[PDF] Clarisse ROBLIN Sep 25 2019 École doctorale

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THÈSE DE DOCTORAT

D"AIX-MARSEILLE UNIVERSITÉ

Spécialité : Microbiologie

École doctorale nº062: Sciences de la vie et de la santé réalisée à l"Institut des Sciences Moléculaires de Marseille - Equipe

BiosCiences et Adisseo

sous la direction de Josette Perrier, Michael Lafond, Estelle Devillard présentée par

Clarisse ROBLIN

pour obtenir le grade de :

DOCTEUR D"AIX-MARSEILLE UNIVERSITÉ

Sujet de la thèse :

Les Ruminococcines C, une nouvelle famille de

sactipeptides comme alternatives aux antibiotiques conventionnels

Thesis subject :

Ruminococcins C, a new family of sactipeptides as

alternatives to conventional antibiotics soutenue le 14 Septembre 2020 devant le jury composé de :

M. Marc FONTECAVE Président du jury

Mme Sylvie REBUFFAT Rapportrice

M. Lhousseine TOUQUI Rapporteur

M. Patrice POLARD Examinateur

Mme Josette PERRIER Directrice de thèse

Mme Estelle DEVILLARD Directrice de thèse

M. Michael LAFOND Directeur de thèse

M. Victor DUARTE Invité

Acknowledgements

First, I would like to express my deepest appreciation to all the jury members, Sylvie Rebuffat, Lhousseine Touqui, Marc Fontecave and Patrice Polard for accepting to read and evaluate this work and I would like to thank them in advance for their presence at the defense. I would also like to thank my committee members, Delphine Destoumieux-Garzon and Aurélie Tasiemski for accepting to review my work along the project and for giving me their helpful insights. I express my gratitude to the head of BiosCiences, Thierry Tron, and the head of the Institute des Sciences Moléculaires de Marseille, Jean Rodriguez, for giving me the opportunity to do research in this lab. In addition, I would like to thank Adisseo for their participation in a CIFRE convention and for financing my thesis project. I would like to express my deepest gratitude to my thesis advisors: Estelle Devillard, Josette Perrier and Mickael Lafond. Estelle, it was always a pleasure to work with you and I learn from you skills that I wouldn"t have been exposed to in a "purely academic PhD", for that I am thankful. Josette and Mickael, I am glad I joined you on this great RumC project and I am grateful for the journey we took together. It took us some time to find our pace, but once we did, I enjoyed every moment we worked together. Thank you for the trust you put in me everyday and for your support. In particular, I want to emphasize my gratitude to Mickael for his constant involvement in this project and for helping me to reach my full potential. Through this project I had the opportunity to discover how competitive research can be and how it can sometimes be an obstacle to our field but above all this project brought to light the best that collaborative work has to offer. It is amazing to see what we can achieve when people from different backgrounds truly work together. Thus, I would like to thank our many collaborators. First, the people from Grenoble: Victor, Hamid, Christian, Sylvie, Yohann and Steve, it was a pleasure to work with you and share this wonderful RumC project with you all these years. It was also a pleasure to travel with you, visit you in Grenoble or welcome you in Marseille and spend time together outside of the frame of work. To Steve, in particular, I am grateful, we had the opportunity to "share" a PhD, never in competition but always supporting each other"s. I hope our professional paths will cross again! Victor, I am thankful you accepted to be in my jury too. You already know most of the content of this manuscript so it might be a little long to read but you might find a few surprises at the end. Then, I would like to thank the people from IMM: Françoise, Olivier and Matthieu. Thank you for your dedication to the resolution of RumC1"s structure and to Olivier, i thanks for the coffee whenever I was at the IMM microscopy platform. Speaking of the microscopy platform, I would like to address a huge thanks to Artemis and Hugo for always welcoming me, with a special mention to Hugo who, not only welcomed me at the platform, but also at the apéro at the end of the day! Many thanks to Gael too, for those long days doing flow cytometry, the great conversations during those days and for your curiosity and your interest in our project. I also want to thank the people from Toulouse for welcoming us for our experiments. I would like to express my gratitude to Leo, Michèle and AJ for letting us use their facil- ities. To Nathalie, Mathieu and Patrice thank you for the warm welcome and I am glad you jumped on board and are now part of the RumC project as well. I would like to thank Patrice for his sharp insight and the interesting discussions on the project, I am sure we will have a lot more during the defense. Nathalie, we clearly have in common the love for fluorescent microscopy and it was a pleasure to spend all these hours visualizing the effects of RumC1 on bacteria with you. Finally, I also want to thank Michel F. for his help on the project and Cédric for his reactivity. I also want to thank all the people from the lab and former members of the lab. Marc and Hamza, it was a pleasure to work with you guys. We did not always agree on every- thing but at the end we improved each other"s projects and my time in the lab wouldn"t have been the same without you. Cendrine, it was always a pleasure to work with you as well and it was never boring, thank you for all the laughs during the hours spending at staring at the shape of pencil stains under the microscope. In addition, thank you for everything you do for this lab. Agnès, thank you for your help during those last months but above all thank you for your pleasant presence in the lab and for feeding me cakes and crepes. Thank you to Elise, Yolande, Olga, Thierry G. for their help on my project. I also want to thank Yasmina, Christophe, Katia and Marius for the interest they took in my progress and globally our project. A special thanks to Pierre for his involvement, although he was not supposed to be a member of our project he ended up being one of the person who helped me the most in the lab. Finally, thanks to all the PhD students and post-doc: Alex, Hugo, David, Julie, Maxime, Raul, Quim, Alessia, Bernadette, Rogelio, Stefani, Mike, Claudio, Robert... I am glad I got to share some parts of this experience with you. My thoughts go to Sybille, as well, I am glad I started this project with you and I wish you the best for your new MCU position. On a more personal note, I would like to thank all my friends who have been with me all along this project and who have been supporting me. A special thanks to those who had to listen daily to my lab stories: my sisters, Lulu and Chloé, and Juju H. before them. Thanks to Juju P. (the voice of reason in the final moments), Lilice, Léon, Etienne for always being here for me. My deepest gratitude goes to Lucas, writing this thesis would have been way more painful without his help. Thanks to all my friends from Paris, Moustakhs and affiliates for the distracting week-ends and holidays. I would also like to thank "La team Marseillaise" for the weekly distraction and JP for his presence when I was writing and for the change of scenery during that period. In addition, I thank two of my friends from the US for always believing in me: Dana and Lindsey. Lindsey, may we always find a way to meet when we"re travelling in each other"s countries for work. Finally, I"d like to thank Lisa and Marie. Lisa, your faith in me never stops to amaze me. Marie, thank you for listening to me almost everyday. ii Finally, I would like to thank my family for supporting me all these years, even though my academic cursus was not always easy to follow and was paved with a few detours. I would like to address a huge thanks to my parents for their advice whenever I was facing an obstacle during this PhD. To my brother, just thank you, for everything. iii iv

List of abbreviations

ABC ATP-Binding Cassette

AMP AntiMicrobial Peptide

AMU Aix-Marseille University

ATP Adenosine TriPhosphate

BGC Biosynthetic Genetic Cluster

CBD Cell wall Binding Domain

CCK Cylic Cystine Knot

CDIClostridium difficileinfection

CERN Centre of Expertise and Research in Nutrition

CTAB Cetyltrimethylammonium bromide

Dha Didehydroalanine

Dhb Didehydrobutyrine

FMT Fecal Matter Transplant

GI Gastro-Intestinal

GlcNAc N-acetylglucosamine

HAI Hopistal-Acquired infection

HIV Human Immunodeficiency Virus

IBD Inflammatory Bowel Disease

IMM Mediterranean Microbiology Institute

ISM2 Institute of Molecular Sciences of Marseille

LAB Lactic Acid Bacteria

Lan Lanthionine

LAP Linear Azol(in)e-containing Peptide

LC Liquid Chromatography

LCBM Laboratory of Metal Chemistry and Biology

LPS LipoPolySaccharide

LTA LipoTeiochic Acid

mAb Mono-clonal Antibody

MBC Minimum Bactericidal Concentration

MDR Multi-Drug Resistant

MeLan Methyl-Lanthionine

MIC Minimum Inhibitory Concentration

mRNA Messenger Ribonucleic Acid

MRSA Methicillin-ResistantStaphylococcus aureus

MS Mass Spectrometry

MS/MS Tandem Mass Spectrometry

MurNAc N-acetylmuramic acid

v

NMR Nuclear Magnetic Resonance

NRP NonRibosomal Peptide

NRPS NonRibosomal Peptide Synthetase

ORF Open Reading Frame

PBP Penicillin-Binding Protein

PBS Phosphate-Buffered Saline

PCAT Peptidase-Containing ATP-binding Cassette

PDB Protein Data Bank

PDR Pan-Drug Resistant

PI Propidium Iodide

PTM Post-Translational Modification

QS Quorum Sensing

RiPP Ribosomally synthesized and Post-translationally modified Peptide

RIT RadioImmunoTherapy

RRE RiPP precursor peptide Recognition Element

rRNA Ribosomal Ribonucleic Acid

RumA Ruminococcin A

RumC Ruminococcin C

Sacti Sulfur toαCarbon Thioether

SAM S-Adenosyl-Methionine

SCFA Short Chain Fatty Acid

SCIFF Six Cysteine In Forty-Five residue

SKF Sporulation Killing Factor

SPPS Solid-Phase Peptide Synthesis

TEM Transmission Electron Microscopy

VAPGH Virion-Associated PeptidoGlycan Hydrolase

VRE Vancomycin-ResistantEnterococcus faecalis

WHO World Health Organization

5"-Ado?5-deoxyadenosyl

vi

List of Figures

1.1 Timeline of antibiotic discovery . . . . . . . . . . . . . . . . . . . . . . . .

2

1.2 Examples from each class of antibiotics . . . . . . . . . . . . . . . . . . . .

6

1.3 Mechanisms of antibiotic resistance . . . . . . . . . . . . . . . . . . . . . .

12

1.4 Timeline of clinical introduction of antibiotics and first identifications of

resistant bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.5 Main causes of deaths worldwide and estimated annual numbers . . . . . .

17

1.6 Phage infection of bacterial cells through lytic or lysogenic cycles . . . . .

19

1.7 Mechanisms of peptidoglycan cleavage by phage lysins . . . . . . . . . . .

21

1.8 Roles of antibodies in bacterial infections . . . . . . . . . . . . . . . . . . .

23

1.9 Action of commensal bacteria or probiotics against pathogens in mam-

malian host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

1.10 Common characteristics and differences of proteinaceous substances active

against microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

1.11 Biosynthesis of NRPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29

2.1 Classification of bacteriocins . . . . . . . . . . . . . . . . . . . . . . . . . .

33

2.2 Structure of class II bacteriocins . . . . . . . . . . . . . . . . . . . . . . . .

34

2.3 Structure of the bacteriolysin lysostaphin . . . . . . . . . . . . . . . . . . .

35

2.4 Sequences and disulfide networks of class IIa microcins MccV and MccL . .

36

2.5 Structure of colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37

2.6 RiPP biosynthesis pathway . . . . . . . . . . . . . . . . . . . . . . . . . . .

39

2.7 RRE of several RiPP maturation enzymes . . . . . . . . . . . . . . . . . .

41

2.8 Hybrid RiPP biosynthetic pathway strategy for novel RiPPs generation . .

42

2.9 Structure of the lanthipeptide nisin A . . . . . . . . . . . . . . . . . . . . .

45

2.10 Structure of the LAP plantazolicin . . . . . . . . . . . . . . . . . . . . . .

45

2.11 Structure of the proteusin polytheonamide B . . . . . . . . . . . . . . . . .

47

2.12 Structure of the linaridin cypemycin . . . . . . . . . . . . . . . . . . . . .

47

2.13 Structure of the bottromycin A2 . . . . . . . . . . . . . . . . . . . . . . . .

47

2.14 Structure of the thiopeptide thiostrepton A . . . . . . . . . . . . . . . . . .

49

2.15 Structure of the class IIb microcin E492 . . . . . . . . . . . . . . . . . . .

49

2.16 Structure of the cyanobactin patellamide A . . . . . . . . . . . . . . . . . .

51

2.17 Structure of the circular bacteriocin acidocin B . . . . . . . . . . . . . . .

51

2.18 Structure of the microviridin B . . . . . . . . . . . . . . . . . . . . . . . .

53

2.19 Structure of the glycocin sublancin 168 . . . . . . . . . . . . . . . . . . . .

53

2.20 Structure of the copper methanobactin produced byMethylocystissp. LW453

2.21 Structure of the class I lasso peptide specialicin . . . . . . . . . . . . . . .

55

2.22 Structure of the autoinducing peptide AIP-1 . . . . . . . . . . . . . . . . .

55

2.23 Structure of the streptide StrA . . . . . . . . . . . . . . . . . . . . . . . .

55
vii

2.24 Structures of the amatoxinα-amanatin and the phallotoxin phalloidin . . .57

2.25 Structure of the borosin omphalotin A . . . . . . . . . . . . . . . . . . . .

57

2.26 Structure of dikaritins . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57

2.27 Structure of the epichloëcyclin A . . . . . . . . . . . . . . . . . . . . . . .

59

2.28 Structure of the cyclotide kalata B1 . . . . . . . . . . . . . . . . . . . . . .

59

2.29 Structure of the orbitide pohlianin A . . . . . . . . . . . . . . . . . . . . .

61

2.30 Structure of the conopeptide contulakin-G . . . . . . . . . . . . . . . . . .

61

2.31 Sequences and thioether networks of sactipeptides . . . . . . . . . . . . . .

65

2.32 Three-dimensional structure of sactipeptides . . . . . . . . . . . . . . . . .

66

2.33 Biosynthetic genetic clusters of sactipeptides . . . . . . . . . . . . . . . . .

67

2.34 Radical SAM-based chemistry . . . . . . . . . . . . . . . . . . . . . . . . .

68

2.35 Radical based mechanism of thioether formation in sactipeptides . . . . . .

69

2.36 Sactipeptide versus ranthipeptide thioether linkages . . . . . . . . . . . . .

73

2.37 Sequences and thioether networks of ranthipeptides . . . . . . . . . . . . .

74

2.38 Chemical structures of GE2270A and its analogs . . . . . . . . . . . . . . .

77

3.1R. gnavusE1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81

3.2 Kinetics of elimination ofC. perfringensCpA in rats feces after inoculation

withR. gnavusE1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82

3.3 Sequence and putative PTMs of RumA . . . . . . . . . . . . . . . . . . . .

83

3.4 Genetic regulon of RumA . . . . . . . . . . . . . . . . . . . . . . . . . . .

84

3.5 Actualized PTMs of RumA . . . . . . . . . . . . . . . . . . . . . . . . . .

85

3.6 Expression of genes from the RumA and RumC regulons . . . . . . . . . .

86

3.7 Regulon of RumC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87

3.8 Amino acid sequences of the 5 isoforms of RumC . . . . . . . . . . . . . .

88

4.1 Objectives, strategies and partners . . . . . . . . . . . . . . . . . . . . . .

92

5.1 Ruminococcin C purification protocol developed by Crost et al., 2011 . . .

94

6.1 Spectra of thermal denaturation of RumC1 . . . . . . . . . . . . . . . . . .

183

6.2 Incorporation of a fluorescent precursor of peptidoglycan . . . . . . . . . .

184
viii

List of Tables

1.1 Examples of generalist definitions of antibiotic and antimicrobial . . . . . .

5

3.1 Molecular weights (Da) of the RumC isoforms isolated from cecal contents

ofR. gnavusE1 mono-associated rats . . . . . . . . . . . . . . . . . . . . .89quotesdbs_dbs27.pdfusesText_33

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