) is an integral part of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway which relays intracellular signals of various cytokines. Tyk2 crucially contributes to host defense mechanisms against microbial pathogens and to tumor surveillance but also facilitates immune pathologies. Here we investigated the impact of Tyk2 on the macrophage proteome using the synthetic double-stranded RNA analog polyinosinic acid-polycytidylic acid (poly(I:C)) as a mimicry of viral infections. By means of 2D-DIGE in connection with PMF obtained by MALDI-MS and sequence tag determination by MS/MS we unambiguously identified eighteen protein spots corresponding to sixteen distinct proteins that are regulated by poly(I:C) and differentially expressed between wildtype (WT) and Tyk2-deficient macrophages. The majority of these proteins are functionally assigned to cellular immune responses and to metabolism. We show for selected metabolic enzymes, i.e. triosephosphate isomerase (TIM), ATP-citrate synthase (ACLY) and long-chain-fatty-acid-CoA ligase 4 (ACSL4), that Tyk2 affects protein expression transcriptionally and post-transcriptionally. We furthermore confirm the involvement of Tyk2 in the regulation of lipid and carbohydrate metabolism at the level of metabolites. Taken together, our results provide new evidence for important functions of Tyk2 at the molecular interface between innate immunity and cellular metabolism.

Comparing the applicability of CGE-on-the-chip and SDS-PAGE for fast pre-screening of mouse serum samples prior to proteomics analysis.

In this comparative study, a set of 13 mouse sera was investigated, which had to be checked in a fast way for homogeneity prior to complex, time‐consuming and expensive proteomics analysis. SDS‐PAGE and CGE‐on‐the‐chip were compared in terms of handling, time consumption and significance of detecting differences. SDS‐PAGE was a system giving good information on the samples at first sight and discrepancies for one sample were observed immediately after staining. The time consumption of up to 20 h was rather high (separation and staining procedure with 14×14 cm gels) in SDS‐PAGE. CGE‐on‐the‐chip system exhibited differences for two samples and, most important, analysis time was reduced to only 1 h. Of importance is that the serum protein pattern obtained by both methods may not represent the same proteins. Sample preparation was equal for both analytical techniques but necessary sample amount was only half of the material in the case of CGE‐on‐the‐chip. Statistical evaluation indicated highly critical values for only one sample in SDS‐PAGE and for two samples in the CGE‐on‐the‐chip system, both in good agreement with visual observations. Three samples showed limited homogeneity with both methods. Five and six samples, respectively, were indicated as noticeable with one of the used methods.

Deciphering host genotype-specific impacts on the metabolic fingerprint of Listeria monocytogenes by FTIR spectroscopy.

Bacterial pathogens are known for their wide range of strategies to specifically adapt to host environments and infection sites. An in-depth understanding of these adaptation mechanisms is crucial for the development of effective therapeutics and new prevention measures. In this study, we assessed the suitability of Fourier Transform Infrared (FTIR) spectroscopy for monitoring metabolic adaptations of the bacterial pathogen Listeria monocytogenes to specific host genotypes and for exploring the potential of FTIR spectroscopy to gain novel insights into the host-pathogen interaction. Three different mouse genotypes, showing different susceptibility to L. monocytogenes infections, were challenged with L. monocytogenes and re-isolated bacteria were subjected to FTIR spectroscopy. The bacteria from mice with different survival characteristics showed distinct IR spectral patterns, reflecting specific changes in the backbone conformation and the hydrogen-bonding pattern of the protein secondary structure in the bacterial cell. Coupling FTIR spectroscopy with chemometrics allowed us to link bacterial metabolic fingerprints with host infection susceptibility and to decipher longtime memory effects of the host on the bacteria. After prolonged cultivation of host-passaged bacteria under standard laboratory conditions, the hosts imprint on bacterial metabolism vanished, which suggests a revertible metabolic adaptation of bacteria to host environment and loss of host environment triggered memory effects over time. In summary, our work demonstrates the potential and power of FTIR spectroscopy to be used as a fast, simple and highly discriminatory tool to investigate the mechanism of bacterial host adaptation on a macromolar and metabolic level.