Oral mucosal lipids are antimicrobial against

153 Pages·2017·4.29 MB·English

University of Iowa
Iowa Research Online
Theses and Dissertations
Spring 2013
Oral mucosal lipids are antimicrobial against
Porphyromonas gingivalis, induce ultrastructural
damage, and alter bacterial lipid and protein
compositions
Carol Lea Fischer
University of Iowa
Copyright 2013 Carol Lea Fischer
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/2494
Recommended Citation
Fischer, Carol Lea. "Oral mucosal lipids are antimicrobial againstPorphyromonas gingivalis,induce ultrastructural damage, and alter
bacterial lipid and protein compositions." PhD (Doctor of Philosophy) thesis, University of Iowa, 2013.
https://doi.org/10.17077/etd.zw858pua
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of theOral Biology and Oral Pathology Commons
ORAL MUCOSAL LIPIDS ARE ANTIBACTERIAL AGAINST PORPHYROMONAS
GINGIVALIS, INDUCE ULTRASTRUCTURAL DAMAGE, AND ALTER
BACTERIAL LIPID AND PROTEIN COMPOSITIONS
by
Carol Lea Fischer
An Abstract
Of a thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Oral Science
in the Graduate College of
The University of Iowa
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May 2013
Thesis Supervisor: Professor Kim A. Brogden
ABSTRACT
Periodontal disease is a chronic inflammation of the gingiva and periodontium
that leads to progressive destruction and irreversible damage to the supportive structures
of the teeth. It affects nearly half of the United States population and is a particular risk
factor in adults older than 65 years of age. Oral microorganisms assemble in plaque as a
polymicrobial biofilm and Porphyromonas gingivalis, an important secondary colonizer
in oral biofilms, has been implicated in periodontal disease. Although the protective
functions of various salivary molecules such as antimicrobial proteins have been
delineated, lipids present in saliva and on the oral mucosa have been largely ignored and
there is growing evidence that the role of lipids in innate immunity is more important
than previously realized. In fact, recent studies suggest that sphingoid bases and fatty
acids, which exhibit potent broad spectrum antimicrobial activity against a variety of
bacteria and fungi, are likely important innate immune molecules involved in the defense
against oral bacterial and fungal infections. However little is known about their spectrum
of activity or mechanisms of action. In addition, the effects of these lipids that are
endogenous to the oral cavity have not been explored against oral bacteria. In this study I
hypothesized that oral mucosal and salivary lipids exhibit dose-dependent antimicrobial
activity against P. gingivalis and alter cell morphology and metabolic events. To test this
hypothesis, I first examined the effects of two fatty acids: sapienic acid and lauric acid,
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and three sphingoid bases: sphingosine, dihydrosphingosine, and phytosphingosine,
against a variety of gram-positive and gram-negative bacteria including P. gingivalis.
Using broth microdilution assays to determine minimum inhibitory and minimum
bactericidal concentrations, I show that antimicrobial activity against bacteria is dose-
dependent, lipid specific, and microorganism specific. Kill kinetics were also variable
across each bacteria-lipid combination. Upon examination of select bacteria-lipid
combinations via scanning and transmission electron microscopy, different morphologies
were evident across all treatments, demonstrating differential activity of each lipid for a
particular bacterium as well as for each bacterium across different lipids. In addition, all
sphingoid bases and fatty acids were taken up and retained in association with P.
gingivalis cells and could be extracted along with bacterial lipids and separated using thin
layer chromatography. Using a combination of two-dimensional in-gel electrophoresis
and Western blots followed by mass spectroscopy and n-terminus degradation
sequencing, I show that sapienic-acid treatment induces a unique stress response in P.
gingivalis, as evidenced by the ability of P. gingivalis to upregulate a set of proteins
involved in fatty acid biosynthesis metabolism and energy production, protein processing,
cell adhesion, and virulence. Finally, utilizing flow cytometry and confocal microscopy,
I assessed the effects of oral antimicrobial lipids against a representative host cell and
describe oral lipid concentrations that are both antimicrobial to P. gingivalis cells and
non-cytotoxic to the representative host cells tested. Combined, these data strongly
suggest that sphingoid bases and fatty acids found within the saliva and on oral mucosa
likely do contribute to the innate antimicrobial activity of saliva, mucosal surfaces, and
skin and this dose-dependent activity is both lipid specific and bacteria specific. This
information adds to current knowledge of the innate functions of endogenous lipids in the
oral cavity. With bacterial resistance to current antibiotics increasing, the exploration of
new antimicrobial agents is important and these lipid treatments may be beneficial for
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prophylactic treatments or therapeutic intervention of infection by supplementing the
natural immune function of endogenous lipids on skin and other mucosal membranes.

Abstract Approved: ____________________________________
Thesis Supervisor
____________________________________
Title and Department
____________________________________
Date
ORAL MUCOSAL LIPIDS ARE ANTIBACTERIAL AGAINST PORPHYROMONAS
GINGIVALIS, INDUCE ULTRASTRUCTURAL DAMAGE, AND ALTER
BACTERIAL LIPID AND PROTEIN COMPOSITIONS
by
Carol Lea Fischer
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Oral Science
in the Graduate College of
The University of Iowa
1

May 2013
Thesis Supervisor: Professor Kim A. Brogden
Copyright by
CAROL LEA FISCHER
2013
All Rights Reserved
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Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Carol Lea Fischer
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Oral Science at the May 2013 graduation.
Thesis Committee: ___________________________________
Kim A. Brogden, Thesis Supervisor
___________________________________
Philip Wertz
___________________________________
David Drake
___________________________________
Georgia Johnson
___________________________________
Mary Wilson
To Benjamin and Samuel, my reasons for living: this document is proof that with
God’s help you can accomplish anything your heart desires. Do not ever let anyone tell
you otherwise. This is the tribute I wish to leave with you.

To Scott, my husband and my best friend: thank you for being my wall, for letting
me lean on you when things got rough, for all your support, encouragement, patience,
dedication to my dreams, and for loving me even when I was too busy to stop and remind
you of how very thankful I am that you’re here.

Words cannot express how much I love each of you.

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ACKNOWLEDGMENTS
I would like to express my deep appreciation and gratitude to Dr. Kim Brogden,
my advisor and friend, for his extraordinary mentorship, support, and patient guidance
throughout this entire process – from application to completion. The combination of Dr.
Brogden’s intellect and genuinely good nature made my time here a pleasure and I am
truly fortunate to have had the opportunity to work with him. This thesis would not have
been possible without the support, guidance, and thought-provoking questions of Dr.
Brogden and the rest of my committee members. Dr. Philip Wertz was a veritable
encyclopedia of lipid knowledge upon which I drew continuously. Dr. Mary Wilson, Dr.
David Drake, and Dr. Georgia Johnson, in addition to giving of their time to serve on my
committee, provided a steady source of support, advice, and encouragement.
Collectively, my committee provided an atmosphere that fostered independent and
critical thinking, learning, and personal growth and I will be forever grateful to each and
every one of them.
I am extremely appreciative of several funding sources supporting my research
including NIH/NIDCR T32 DE014678, NIH/NIDCR R01 DE018032 (Dr. Wertz’ R01),
and NIH/NIDCR R01 DE014390 (Dr. Brogden’s R01). I also want to thank Dr. Leslie
Mehalick for the use of one of her figures and data. I am indebted to the College of
Dentistry, my lab members, and all my friends here, for their continuous moral support
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and encouragement, for listening to me talk about my research, and for providing a
comfortable family atmosphere that contributed to a positive learning environment.
Many thanks also to Dr. Deborah Dawson and Derek Blanchette for all their statistical
expertise and for answering a million questions. I would also like to thank my graduate
student and post-grad friends for their support, feedback, and friendship.
Several of my undergraduate professors, Dr. Kenneth Andrews, Dr. Michael Bay,
Dr. Charlie Biles, and Dr. Terry Cluck, who contributed to my love of science and
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encouraged me to pursue a doctorate, also deserve my gratitude for helping me find my
way here and showing support and encouragement as I pursued this degree. I would also
like to thank my large group of family and friends for their love and support, for
accepting nothing less than completion from me, and for being there when I needed
encouragement or a nudge in the right direction. Specifically I want to thank Benjamin
and Samuel for their willingness to share me with “school” for the past eleven years
while I completed both my undergraduate and graduate degrees. Their understanding,
support, encouragement, and unfailing love kept me going when things were difficult.
And last, but certainly not least, a special thanks to my husband, Scott for being my solid
wall of strength and for shouldering more than his fair share of the household duties
during comps and the writing of this dissertation. Most importantly, I am thankful for my
faith in God, who is the ultimate provider of all my strength and everything good in my
life.
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