UN1VERSITY OF HAWAl'l LIBRARY SYMBIONT-INDUCED CHANGES IN HOST GENE EXPRESSION: THE SQUID VmRIO SYMBIOSIS A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOMEDICAL SCIENCES (CELL AND MOLECULAR BIOLOGy) DECEMBER 2003 By Jennifer Loraine Kimbell Dissertation Committee: Margaret McFall-Ngai, Chairperson Edward Ruby Tom Humphreys AlanLau Katalin Csiszar Karen Glanz ABSTRACT All animals exist in lifelong relations with a complement ofbacteria. Because of the ubiquity ofthese symbioses as well as the derived biomedical applications, the study ofboth beneficial and pathogenic host-microbe associations has long been established. The monospecific light organ association between the Hawaiian sepiolid squid Euprymrw se%pes and the marine luminous bacterium Vibrio fiseheri has been used as a experimental model for the study ofthe most common type ofanimal-bacterial interaction, i.e., the association ofcoevolved Gram-negative bacteria with the extracellular apical surfaces ofpolarized epithelia. A fundamental step for understanding the mechanisms ofhost-symbiont associations lies in defining the genetic components involved; specifically defining changes in host gene expression. The studies presented in this dissertation identify and characterize V. fiseheri-induced changes in host gene expression at both the transcript and protein level. iii TABLE OF CONTENTS Abstract. ...................................... ........................................... iii List ofTables............ vii List ofFigures......... viii Chapter 1. Generallntroduetion......... I Chapter 2. A Glimpse ofthe host transcriptome...................................... 8 Introduction. ..................................•.. .......................... 8 Materials and Methods...... 10 RNA isolation....................................................... 10 Construction ofphage cDNA libraries........ II Subtractive hybridization.. ....... ..... ..................... 12 Results and Discussion............... 14 RNA isolation.......................................... 14 Construction ofphage cDNA libraries 14 Resultant cDNA phage libraries and clone characteristics......... 17 Resultant clones from the subtractive hybridization............ 20 Euprymna se%pes codon usage....... 20 Conclusion... ....................................................... .......... 20 Chapter 3. The role ofthe host proteasome in the squid-vibrio symbiosis......... 24 Abstract...................... 24 Introduction... ......................... .................................. .• .. 25 Materials and Methods... ......................................................... 29 GeneralJlroced~s....................................................... 29 RNA isolation... 30 cDNA phage library construction....................................... 30 Subtractive hybridization... ............................................. 31 Real time RT·PCR........................................................ 31 In situ hybridization...................................................... 32 Western·blot analyses.................................... 33 Protease inhibition........................................................ 34 Regression ofthe ciliated epithelial fields................... 34 Results 35 The C8 subunit is an abundant transcript in the 12-h symbiotic light organ............... 35 C8 mRNA is more abundant in symbiotic light organs.............. 36 C8 mRNA localizes to hemocytes in the light organ appendage sinuses 36 Differences in proteasome protein levels were not detected......... 38 Proteasome inhibitors induced or exJledited CEF regression 38 C8 transcript is more abundant in light organs treated with the proteasome inhibitor, Iactacystin 41 Discussion " .... 44 iv Chapter 4. Symbiont-induced changes host actin during the onset ofa beneficial animal-bacterial association 48 Abstract. ....•..•, , " 48 Introduction. ..... .. .................. .... .. ....... .. ......... ...... .. .... ... 50 Material and Methods......................................................... 54 General procedures............. .. .. 54 Confocal microscopy. .... ............. ... ............ .. ..... 55 Two-dimensional polyacrylamide gel electrophoresis........... 56 Western-blot analyses................................................ 57 lmmunocytoehemistry......... 57 Detennination oflight organ mRNA levels.. 58 In situ hybridization.............................. 59 Labeling with phalloidin............ 61 Measurements ofduct dimensions and determinations ofduct epithelial cell number and shape............... ............ .. .. ...... 61 Results............................................................................ 62 Actin synthesis increased during the onset ofthe symbiosis. ..... 62 Increases in actin abundance were localized to the apical surfaces ofthe duct epithelia.......................................... 62 No changes in actin mRNA levels were detectable during the onset ofthe symbiosis.................................... 66 Persistent colonization with V. [lScheri was required to maintain the changes in light organ actin " ,. 66 Increases in duct actin correlated with constriction ofthe ducts.. 69 Discussion.................. 72 Appendix I. Protocol for subtractive hybridization............... .. .. .. .. .. 77 Protocol overview '" .. 77 Preparation ofthe tester (antisense) single stranded DNA................... 77 Preparation ofthe driver (sense) template RNA strand....................... 79 S~~oJ1hybridization ................................•......•..•.............. 82 Appendix n. Two genetically distinct populations ofthe bobtail squid, Euprymna scolopes, exist on the island ofOahu......... ..........•. 85 Abstract..................................................................... 85 Introduction " '" .. ... .. .......... .. 86 Material and Methods... 88 Capture and maintenance ofspecimens...... 88 Morphometric data ". .. ... ........... 88 Allozymes: cellulose acetate gel electrophoresis... 90 Mitochondrial DNA: Cytochrome oxidase I.......................... 91 Jlltyiogeo~canalysis ...............•...........................•.... 92 Population analysis...... .. 92 Results........................... ...........•.................. 93 Discussion............... 99 Appendix DI. The squid-vibrio symbioses: from demes to genes.................... 101 Abstract...... 101 v Introduction , " ,.... 102 The ecology and evolutionary biology ofsepiolid squid-vibrio symbioses 102 A closer look: the Euprymna sc%pes-Vibrio fischeri symbiosis............ 105 Symbiont-induced changes in host protein production and gene expression " ". 110 What changes do V. fischer; cells induce in the proteome ofthe host light organ? 110 Does V. fischer; induce changes in the light organ transeriptome? III Conclusion..................... 116 References '" ... ... ..... .... ... ...... ..... ... ............ .... .. 118 vi LIST OF TABLES 2.1 Characteristics ofRNA extractions from E. seolopes light organs......... ..... 16 2.2 Characteristics ofresultant eDNA libraries......... 18 2.3 Resultant cDNA clones with homology to genes in the NCBI database....... 19 2.4 Resultant cDNAs from the subtractive hybridization........................... 21 2.5 E. seolopes codon usage......................................................... ..... 22 3.1 Protease inhibitors used to determine the effect on ciliated epithelial. ........ 40 field regression vii LIST OF FIGURES Figure 1.1 Light organ ofjuvenile E. seD/opes................................................ 5 1.2 Early events in the establishment ofthe E. seolopes- V. fischerl symbiosis... 7 2.1 Flow chart ofthe subtractive hybridization protocol used...... 13 2.2 Examples oftotal RNA, mRNA, and cDNA run out on a 1% agarose gel.... 15 3.1 The proteasome...... 26 3.2 C8 mRNA abundance and localization 37 3.3 Abundance ofalpha proteasome protein in 12-h light organs................... 39 3.4 Representative SEMs the effect ofthe proteasome inhibitor lactaeystin on CEF regression......................................................................... 42 3.5 Confocal micrographs showing the localization ofC8 mRNA by in situ hybridization............... 43 4.1 V. fischerl travel down ciliated duets to colonize the light organ................ 52 4.2 Actin protein was more actively synthesized in 12-h symbiotic light organs... 63 4.3 The V. fischerl-induced changes in actin protein levels occurred in the duct epithelia 65 4.4 mRNA abundance in the light organ ofaposymbiotic and symbiotic light organs was not significantly different during the onset ofthe symbiosis....... 67 4.5 Constant exposure to V. fiseheri was required to maintain the symbiont- induced changes in actin........................... 68 4.6 Persistent colonization by V. flScheri induced changes in medial duct circumference , '"... 70 4.7 Cells lining the medial ducts of48-hour symbiotic animals were fewer in number in aposymbiotic animals 71 viii CHAPTER ONE: GENERAL INTRODUCTION All animals exist in lifelong associations with a complement of bacteria. Because of the ubiquity of these symbioses as well as the derived biomedical applications, the study of both beneficial and pathogenic host-microbe interactions has long been established. Although for the past century scientists have diligently described morphological, biochemical, and physiological aspects of animal-bacterial associations, the understanding of the molecular mechanisms