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Öğe Development of a larval zebrafish infection model for clostridioides difficile(Journal Of Visualized Experiments, 2020) Li, Junkai; Ünal, Can Murat; Namikawa, Kazuhiko; Steinert, Michael; Koester, Reinhard W.Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.Öğe FKBPs in bacterial infections(Elsevier Science Bv, 2015) Ünal, Can Murat; Steinert, MichaelBackground: FK506-binding proteins (FKBPs) contain a domain with peptidyl-prolyl-cis/trans-isomerase (PPlase) activity and bind the immunosuppressive drugs FK506 and rapamycin. FKBPs belong to the immunophilin family and are found in eukaryotes and bacteria. Scope of review: In this review we describe two major groups of bacterial virulence-associated FKBPs, the trigger factor and Mip-like PPIases. Moreover, we discuss the contribution of host FKBPs in bacterial infection processes. Major conclusions: Since PPIases are regarded as alternative antiinfective drug targets we highlight current research strategies utilizing pipecolinic acid and cycloheximide derivatives as well as substrate based inhibitors. General significance: The current research strategies suggest a beneficial synergism of drug development and basic research. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets. (C) 2014 Elsevier B.V. All rights reserved.Öğe Microbial peptidyl-prolyl cis/trans isomerases (ppIases): virulence factors and potential alternative drug targets(Amer Soc Microbiology, 2014) Ünal, Can Murat; Steinert, MichaelInitially discovered in the context of immunomodulation, peptidyl-prolyl cis/trans isomerases (PPIases) were soon identified as enzymes catalyzing the rate-limiting protein folding step at peptidyl bonds preceding proline residues. Intense searches revealed that PPIases are a superfamily of proteins consisting of three structurally distinguishable families with representatives in every described species of prokaryote and eukaryote and, recently, even in some giant viruses. Despite the clear-cut enzymatic activity and ubiquitous distribution of PPIases, reports on solely PPIase-dependent biological roles remain scarce. Nevertheless, they have been found to be involved in a plethora of biological processes, such as gene expression, signal transduction, protein secretion, development, and tissue regeneration, underscoring their general importance. Hence, it is not surprising that PPIases have also been identified as virulence-associated proteins. The extent of contribution to virulence is highly variable and dependent on the pleiotropic roles of a single PPIase in the respective pathogen. The main objective of this review is to discuss this variety in virulence-related bacterial and protozoan PPIases as well as the involvement of host PPIases in infectious processes. Moreover, a special focus is given to Legionella pneumophila macrophage infectivity potentiator (Mip) and Mip-like PPIases of other pathogens, as the best-characterized virulence-related representatives of this family. Finally, the potential of PPIases as alternative drug targets and first tangible results are highlighted.Öğe Novel cycloheximide derivatives targeting the moonlighting protein Mip exhibit specific antimicrobial activity against Legionella pneumophila(Frontiers Media Sa, 2015) Rasch, Janine; Theuerkorn, Martin; Ünal, Can Murat; Heinsohn, Natascha; Tran, Stefan; Fischer, Gunter; Steinert, MichaelMacrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180nM and 1.7 mu M, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 mu M, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.Öğe Novel therapeutic strategies for Clostridium difficile infections(Taylor & Francis Ltd, 2016) Ünal, Can Murat; Steinert, MichaelIntroduction: In recent years, Clostridium difficile has become the primary cause of antibiotic-associated diarrhea and pseudomembranous colitis, resulting in long and complicated hospital stays that represent a serious burden for patients as well as health care systems. Currently, conservative treatment of C. difficile infection (CDI) relies on the antibiotics vancomycin, metronidazole or fidaxomicin, or in case of multiple recurrences, fecal microbiota transplantation (FMT).Areas covered: The fast-spreading, epidemic nature of this pathogen urgently necessitates the search for alternative treatment strategies as well as antibiotic targets. Accordingly, in this review, we highlight the recent findings regarding virulence associated traits of C. difficile, evaluate their potential as alternative drug targets, and present current efforts in designing inhibitory compounds, with the aim of pointing out possibilities for future treatment strategies.Expert opinion: Increased attention on systematic analysis of the virulence mechanisms of C. difficile has already led to the identification of several alternative drug targets. In the future, applying state of the art omics' and the development of novel infection models that mimic the human gut, a highly complex ecological niche, will unveil the genomic and metabolic plasticity of this pathogen and will certainly help dealing with future challenges.Öğe Peptidylprolyl cis-trans isomerases of Legionella pneumophila: virulence, moonlighting and novel therapeutic targets(Portland Press Ltd, 2014) Rasch, Janine; Ünal, Can Murat; Steinert, MichaelLegionella pneumophila, typically a parasite of free-living protozoa, can also replicate in human alveolar macrophages and lung epithelial cells causing Legionnaires' disease in humans, a severe atypical pneumonia. The pathogen encodes six peptidylprolyl cis-trans isomerases (PPIases), which generally accelerate folding of prolyl peptide bonds, and influence protein folding. PPIases can be divided into three classes, cyclophilins, parvulins and FK506-binding proteins (FKBPs). They contribute to a multitude of cellular functions including bacterial virulence. In the present review, we provide an overview of L. pneumophila PPIases, discussing their known and anticipated functions as well as moonlighting phenomena. By taking the example of the macrophage infectivity potentiator (Mip) of L. pneumophila, we highlight the potential of PPIases as promising drug targets.Öğe Pleiotropic clostridioides difficile cyclophilin PpiB controls cysteine-tolerance, toxin production, the central metabolism and multiple stress responses(Frontiers Media Sa, 2019) Ünal, Can Murat; Karagoz, Mustafa Safa; Berges, Mareike; Priebe, Christina; de Acuna, Jose Manuel Borrero; Wissing, Josef; Steinert, MichaelThe Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPlases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPlase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, 0-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteinemediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPlase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.Öğe Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection(Elsevier Gmbh, Urban & Fischer Verlag, 2014) Shevchuk, Olga; Paegelow, Dennis; Rasch, Janine; Döhrmann, Simon; Günther, Gabriele; Hoppe, Julia; Steinert, Michael; Ünal, Can Murat; Bronietzki, Marc; Gutierrez, Maximiliano GabrielL. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their colocalization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease. (C) 2014 Elsevier GmbH. All rights reserved.Öğe PrsA2 (CD630_35000) of clostridioides difficile Is an Active Parvulin-Type PPlase and a virulence modulator(Frontiers Media Sa, 2018) Ünal, Can Murat; Berges, Mareike; Smit, Nathiana; Schiene-Fischer, Cordelia; Priebe, Christina; Strowig, Till; Steinert, MichaelClostridioides difficile is the main cause for nosocomial antibiotic associated diarrhea and has become a major burden for the health care systems of industrial countries. Its main virulence factors, the small GTPase glycosylating toxins TcdA and TcdB, are extensively studied. In contrast, the contribution of other factors to development and progression of C. difficile infection (CDI) are only insufficiently understood. Many bacterial peptidyl-prolyl-cis/trans-isomerases (PPlases) have been described in the context of virulence. Among them are the parvulin-type PrsA-like PPlases of Gram-positive bacteria. On this basis, we identified CD630_35000 as the PrsA2 homolog in C. difficile and conducted its enzymatic and phenotypic characterization in order to assess its involvement during C. difficile infection. For this purpose, wild type CdPrsA2 and mutant variants carrying amino acid exchanges mainly in the PPlase domain were recombinantly produced. Recombinant CdPrsA2 showed PPlase activity toward the substrate peptide Ala-Xaa-Pro-Phe with a preference for positively charged amino acids preceding the proline residue. Mutation of conserved residues in its active site pocket impaired the enzymatic activity. A PrsA2 deficient mutant was generated in the C. difficile 6300erm background using the ClosTron technology. Inactivation of prsA2 resulted in a reduced germination rate in response to taurocholic acid, and in a slight increase in resistance to the secondary bile acids LCA and DCA. Interestingly, in the absence of PrsA2 colonization of mice by C. difficile 630 was significantly reduced. We concluded that CdPrsA2 is an active PPlase that acts as a virulence modulator by influencing crucial processes like sporulation, germination and bile acid resistance resulting in attenuated mice colonization.Öğe The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions(Royal Soc, 2015) Xiong, Qiuhong; Ünal, Can Murat; Matthias, Jan; Steinert, Michael; Eichinger, LudwigMacroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG160(-) and ATG9(-)/16(-) cells and compare them to the previously reported ATG9(-) mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9(-) and ATG16(-) cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9(-)/16(-) double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiqui-tin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9(-)/16(-) cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9(-)/16(-) cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.Öğe Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation(Wiley, 2021) Scheithauer, Lina; Thiem, Stefanie; Schmelz, Stefan; Dellmann, Ansgar; Buessow, Konrad; Brouwer, Rene M. H. J.; Ünal, Can Murat; Blankenfeldt, Wulf; Steinert, MichaelProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.
