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Oral mucosal lipids are antimicrobial against PDF

153 Pages·2017·4.29 MB·English
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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 1 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, 1 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 2 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 2 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. 2 ii 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 3 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 iii 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. 4 iv

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Follow this and additional works at: http://ir.uiowa.edu/etd the buccal region, underside of the tongue, and floor of the mouth do not have a stratum.
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