• Heparin 2.0: A New Approach to the Infection Crisis.

      Seffer, Malin-Theres; Cottam, Daniel; Forni, Lui G; Kielstein, Jan T; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Karger AG, 2020-07-02)
      In April 2020, the US Food and Drug Administration granted emergency use authorization for certain medical devices to be used in patients with coronavirus disease 2019 (CO-VID-19). This included extracorporeal blood purification devices. This narrative review will give a brief overview regarding some of the extracorporeal devices that could be used to treat COVID-19 patients, including the Seraph® 100 Microbind® Affinity Blood Filter, produced by ExThera Medical (Martinez, CA, USA), first licensed in the European Economic Area in 2019. The Seraph® 100 contains ultrahigh molecular weight polyethylene beads with end point-attached heparin and is approved for the reduction of pathogens from the bloodstream either as a single agent or as an adjunct to conventional anti-infective agents. Bacteria, viruses, fungi, and toxins have been shown to bind to the immobilized heparin in a similar way to the interaction with heparan sulfate on the cell surface. This binding is nonreversible and as such, the pathogens are removed from the bloodstream. In this review, we describe the pathophysiological basis and rationale for using heparin for pathogen removal from the blood as well as exploring the technology behind the adaptation of heparin to deprive it of its systemic anticoagulant activity. In addition, we summarize the in vitro data as well as the available preclinical testing and published clinical reports. Finally, we discuss the enormous potential of this technology in an era of increasing antibiotic resistance and high mortality associated with sepsis and consider the application of this as a possible treatment option for COVID-19.
    • Hepatic Transcriptome Analysis Identifies Divergent Pathogen-Specific Targeting-Strategies to Modulate the Innate Immune System in Response to Intramammary Infection.

      Heimes, Annika; Brodhagen, Johanna; Weikard, Rosemarie; Seyfert, Hans-Martin; Becker, Doreen; Meyerholz, Marie M; Petzl, Wolfram; Zerbe, Holm; Hoedemaker, Martina; Rohmeier, Laura; et al. (Frontiers, 2020-04-29)
      Mastitis is one of the major risks for public health and animal welfare in the dairy industry. Two of the most important pathogens to cause mastitis in dairy cattle are Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). While S. aureus generally induces a chronic and subclinical mastitis, E. coli is an important etiological pathogen resulting in an acute and clinical mastitis. The liver plays a central role in both, the metabolic and inflammatory physiology of the dairy cow, which is particularly challenged in the early lactation due to high metabolic and immunological demands. In the current study, we challenged the mammary glands of Holstein cows with S. aureus or E. coli, respectively, mimicking an early lactation infection. We compared the animals' liver transcriptomes with those of untreated controls to investigate the hepatic response of the individuals. Both, S. aureus and E. coli elicited systemic effects on the host after intramammary challenge and seemed to use pathogen-specific targeting strategies to bypass the innate immune system. The most striking result of our study is that we demonstrate for the first time that S. aureus intramammary challenge causes an immune response beyond the original local site of the mastitis. We found that in the peripheral liver tissue defined biological pathways are switched on in a coordinated manner to balance the immune response in the entire organism. TGFB1 signaling plays a crucial role in this context. Important pathways involving actin and integrin, key components of the cytoskeleton, were downregulated in the liver of S. aureus infected cows. In the hepatic transcriptome of E. coli infected cows, important components of the complement system were significantly lower expressed compared to the control cows. Notably, while S. aureus inhibits the cell signaling by Rho GTPases in the liver, E. coli switches the complement system off. Also, metabolic hepatic pathways (e.g., lipid metabolism) are affected after mammary gland challenge, demonstrating that the liver restricts metabolic tasks in favor of the predominant immune response after infection. Our results provide new insights for the infection-induced modifications of the dairy cow's hepatic transcriptome following mastitis.
    • Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins.

      Romero Pastrana, Francisco; Neef, Jolanda; Koedijk, Dennis G A M; de Graaf, Douwe; Duipmans, José; Jonkman, Marcel F; Engelmann, Susanne; van Dijl, Jan Maarten; Buist, Girbe; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-02-19)
      Human antibody responses to pathogens, like Staphylococcus aureus, are important indicators for in vivo expression and immunogenicity of particular bacterial components. Accordingly, comparing the antibody responses to S. aureus components may serve to predict their potential applicability as antigens for vaccination. The present study was aimed at assessing immunoglobulin G (IgG) responses elicited by non-covalently cell surface-bound proteins of S. aureus, which thus far received relatively little attention. To this end, we applied plasma samples from patients with the genetic blistering disease epidermolysis bullosa (EB) and healthy S. aureus carriers. Of note, wounds of EB patients are highly colonized with S. aureus and accordingly these patients are more seriously exposed to staphylococcal antigens than healthy individuals. Ten non-covalently cell surface-bound proteins of S. aureus, namely Atl, Eap, Efb, EMP, IsaA, LukG, LukH, SA0710, Sle1 and SsaA2, were selected by bioinformatics and biochemical approaches. These antigens were recombinantly expressed, purified and tested for specific IgG responses using human plasma. We show that high exposure of EB patients to S. aureus is mirrored by elevated IgG levels against all tested non-covalently cell wall-bound staphylococcal antigens. This implies that these S. aureus cell surface proteins are prime targets for the human immune system.
    • In vivo model to study the impact of genetic variation on clinical outcome of mastitis in uniparous dairy cows.

      Rohmeier, L; Petzl, W; Koy, M; Eickhoff, T; Hülsebusch, A; Jander, S; Macias, L; Heimes, A; Engelmann, S; Hoedemaker, M; et al. (BioMed Central (BMC), 2020-01-31)
      BACKGROUND: In dairy herds, mastitis causes detrimental economic losses. Genetic selection offers a sustainable tool to select animals with reduced susceptibility towards postpartum diseases. Studying underlying mechanisms is important to assess the physiological processes that cause differences between selected haplotypes. Therefore, the objective of this study was to establish an in vivo infection model to study the impact of selecting for alternative paternal haplotypes in a particular genomic region on cattle chromosome 18 for mastitis susceptibility under defined conditions in uniparous dairy cows. RESULTS: At the start of pathogen challenge, no significant differences between the favorable (Q) and unfavorable (q) haplotypes were detected. Intramammary infection (IMI) with Staphylococcus aureus 1027 (S. aureus, n = 24, 96 h) or Escherichia coli 1303 (E. coli, n = 12, 24 h) was successfully induced in all uniparous cows. This finding was confirmed by clinical signs of mastitis and repeated recovery of the respective pathogen from milk samples of challenged quarters in each animal. After S. aureus challenge, Q-uniparous cows showed lower somatic cell counts 24 h and 36 h after challenge (P < 0.05), lower bacterial shedding in milk 12 h after challenge (P < 0.01) and a minor decrease in total milk yield 12 h and 24 h after challenge (P < 0.01) compared to q-uniparous cows. CONCLUSION: An in vivo infection model to study the impact of genetic selection for mastitis susceptibility under defined conditions in uniparous dairy cows was successfully established and revealed significant differences between the two genetically selected haplotype groups. This result might explain their differences in susceptibility towards IMI. These clinical findings form the basis for further in-depth molecular analysis to clarify the underlying genetic mechanisms for mastitis resistance.
    • Metaproteomics to unravel major microbial players in leaf litter and soil environments: challenges and perspectives.

      Becher, Dörte; Bernhardt, Jörg; Fuchs, Stephan; Riedel, Katharina; Ernst-Moritz-Arndt-University of Greifswald, Institute of Microbiology, Greifswald, Germany. (2013-10)
      Soil- and litter-borne microorganisms vitally contribute to biogeochemical cycles. However, changes in environmental parameters but also human interferences may alter species composition and elicit alterations in microbial activities. Soil and litter metaproteomics, implying the assignment of soil and litter proteins to specific phylogenetic and functional groups, has a great potential to provide essential new insights into the impact of microbial diversity on soil ecosystem functioning. This article will illuminate challenges and perspectives of current soil and litter metaproteomics research, starting with an introduction to an appropriate experimental design and state-of-the-art proteomics methodologies. This will be followed by a summary of important studies aimed at (i) the discovery of the major biotic drivers of leaf litter decomposition, (ii) metaproteomics analyses of rhizosphere-inhabiting microbes, and (iii) global approaches to study bioremediation processes. The review will be closed by a brief outlook on future developments and some concluding remarks, which should assist the reader to develop successful concepts for soil and litter metaproteomics studies.
    • Mixture Effects of Estrogenic Pesticides at the Human Estrogen Receptor α and β.

      Seeger, Bettina; Klawonn, Frank; Nguema Bekale, Boris; Steinberg, Pablo; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Consumers of fruits and vegetables are frequently exposed to small amounts of hormonally active pesticides, some of them sharing a common mode of action such as the activation of the human estrogen receptor α (hERα) or β (hERβ). Therefore, it is of particular importance to evaluate risks emanating from chemical mixtures, in which the individual pesticides are present at human-relevant concentrations, below their corresponding maximum residue levels. Binary and ternary iso-effective mixtures of estrogenic pesticides at effect concentrations eliciting a 1 or 10% effect in the presence or absence of 17β-estradiol were tested experimentally at the hERα in the yeast-based estrogen screen (YES) assay as well as in the human U2-OS cell-based ERα chemical-activated luciferase gene expression (ERα CALUX) assay and at the hERβ in the ERβ CALUX assay. The outcome was then compared to predictions calculated by means of concentration addition. In most cases, additive effects were observed with the tested combinations in all three test systems, an observation that supports the need to expand the risk assessment of pesticides and consider cumulative risk assessment. An additional testing of mixture effects at the hERβ showed that most test substances being active at the hERα could also elicit additive effects at the hERβ, but the hERβ was less sensitive. In conclusion, effects of the same ligands at the hERα and the hERβ could influence the estrogenic outcome under physiological conditions.
    • smORFer: a modular algorithm to detect small ORFs in prokaryotes.

      Bartholomäus, Alexander; Kolte, Baban; Mustafayeva, Ayten; Goebel, Ingrid; Fuchs, Stephan; Benndorf, Dirk; Engelmann, Susanne; Ignatova, Zoya; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Oxford Academic, 2021-06-14)
      Emerging evidence places small proteins (≤50 amino acids) more centrally in physiological processes. Yet, their functional identification and the systematic genome annotation of their cognate small open-reading frames (smORFs) remains challenging both experimentally and computationally. Ribosome profiling or Ribo-Seq (that is a deep sequencing of ribosome-protected fragments) enables detecting of actively translated open-reading frames (ORFs) and empirical annotation of coding sequences (CDSs) using the in-register translation pattern that is characteristic for genuinely translating ribosomes. Multiple identifiers of ORFs that use the 3-nt periodicity in Ribo-Seq data sets have been successful in eukaryotic smORF annotation. They have difficulties evaluating prokaryotic genomes due to the unique architecture (e.g. polycistronic messages, overlapping ORFs, leaderless translation, non-canonical initiation etc.). Here, we present a new algorithm, smORFer, which performs with high accuracy in prokaryotic organisms in detecting putative smORFs. The unique feature of smORFer is that it uses an integrated approach and considers structural features of the genetic sequence along with in-frame translation and uses Fourier transform to convert these parameters into a measurable score to faithfully select smORFs. The algorithm is executed in a modular way, and dependent on the data available for a particular organism, different modules can be selected for smORF search.
    • Specific serum IgG at diagnosis of Staphylococcus aureus bloodstream invasion is correlated with disease progression.

      Stentzel, Sebastian; Sundaramoorthy, Nandakumar; Michalik, Stephan; Nordengrün, Maria; Schulz, Sarah; Kolata, Julia; Kloppot, Peggy; Engelmann, Susanne; Steil, Leif; Hecker, Michael; et al. (2015-07-05)
      Although Staphylococcus aureus is a prominent cause of infections, no vaccine is currently available. Active vaccination relies on immune memory, a core competence of the adaptive immune system. To elucidate whether adaptive immunity can provide protection from serious complications of S. aureus infection, a prospective observational study of 44 patients with S. aureus infection complicated by bacteremia was conducted. At diagnosis, serum IgG binding to S. aureus extracellular proteins was quantified on immunoblots and with Luminex-based FLEXMAP 3D™ assays comprising 64 recombinant S. aureus proteins. Results were correlated with the course of the infection with sepsis as the main outcome variable. S. aureus-specific serum IgG levels at diagnosis of S. aureus infection were lower in patients developing sepsis than in patients without sepsis (P<0.05). The pattern of IgG binding to eight selected S. aureus proteins correctly predicted the disease course in 75% of patients. Robust immune memory of S. aureus was associated with protection from serious complications of bacterial invasion. Serum IgG binding to eight conserved S. aureus proteins enabled stratification of patients with high and low risk of sepsis early in the course of S. aureus infections complicated by bacteremia.
    • Staphylococcal serine protease-like proteins are pacemakers of allergic airway reactions to Staphylococcus aureus.

      Stentzel, Sebastian; Teufelberger, Andrea; Nordengrün, Maria; Kolata, Julia; Schmidt, Frank; van Crombruggen, Koen; Michalik, Stephan; Kumpfmüller, Jana; Tischer, Sebastian; Schweder, Thomas; et al. (2017-02)
      A substantial subgroup of asthmatic patients have "nonallergic" or idiopathic asthma, which often takes a severe course and is difficult to treat. The cause might be allergic reactions to the gram-positive pathogen Staphylococcus aureus, a frequent colonizer of the upper airways. However, the driving allergens of S aureus have remained elusive.
    • A systematic proteomic analysis of Listeria monocytogenes house-keeping protein secretion systems.

      Halbedel, Sven; Reiss, Swantje; Hahn, Birgit; Albrecht, Dirk; Mannala, Gopala Krishna; Chakraborty, Trinad; Hain, Torsten; Engelmann, Susanne; Flieger, Antje (2014-11)
      Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein.
    • Towards the characterization of the hidden world of small proteins in Staphylococcus aureus, a proteogenomics approach.

      Fuchs, Stephan; Kucklick, Martin; Lehmann, Erik; Beckmann, Alexander; Wilkens, Maya; Kolte, Baban; Mustafayeva, Ayten; Ludwig, Tobias; Diwo, Maurice; Wissing, Josef; et al. (PLOS, 2021-06-01)
      Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 proteins with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.
    • Within-Host Adaptation of in a Bovine Mastitis Infection Is Associated with Increased Cytotoxicity.

      Mayer, Katharina; Kucklick, Martin; Marbach, Helene; Ehling-Schulz, Monika; Engelmann, Susanne; Grunert, Tom; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-08-17)
      Within-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen's strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype-phenotype associations under different infection-relevant growth conditions.