Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences Julius-Maximilians-Universität Würzburg Section: Infection and Immunity submitted by Srikkanth Balasubramanian

Novel anti-infectives against pathogenic bacteria Neue Antiinfectiva gegen pathogene Bakterien Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences Julius-Maximilians-Universität Würzburg Section: Infection and Immunity submitted by Srikkanth Balasubramanian from Chennai, India Würzburg, 2018 -1- Submitted on: ………………………………………………………….…… Office stamp Members of the Thesis Committee: Chairperson: Prof Dr Thomas Dandekar Primary Supervisor: Dr Tobias Ölschläger Supervisor (Second): Prof Dr Ute Hentschel-Humeida Supervisor (Third): Prof Dr Ulrike Holzgrabe Supervisor (Fourth): Dr Usama Ramadan Abdelmohsen Date of Public Defence: Date of Receipt of Certificates: -2- Affidavit I hereby confirm that my thesis entitled “Novel anti-infectives against pathogenic bacteria” is the result of my own work I did not receive any help or support from commercial consultants All source and/or materials applied are listed and specified in the thesis Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form Würzburg, Srikkanth Balasubramanian -3- Eidesstattliche Erklärung Hiermit erkläre ich an Eides statt, die Dissertation "Neue Antiinfectiva gegen pathogene Bakterien" eigenständig, d h insbesondere selbstständig und ohne Hilfe eines kommerziellen Promotionsberaters, angefertigt und keine anderen als die von mir angegebenen Quellen und Hilfsmittel verwendet zu haben Ich erkläre außerdem, dass die Dissertation weder in gleicher noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat Würzburg, Srikkanth Balasubramanian -4- Dedication This dissertation is dedicated to My Parents -5- Acknowledgements I wish to thank the following persons: My mentor and advisor Dr Tobias Ölschläger for giving me this wonderful opportunity to work in his research group for my PhD thesis I am grateful to him for his excellent guidance and insightful scientific discussions I especially would like to thank him for encouraging me to participate in international conferences and workshops My second supervisor Prof Dr Ute Hentschel-Humeida for giving me the opportunity to work on this applied research project involving marine sponge-associated actinomycetes I thank her for all her critical scientific inputs on the projects and manuscripts preparation My gratitude also goes to all the present and former members of AG Hentschel, especially Dr Cheng Cheng for providing the actinomycetes strains and Christine Gernert for technical assistance, Dr Hannes Horn, Dr Lucas Moitinho-Silva, Dr Lucía Pita Galán, Dr Beate Slaby, Dr Kristina Bayer and Martin Jahn for the nice time we spent together at Department of Botany-II My thesis committee member Prof Dr Ulrike Holzgrabe for all her guidance in isolation of active compound from the crude extract I thank her for all the scientific and professional support I am thankful to Joseph Skaf for his assistance in fractionation experiments and analyses I further thank all the members of AG Holzgrabe for the productive and nice atmosphere in the lab My thesis committee member Dr Usama Ramadan Abdelmohsen who has been a great support in my PhD thesis project I thank him for all his valuable guidance, encouragements, help in data analysis and manuscripts preparation Dr Wilma Ziebuhr and Dr Knut Ohlsen for providing their scientific expertise in the field of staphylococcal biofilms Dr Konrad Förstner and Dr Richa Bharti (Core Unit Systems Medicine at the University Hospitals of Würzburg) for their extended bioinformatics support I am grateful to them for helping me with the analysis of transcriptome data Ms Daniela Bunsen, Ms Claudia Gehrig and Ms Hilde Merkert for assisting me with scanning electron and confocal microscopy experiments Ms Mona Alzheimer for I introducing me to cell culture handling and Dr Eman Maher Othman for performing toxicity evaluations on human corneal epithelial cell lines Dr Mathias Grüne and Ms Juliane Adelmann (Institute of Organic Chemistry, University of Würzburg) for the LC-MS and NMR measurements Prof Dr Rolf Müller (Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany) for his collaboration in structure elucidation of the bioactive compound SKC3 My DAAD-RISE intern student Ms Brinkley Raynor (North Carolina State University, USA) for assisting me in biofilm experiments My PhD buddies Susi and Mano for all the discussions, laughs, lunches and good times we had together Especially, Susi for writing the zusammenfassung for this thesis Present and former members of AG Ölschläger, Rebekka, Juna, Laura, Sharon, Stefan, Simon and Christian for all the enjoyable times we had in lab Members of AG Ziebuhr (Abishek, Freya, Gabri and Sonja) for all the discussions, gettogethers and time spent together at the office All the present and former members of the Graduate School of Life Sciences (Dr Gabriele Blum-Oehler, Ms Jennifer Heilig, Ms Felizitas Berninger, Mr Vikas Dalal, Ms Katrin Lichosik) for their administrative support Graduate School of Life Sciences (GSLS), University of Würzburg for providing me the financial support through GSLS fellowship I am also thankful to the GSLS for offering the wide range of transferable skill workshops SFB630 (TPA5 and Z1) consortium for the monetary support of Anti-Shiga toxin compound discovery project My best friends Krishna, Kapilesh, Surendhar, Amarto, Lavanya and Gi for all their encouragements and support through endless Skype and telephonic conversations All friends in Würzburg who made my PhD life enjoyable (Mohindar, Ravi, Suhail, Aparna and others) I can never forget the fun-filled evenings, birthdays, movies, Indian chai and other memorable moments I had with them My whole family and friends in India Especially, my amma and appa (Lalitha and Balasubramanian) who did everything they can to help me reach here with their everlasting support, and my cousin Chintoo for her unconditional love Thank you all! II Table of contents Summary V Zusammenfassung VIII General introduction 1.1 Infectious diseases and antibiotic resistance 1.2 Anti-virulence strategies 1.3 Enterohemorrhagic Escherichia coli and Shiga toxin 1.4 Staphylococci and biofilms 10 1.4.1 Initial attachment and microcolony formation 11 1.4.2 Accumulation 12 1.4.3 Structuring and maturation of biofilms 13 1.4.4 Detachment 13 1.5 Bioactive potential of Marine Natural Products 15 1.5.1 Marine sponges and their microbial consortia 16 1.5.2 Marine sponge-associated actinomycetes 18 1.6 Scope of the study 23 Inhibitory potential of strephonium A in restraining Shiga toxin production in EHEC strain EDL933 25 Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation 32 A new bioactive compound from marine sponge-derived Streptomyces sp SBT348 inhibits staphylococcal growth and biofilm formation 47 General discussion 92 5.1 A retrospective of the bioactive potential of sponge-associated actinomycetes 92 5.2 Anti-Stx approaches: state-of-the-art 97 5.2.1 Quorum sensing inhibitors 98 5.2.2 Pyocins 98 5.2.3 Vaccines and immunotherapy 99 5.2.4 Toxin binding inhibitors 99 5.2.5 Probiotics 99 III 5.2.6 Anti-Stx NPs 100 5.3 Anti-biofilm approaches: state-of-the-art 101 5.3.1 Prevention 102 5.3.2 Weakening 102 5.3.3 Disruption 103 5.3.4 Killing 103 Conclusion and future perspectives 113 Bibliography (introduction and discussion) 114 Appendix 142 List of abbreviations and symbols 142 List of figures (chapter-wise) 147 List of tables (chapter-wise) 149 Statement of author contributions 150 List of publications 154 Poster presentations at conferences and symposia 155 Selected workshops 156 Curriculum vitae 157 IV Summary Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria However, not much is known about their antiinfective or anti-virulence potential against human pathogens This Ph.D project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes Diarrheal infections pose a huge burden in several developing and developed countries Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit It was found that treatment with the extracts did not lead to significant reduction in Stx production However, strepthonium A isolated from the culture of Streptomyces sp SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 µM concentration) in EHEC strain EDL933 without affecting the bacterial growth The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments This demonstrated the possible application of strepthonium A in restraining EHEC infections V TGN trans Golgi network ER endoplasmic reticulum DRIs device-related infections nm nanometer cm centimeter UV ultraviolet CoNS coagulase negative staphylococci CoPS coagulase positive staphylococci AtlE autolysin Aap accumulation-associated protein Bap biofilm-associated protein PNAG poly-N-acetylglucosamine GlcNAc N-acetylglucosamine FnBP fibronectin binding protein WTA wall teichoic acid LTA lipoteichoic acid QS quorum sensing PSM phenol soluble modulins CW cell wall CM cell membrane PG peptidoglycan µm micrometer NPs natural products MNPs marine natural products kg kilogram l liter rRNA ribosomal RNA DGGE denaturing gradient gel electrophoresis FISH Fluorescent In Situ Hybridization GC Guanine-cytosine NCBI National Center for Biotechnology Information BD bloody diarrhea m/z mass to charge ratio MAbs monoclonal antibodies 143 δC chemical shift (ppm), 13C-NMR δH chemical shift (ppm), 1H-NMR ºC degree Celsius rpm revolutions per minute ASW artificial sea water 13C-NMR carbon nuclear magnetic resonance spectroscopy COSY correlation spectroscopy DMSO Dimethyl sulfoxide HMBC heteronuclear multiple bond correlation 1H-NMR proton nuclear magnetic resonance spectroscopy HSQC heteronuclear single quantum coherence Hz hertz MHz megahertz MeOH methanol EtOAc ethylacetate PC positive control µM micromolar LB Luria broth NIH National Institute of Health eDNA extracellular DNA TSB Tryptic soy broth v/v volume per unit volume w/v weight per unit volume g/l gram per liter µg/l microgram per liter % percentage mM millimolar ml milliliter ISP2 International Streptomyces project medium OD pptical density at a particular wavelength µg microgram PBS phosphate buffer saline ATCC American type culture collection mm millimeter 144 HCEC human corneal epithelial cells U/ml units/millilter µl microliter BIC50 50% biofilm inhibitory concentration A260/A280 absorbance at 260 nm to absorbance at 280 nm qPCR quantitative real time PCR PCR polymerase chain reaction Ct cycle threshold DHFR dihydrofolate reductase DCM dichloromethane MeCN acetonitrile kV kilovolts LC-MS liquid chromatography-mass spectrometry SEM scanning electron microscopy CLSM confocal laser scanning microscopy IOLs intraocular lenses mRNA messenger RNA Rt retention time FTIR fourier transform infra-red spectroscopy MRSA methicillin resistant S aureus MSSA methicillin sensitive S aureus VRSA vancomycin resistant S aureus VRE vancomycin resistant enterococci ND not determined NC no cytotoxicity ABC ATP binding cassette SMR small molecule resistance CSSSI somplicated skin and skin structure infections ABSSI acute bacterial skin and skin structure infections ISP2_F International Streptomyces project medium 2_filamentous BLAST Basic Local Alignment Search Tool antiSMASH Antibiotics and Secondary Metabolite Analysis Shell MIBiG Minimum Information about a Biosynthetic Gene Cluster NaPDoS Natural Product Domain Seeker 145 LPS lipopolysaccharide sRNA small RNA PCA principal component analysis MC medium control UT untreated t time MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide M molecular weight ladder bp base pair HT heat treated TT trypsin treated PKT proteinase K treated DOSY Diffusion Ordered Spectroscopy ns not significant (statistically); p>0.05 *, **, ***, **** p