• 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.