Unraveling the muco-adhesion of Lactococcus lactis

226 Pages·2017·11.64 MB·English

Unraveling the muco-adhesion of Lactococcus lactis:
development of biophysical approaches
Thi-Ly Tran
To cite this version:
Thi-Ly Tran. Unraveling the muco-adhesion of Lactococcus lactis: development of biophysical ap-
proaches. Biochemistry,MolecularBiology. INSAdeToulouse,2013. English. ￿NNT:2013ISAT0029￿.
￿tel-01127035￿
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TTHHÈÈSSEE

En vue de l'obtention du

DDOOCCTTOORRAATT DDEE LL’’UUNNIIVVEERRSSIITTÉÉ DDEE TTOOUULLOOUUSSEE

Délivré par :

Institut National des Sciences Appliquées de Toulouse (INSA de Toulouse)

Présentée et soutenue par :
TRAN Thi Ly
Le : jeudi 12 décembre 2013
Titre :

Unraveling the muco-adhesion of Lactococcus lactis : development of
biophysical approaches

École doctorale et discipline ou spécialité :
ED SEVAB : Ingénieries microbienne et enzymatique

Unité de recherche :
Laboratoire Ingénierie des Systèmes Biologiques et des Procédés UMR 5504 CNRS - UMR 792 INRA

Directeur(s) de Thèse :
MERCIER-BONIN Muriel

JURY :
M. SCHMITZ Philippe, Professeur INSA de Toulouse, Président
M. KULAKAUSKAS Saulius, Directeur de Recherche INRA, Rapporteur
M. Di MEGLIO Jean-Marc, Professeur Université Denis Diderot Paris 7, Rapporteur
Mme MERCIER-BONIN Muriel, Chargée de Recherche INRA, Directrice de thèse

M. CASTELAIN Mickaël, Chargé de Recherche INRA, Co-encadrant de thèse

ACKNOWLEDGEMENTS
First of all, I would like to thank Mr. Nic LINDLEY, Director of the
“Laboratoirie d'Ingénierie des Systèmes Biologiques et Procédés” of the “Institut National
des Sciences Appliquées” (INSA) in Toulouse, where this work was carried out from
November 2010 to December 2013, for having welcomed me to his laboratory and provided
the materials and equipments that I have needed to produce and complete my thesis.
I would like to thank all members of the jury for agreeing to assess this manuscript and to
participate in the thesis defence:
• Mr. Philippe SCHMITZ, Professor of the INSA in Toulouse, the chairman of the jury,
for his encouragement, valuable discussions, and good humor.
• Mr. Saulius KULAKAUSKAS, Research Director of the INRA Jouy-en-Josas, for his
great enthusiasm and time spent in reading, analyzing, assessing and writing the
report.
• Mr. Jean-Marc Di MEGLIO, Professor of “Université Denis Diderot Paris 7”, for his
particularly rigorous reading, constructive criticism and time spent in writing the
report.
I would especially like to express my most sincere gratitude to my supervisor, Mrs. Muriel
MERCIER-BONIN, for her excellent guidance, advice and support in all stages of this thesis.
I would also like to thank her for helping me to shape my interest and ideas, painstakingly
and patiently correct my writing. She cared much about my work and responded to my
questions and queries so promptly. Additionally, I will forever be thankful to Mr. Mickaël
CASTELAIN, who was willing to provide me a great knowledge of biophysics as well as
spend his time and effort during the final year of my PhD. One simply could not wish for a
better and friendlier advisor.
I would like to thank Mr. Nicolas DIETRICH, researcher of the “Transfer, Interfaces,
Mixing” (TIM) group of the LISBP laboratory in Toulouse, who let me experience the
Diffusion Front Tracking method and helped me analyze data using the Monte Carlo Markov
Chain program. His motivating collaboration as well as his participation in my doctoral
committee meetings was greatly appreciated. I would also like to thank Mrs. Dominique
ANNE-ARCHARD, researcher of the “'Institut de Mécanique des Fluides de Toulouse”
(IMFT) in Toulouse, for her particular interest in my work and for taking part in my doctoral
committee meetings.
The major part of this thesis is the fruitful result of collaboration with the MICALIS Institute
in Jouy-en-Josas, France. I would like to thank Marie-Pierre CHAPOT-CHARTIER and
Mickaël MEYRAND for their Lactococcus lactis wild-type, mutant and plasmid-cured
derivative strains.
I would also like to thank Etienne DAGUE from the “Laboratoire d'Analyse et d'Architecture
des Systèmes” (LAAS) in Toulouse for the Atomic Force Microscopy measurement, Yann
GUERARDEL from the “Unité de Glycobiologie Structurale et Fonctionnelle” (UGSF) in
Lille for pig gastric mucin fractions.
I would particularly and gratefully thank to Mrs. HO Phu Ha and Mr. CHU-KY Son from
“Hanoi University of Science and Technology” (HUST), for their confidence in me,
recommending me to this project and constant encouragement during my study. Additionally,
I would like to thank the “Université des Sciences et Technologies de Hanoï” (USTH), for
providing the funding which allowed me to undertake this research. I would also like to thank
them for giving me the opportunity to attend the annual USTH workshops which provided a
number of the diverse and interesting training courses as well as the career guidance as a
teacher-researcher.
I am deeply grateful to Ms. Marie-Pierre DUVIAU who was there for helping me set up the
Shear Stress Flow Chamber experiments when I came to the laboratory at the beginning, for
her enthusiasm for discussing, caring and giving advices during my thesis.
I would like to thank all the members of my group EAD4 who are extremely friendly, humor
and helpful. I would particularly like to thank Mr. Pascal LOUBIÈRE, head of the team, for
receiving me to his group. I’m so glad to have worked with the wonderful colleagues. Special
thanks go to my young, funny and cheerful office mates (Manon, Marie, Jade, Thomas,
Arthur, Pauline, Amandine, Stéphanie, Anne-Laure and Jérôme). I just wish them the best of
luck for their thesis and future career.
I would like to thank my countryman, LE Doan Thanh Lam, for his constant help and
encouragement while he was doing his thesis and even after he left the laboratory. He was the
one whom I wrote up for asking so many questions of science and society. I think of him as a
big brother.
Loving thanks to my friends for providing support and friendship that I needed. I will not
forget the beautiful moments that we shared in France.
I especially thank my mom, dad and brothers. My hard-working parents have sacrificed their
lives for their children and provided unconditional love and care. I know I always have my
family to count on when times are rough.
At last, not at least, special thanks to my best friend, soul-mate and boyfriend, Jérémy, who is
the only person can appreciate my quirkiness and sense of humor, and his family who all
have been supportive and caring.

SUMMARY OF THE THESIS
Context and objectives. The digestive epithelium is covered with a protective mucus layer,
regarded as a viscoelastic and permeable hydrogel. This layer serves as an ecological niche
for commensal and probiotic bacteria, and plays a role in the defense against pathogens. The
mucus layer is described as a secreted mucin-fiber scaffold. Mucins are large glycoproteins
with a serine and threonine-rich protein backbone, linked to a wide variety of O-linked
oligosaccharide side chains arranged in a bottle-brush configuration. Such O-glycans are
nutritive sources for bacteria and/or potential ligands for bacterial adhesins, probably
contributing in this way to the selection of the species-specific microbiota.

Many studies on bacterial muco-adhesion have been carried out with commensal
Lactobacillus species, with the aim to select probiotics based on their ability to persist within
the gut. In contrast, little is known about the structural and functional factors involved in the
muco-adhesion of Lactococcus lactis, the model for Lactic Acid Bacteria.

In this thesis, we focused on unraveling multi-scale interactions between a vegetal L. lactis
subsp. lactis isolate, TIL448 and a model mucin, Pig Gastric Mucin (PGM). In a previous
study, L. lactis TIL448 was shown to expose at its surface both pili and mucus-binding
protein. In contrast to the other tested L. lactis strains, specific adhesion to Caco-2 human
intestinal epithelial cells was reported. However, no data was available of the TIL448 muco-
adhesive phenotype. To address such questions, different biophysical approaches were
implemented. Our work was achieved in close collaboration with Micalis Institute in Jouy-
en-Josas, France.

Results. In a first part, we performed single-cell scale AFM measurements with dedicated
lacto-probes and shear stress flow chamber experiments at the bacterial population level,
under laminar flow conditions on the wild type L. lactis TIL448. We also tested the plasmid-
cured strain and two mutants, obtained by disruption of the genes encoding the major pilin
and the mucus-binding protein. Bacterial cells were put in adhesive contact with a biomimetic
PGM-coated surface, under static or shear-flow conditions. AFM experiments on TIL448
revealed a high proportion of specific adhesive events (60 %) and a low level of non-adhesive
ones (2 %). The strain muco-adhesive properties were confirmed by the weak detachment of
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bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were
detected at short (100-200 nm) and long distances (up to 600-800 nm). AFM measurements
on pili and mucus-binding protein defective mutants demonstrated the comparable role
played by these two surface proteinaceous components in adhesion to PGM under static
conditions. Under shear flow, a more important contribution of the mucus-binding protein
than the pili one was observed. Both methods differ by the way of probing the adhesion force.
AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated
that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM.
Then, the diffusion ability of L. lactis was determined by implementing a novel method,
named Diffusion Front Tracking (DFT). It consists of tracking the diffusion front of stained
cell suspensions over time within the PGM network. The technique involved tracking a
liquid-liquid interface in a Hele-Shaw cell using a digital camera. A suspension of bacterial
cells, stained with fuchsine and injected on the top, was allowed to diffuse down to the PGM
hydrogel. The feasibility of the method was demonstrated. Then, the diffusion coefficient was
determined for all the strains under study, which required solely the diffusing front position
over time. A mathematical analysis was developed to solve this problem. Diffusion of
bacterial cells follows the Fick’s law and was fitted to a model using Monte-Carlo algorithm.
The latter allows checking the presence of convection or sedimentation artifacts.

In a second part, in order to have a more thorough understanding of the L. lactis muco-
adhesive and diffusive ability, the microstructure and mechanical properties of PGM were
determined. Gel microstructure for varying PGM concentration was probed by the analysis of
diffusivities of 200-nm and 500-nm fluorescent nanoparticles with different surface
properties (carboxyl-terminated, negatively charged tracers, with and without PEG coating;
amine-terminated, positively charged tracers), using fluorescence Multiple-Particle Tracking.
The pore size of the PGM network was evaluated between 240 and 470 nm, depending on the
PGM concentration. A high heterogeneity of the mesh pore size was also highlighted.
Characterization of the PGM rheological properties was achieved by combining classical
bulk rheometry and one-point and two-point microrheometry approaches.

Conclusion. Our study, based on the combination of different biophysical approaches and
tools, has allowed dissecting the muco-adhesive and diffusive phenotype of L. lactis TIL448,
in relation with the nature of the bacterial surface determinants and the structural, mechanical
and rheological properties of the PGM network.
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