Comprehension of the mechanisms of action of antimicrobial molecules using nanobiotechnologies

341 Pages·2017·35.06 MB·English

Comprehension of the mechanisms of action of
antimicrobial molecules using nanobiotechnologies
Cécile Formosa-Dague
To cite this version:
Cécile Formosa-Dague. Comprehension of the mechanisms of action of antimicrobial molecules using
nanobiotechnologies. Micro and nanotechnologies/Microelectronics. Université de Toulouse III - Paul
Sabatier, 2014. English. ￿NNT: ￿. ￿tel-01080090￿
HAL Id: tel-01080090
https://theses.hal.science/tel-01080090
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UniversitéToulouse3PaulSabatier(UT3PaulSabatier)

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CécileFORMOSA
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lundi22septembre2014
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Comprehensionofthemechanismsofactionofantimicrobialmoleculesusing
nanobiotechnologies
et discipline ou spécialité
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EDSDM:Nano-physique,nano-composants,nano-mesures-COP00
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LAAS-CNRSUPR8001-SRSMC-CNRSUMR7565
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Dr.EtienneDAGUE
Prof.RaphaëlE.DUVAL
Jury:
Dr.SandorKASAS,scientificcollaborator,EPFL,Switzerland
Prof.JohnR.DUTCHER,professor,UniversityofGuelph, Ontario,Canada
Dr.DavidALSTEENS,postdoctoralfellow,ETHZ,Switzerland
Prof.ChristineROQUES,professor,UniversityofToulouse,France
Dr.MarionGRARE,scientificcollaborator,UniversityofToulouse,France

Abstract

My PhD work consists in using Atomic Force Microscopy (AFM) techniques to study
pathogenic microorganisms, and to probe their interactions with antimicrobials. During the last
three decades, microbial resistance has dramatically increased and spread around the world.
Pathogenic bacteria and yeasts have developed several ways to resist against almost all
antimicrobials used. These pathogens can cause a wide range of superficial infections, but are
also the cause of life-threatening infections in some patients. There are therefore two
emergencies; the first one is to find new antimicrobial molecules, with an innovative mechanism
of action. To reach this goal, it is mandatory to get further fundamental knowledge on the
microbial cell wall, in order to identify original targets at the cell surface for new molecules.
Therefore the second emergency is to develop techniques to explore microbial surfaces from a
different angle, which requires an original experimental approach. In this context, biophysical
approaches still remain underexploited in clinical microbiology. In my PhD, we took advantage
of a technology coming from physics, and adapted to biological conditions, AFM. Its principle
relies on the measure of a force between a sharp tip and a surface; by keeping this force constant
while scanning the sample, it is possible to get a three dimensional image of it. An advantage of
AFM is the possibility to work in liquid on living cells, which allowed us to image the elongation
of live bacterial cells of P. aeruginosa treated by ticarcillin, a cell wall targeting antibiotic, and
the removal of capsular polysaccharides from K. pneumoniae upon treatment with colistin, a last
chance antibiotic. Nevertheless, sample immobilization is often a challenge that must be
addressed for each kind of microorganisms studied. It represents an entire field of research, and
led us to engineer a microstructured polydimethylsiloxane (PDMS) stamp to immobilize round
cells of different sizes such as yeasts. Once this step was accomplished, AFM can be used in
classic imaging and force spectroscopy modes (contact mode, oscillating mode, force volume
mode), but also in advanced modes in order to acquire multiparametric sets of data on living
cells. Indeed, AFM is also a highly sensitive force machine, able to record force distance curves
that give access to nanomechanical and adhesive properties of living cells. We therefore used the
multiparametric imaging mode, QITM from JPK Instruments, to image and quantify the
nanomechanical/adhesive properties of microorganisms as well as mammalian cells and their
isolated nucleus. We could then observe the modifications of the adhesive properties of the yeast
C. albicans, treated or not by caspofungin, a last chance antifungal. Finally, to get further in the
architecture of the cell wall of microorganisms, it is possible to functionalize AFM tips with
biomolecules. A strategy that we have developed has consisted of grafting antibodies targeted
against a peptide on the AFM tip while tagging proteins with the same peptide at the surface of
living cells. This allowed mapping the localization of specific proteins at the surface of living
yeasts and mammalian cells. Another strategy we developed has consisted in directly grafting on
the AFM tip a biomolecule that naturally interacts with cell wall components. We used this last
strategy to probe the bacterial cell wall of P. aeruginosa submitted to treatment by an innovative
antibacterial, Cx1, and better understand the structure of its peptidoglycan. In conclusion, during
my PhD, we especially addressed the contribution of biophysics in clinical microbiology. We
developed original techniques to immobilize samples, to functionalize AFM tips, and used
advanced multiparametric AFM modes to acquire original data on the surface of pathogenic
microorganisms, in native conditions or in interaction with antimicrobials.

Acknowledgments

I have many people to thank for this PhD, because we have all been working as a team, because I
could not have done any of this without this team.

Thank you to my fundings

I would like to thank the Agence Nationale pour la Recherche (ANR) and the Direction Générale
de l’Armement (DGA) for funding this PhD. I particularly thank Emmanuelle Guillot-Combe,
Xavier Dramard, and Gilles Vergnaud from DGA for following my work and supporting me
throughout these three years.

Thank you to the people of the laboratories

First I need to thank the SRSMC and LAAS-CNRS for hosting me, and my microorganisms and
for providing me with the equipment I needed to perform my experiments. I would also like to
thank ITAV and Childérick Severac for technical support on AFMs of the Nanobiotechnologies
platform, as well as LISBP EAD5 for hosting me and advising me on the yeast part. Thank you
also to Sébastien Noailles for giving me access to the P2 laboratory of the LISBP. Thank you to
Charline Blatché and Sandrine Assié-Souleille, for technical assistance in LAAS, and for trusting
me with my microorganisms and my equipment. Thank you to Adrian Laborde for technical work
in the clean room. Thank you finally to Jean-Bernard Regnoufs-de-Vains for the Cx1 synthesis.

Thank you to the people with whom I have collaborated

To Marion Grare, my very first collaborator, nothing at all on bacteria would have been done
without your work. Thank you for letting me finish what you had started, thank you for letting
Cx1 be my subject. I hope we will continue working together; we have not finished everything
yet on Cx1, and on MDR bugs…!

To Jean-Marie François, thank you for providing me with yeasts strains, and with fundamental
questions about the yeast cell wall. Thank you also for your advises on papers and on future
plans.

To Hélène Martin-Yken, thank you for working with me on yeast subjects, thank you for your
patience in front of the AFM, and thank you for answering all the questions I had!

To Marie-Pierre Rols, thank you for trusting me with the CHO cells, which is such a perfect
model for AFM single-molecule experiments. Thank you also for your support, I really did
appreciate it.

To Céline Galès, thank you for your constant enthusiasm for everything that I have been doing,
thank you for making me benefit from you technical skills, and thank you for this topic, GPCRs,
that is so full of perspectives! Thank you also for showing me that by working, we can succeed. I
really do wish to keep working with you in the future, on microbes and heart issues, why not?
Thank you to my friends and coworkers

To Eric Jauvert, thank you for teaching me all you knew about AFM on bacteria, and for the
functionalization protocol that you built, and that we still use, and will use for a while! You have
been very nice and patient with me, and I am grateful for that.

To Louise Chopinet, I was so sad when you left… I really did love working with you and sharing
my life with you at the lab. Thank you for helping me get my funding for this PhD, and for all
you advices. Thank you for listening to me. You were a strong support, and a really good friend. I
wish you could have stayed longer. I really did miss you.

To Flavien Pillet, alias Flavine, thank you for joining us on your postdoc, thank you for being so
funny, and for being such a good imitator of various animals’ noises. You have been there from
the beginning, from the master’s internship, to the end of the PhD, and I hope you will still be
there in the future. You have personally and professionally contributed to my life, and I would
like to thank you for that. I wish the best for you, and success in your future plans.

To Marion Schiavone, we have been sharing the same office, the same technical problems on
AFM and yeasts cells, we have been breaking cell walls together (so many!), and chasing spiders
in the AFM room. You have been listening to me and supporting me at all times, I hope I could
do the same for you. Thank you for being my friend.

To Véronique Lachaize, thank you for letting me teach you all I knew, you are the person who
makes my work useful. Thank you for all the fun stories, thank you for making me run with you,
I got good thanks to you! I am sure you will be good at your PhD, and I hope to be there the day
you will defend (yes, yes, it will happen, get ready!)

To Carlos Quintero, my favorite Colombian person! Thank you for not freaking out after one day
in our office, thank you for all the explanations on photolithography, etching, units, and
calculations!! But most of all, thank you for being such a nice person, for your advices, and for
making our office smell like science by occupying half of the white board with big equations! I
really do wish the best for you.

I do not forget of course Stéphane Lemonnier, the first person I talked to about AFM, and who
encouraged me to come work with AFM. Thank you Majorie Petitjean for making the
experiments at LISBP fun moments that we could share; you have a great career ahead of you,
keep fighting to make it happen! Thank you to Marie-Ange Teste, for welcoming me in the lab.
Thank you to Damien Veau, you were a superhero to me when I started, and a great friend. Thank
you Annaick Desmaison, for all the laughing during my internship; I can still hear you laughing
from the other way of the hall! Thank you Angélique Coutable for your help on experiments, and
thank you Amandine Egea for sharing my opinions on various subjects. Thank you Emmanuelle
Trevisiol for all you advices in chemistry and for the fun conversations.
Thank you to my interns

Thank you Estelle Yvard and Claire Murzeau, for being the best interns a PhD student can have!
You were great girls, you did good, and I am sure you will succeed in the future! Thank you for
trusting me and letting me teach you what I knew.

Thank you to my PhD directors

I wish to thank my PhD directors, Etienne Dague and Raphaël E. Duval. I have bet on you two by
starting the PhD with you, and I was right, it was really worth it. Raphaël, you have always been
enthusiastic about my work, you were always there for telling me that what I was doing was
good, and for giving me self-confidence. You gave a lot to me, brilliant ideas for experiments,
and perspectives for the future. You are a great person, and I want to thank you for believing in
me always. I hope this is not over and that we will keep working together.

And Etienne, we have been working together, as partners, during three and a half years. You
were right about a lot of things, first of all, the PhD is not a straight way, I got it now that it is
over! Thank you for sharing all your expertise with me and your experience. I also would like to
thank you for letting me be myself and for pushing me forward. I did not give you the “best
advisor” cup of coffee, but you really do deserve it.

Thank you both of you for making me a good researcher, and for preparing me to become
independent.

Thank you to my family and friends

And finally, thank you to my family and friends, who never understood what I was talking about
but always found it wonderful and great. Thank you for supporting me all the way and for being
proud of me now.

Summary

List of publications p4
List of communications p6
List of abbreviations p8
List of figures p10

Introduction p12

Chapter 1: General Overview p18

Chapter 1.1: General context, the return to the “pre-antibiotic era” p19

1.1.1 Antimicrobials and pathogens resistance p19

1.1.2 How to get out of this situation? p24

1.1.3 Technologies to study pathogens p26

Chapter 1.2: Review of the PhD work: technological developments and
fundamental studies of microorganism’s cell walls. p28

1.2.1 Introduction p28

1.2.2 Technological developments to study the cell wall of
microorganisms by AFM p31

1.2.3 A nanoscale view of the yeast cell wall of Candida albicans
and Saccharomyces cerevisiae p40

1.2.4 Nanoscale behavior of the bacterial cell wall submitted
to antibacterials p47

1.2.5 AFM to understand the mechanism of action of a new
antibacterial molecule p55

Chapter 1.3: Discussion: contribution of “nanomicrobiology” to clinical
microbiology; does it help? p58

1.3.1 AFM as a useful tool for clinical microbiology p58

1.3.2 Cell surface properties of Candida albicans p60

1.3.3 Probing the effects of antibiotics on the cell wall of bacteria p64

1