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«THE ROLE OF REGULATORY CHANGES AND QUORUM SENSING DURING SALMONELLA COLONIZATION OF NON-TRADITIONAL HOSTS By CLAYTON E. COX A DISSERTATION PRESENTED ...»

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THE ROLE OF REGULATORY CHANGES AND QUORUM SENSING DURING

SALMONELLA COLONIZATION OF NON-TRADITIONAL HOSTS

By

CLAYTON E. COX

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

© 2012 Clayton E. Cox To my family for their continued support of my education

ACKNOWLEDGMENTS

I would like to thank the National Science Foundation for the generous support provided by their Graduate Research Fellowship. I thank the University of Florida and the School of Natural Resources and Environment for the generous support provided by their Graduate Alumni Award. I would also like to thank the Howard Hughes Medical Institute and the Group Advantaged Training of Research Program for their support and wonderful opportunity to improve my mentoring abilities.

I thank Tommy Ward for graciously providing the oysters used during these studies and the students of the Wright lab who cared for them prior to my use. I also thank Marianne Fatica and the Schneider lab for their supply of green tomatoes. I appreciate the members of the Teplitski lab for their innumerous small favors, willingness to troubleshoot or lend advice as well as the general camaraderie which have been tremendously appreciated over the past 5 years. I especially thank Beth Creary for the constant support and humor she lent the lab. Finally, I thank my advisor Max Teplitksi for always encouraging me to focus on what matters.

TABLE OF CONTENTS

page ACKNOWLEDGMENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

Abstract

CHAPTER

1 ALTERNATIVE HOSTS AND THEIR ROLE IN THE LIFESTYLE OF

SALMONELLA

Introduction

Environmental Survival

Survival of Salmonella in Aquatic Environments

Survival of Salmonella in Mollusks

Survival of Salmonella in Soil

Survival of Salmonella in Manure

Survival of Salmonella on Plants and Produce

Survival of Salmonella in Single Celled Hosts

Interactions with Host Associated Microbial Communities

Role of Diversity

Self Destruction Cooperation

The Role of Quorum Sensing

Quorum Quenching

gacS/gacA

Project Rationale

2 COMMON MATERIALS AND METHODS

Growth Conditions

Media

Strain Storage

Cell Washing

DNA Techniques

DNA Isolation

DNA Imaging

DNA Amplification via PCR

Cloning and Mutant Construction

Subcloning

Preparation of other Plasmid Vectors

Heat Shock Transformation of Ligated Vectors

Electroporation

Phage Transductions

Deletion Mutants

RIVET Reporters

Handling of Oysters

3 A HIGH-THROUGHPUT SCREEN FOR INHIBITORS OF THE GACS/GACA

TWO- COMPONENT SIGNALING SYSTEM

Introduction

The Ecological Role of the GacS/GacA Two-Component System

Potential Effects of GacS/GacA Two-Component System Inhibitors................ 58 Materials and Methods

Bacterial Strains and Growth Conditions

Chemical Libraries

Construction and Selection of Reporter Plasmids

PcsrB-LUX Screens

Statistical Comparison of Assay Runs

Biofilm Formation Assays

Results

Selection of a PcsrB-LUX Reporter Plasmid

Initial Screens of the LOPAC Library

Dilution Series Screens of the LOPAC Library

LOPAC Biofilm Assays

HBOI Library Screens

Discussion

4 PROMOTER PROBE LIBRARY SCREEN OF SALMONELLA ENTERICA SV.

TYPHIMURIUM FOR GENES ASSOCIATED WITH PERISTENCE IN THE

EASTERN OYSTER, CRASSOSTREA VIRGINICA

Introduction

Materials and Methods

Bacterial Strains and Culture

Oyster Maintenance

gfp Labeled Promoter Probe Library Screen

Promoter Expression in Live Oysters Measured via RIVET Assays............... 101 Competitive Co-Infection of Deletion Mutants in Live Oysters

Hemocyte Assays

Results

Identification of Oyster-Specific Promoters using a gfp Promoter Probe Library

Confirmation of Oyster Specific Promoter Activity using RIVET Reporters.... 104 ssrB Increases Competitive Fitness but does not Regulate Hemocyte Invasion in Oysters

Discussion

5 THE ROLE OF QUORUM SENSING DURING THE ESTABLISHMENT OF

SALMONELLA ENTERICA SV. TYPHIMURIUM WITHIN THE NATIVE

MICROBIOTA OF THE EASTERN OYSTER, CRASSOSTREA VIRGINICA....... 132 Introduction

Materials and Methods

Bacterial Strains and Culture

Oyster Maintenance

Construction of QS System RIVET Reporters

Activity of QS Related Promoters in Live Oysters Measured via RIVET Assays





Response of the lsrG-tnpR Reporter to Exogenous AI-2

Expression of sdiA-tnpR in response to NaCl concentration

Fitness Phenotype as Determined by Competitive Co-Infection of Deletion Mutants in Live Oysters

Confirmation of AI-2 Production via the Vibrio harveyi LUX Assay................. 144 Results

Confirmation of the lsrG-tnpR Reporter

Activity of QS Systems during Colonization of Oysters

Fitness Phenotypes Associated with QS during Oyster Colonization............. 146 Effect of Environmental Conditions on sdiA Activity

Discussion

6 ANALYSIS OF SALMONELLA LUXS AND LSR OPERON MUTANTS REVEAL

NO ROLE FOR AI-2 SIGNALING DURING COLONIZATION OF TOMATOES

IN THE PRESENCE OR ABSENCE OF THE SOFT-ROT PATHOGEN

PECTOBACTERIUM CAROTOVORUM

Introduction

Materials and Methods

Strains and Culture Conditions

Confirmation of AI-2 Production via the Vibrio harveyi LUX Assay................. 170 In vitro Reception of Pectobacterium AI-2 Signaling by Salmonella............... 170 In vivo Competition Assays in the Presence and Absence of Soft-Rot........... 171 In vivo Promoter Expression Measured via RIVET assays

Results

In vitro Perception of the Pectobacterium AI-2 Signal by Salmonella............. 173 In vivo Promoter Expression Measured via RIVET Assays

In vivo Competition Assays in the Presence and Absence of Soft-Rot........... 176 Discussion

7 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS

Conclusions

Future Directions

APPENDIX A COMPOSITION OF COMMON GROWTH MEDIA

B COMPOUND KEY FOR LOPAC PLATE 1

C COMPOUND KEY FOR LOPAC PLATE 2

D COMPOUND KEY FOR LOPAC PLATE 3

E COMPOUND KEY FOR LOPAC PLATE 4

F COMPOUND KEY FOR HBOI PURE PLATE #1 LIBRARY

LIST OF REFERENCES

BIOGRAPHICAL SKETCH

–  –  –

1-1 Salmonella contamination rates of selected studied watersheds

3-1 List of bacterial strains used in Chapter 3

3-2 List of plasmids used in Chapter 3

3-3 List of primers used in Chapter 3

4-1 List of bacterial strains used in Chapter 4

4-2 List of bacterial strains constructed for use in Chapter 4

4-3 List of plasmids used in Chapter 4

4-4 List of primers used in Chapter 4

4-5 Oyster active promoters identified by the promoter probe library screen.......... 123 5-1 Common culturable oyster-associated bacteria

5-2 List of bacterial strains used in Chapter 5

5-3 List of plasmids used in Chapter 5

5-4 List of primers used in Chapter 5

6-1 List of bacterial strains used in Chapter 6

–  –  –

3-1 Methodology for the PcsrB-LUX screen

3-2 Results of PcsrB-LUX reporter plasmids selection trials in Salmonella hosts....... 76 3-3 Results of the first round LOPAC library screen.

3-4 Results of the second round LOPAC screen.

3-5 Results of the third round dilution series for the 9 compounds selected as potentially inhibitory

3-6 Biofilm assay results for the 9 compounds selected as potentially inhibitory...... 92 3-7 PcsrB-LUX dilution series for Diiscyanoamphilectin, identified as possibly inhibitory during the screen of the HBOI library

4-1 Construction of RIVET reporters

4-2 FACS sorts of a gfp labeled Salmonella promoter probe library in live oysters and relevant controls.

4-3 Percent resolution of RIVET reporters during 24 hour incubation in live oysters or on agar controls at 22 ˚C

4-4 Competitive co-infections of selected defined mutants vs. wild type S.

enterica 14028 in live oysters. log CI (competive index) was calculated using Equation 4-1

4-5 24-hour Infection of oyster hemocytes with gfp labeled S. enterica 14028 wild type and MJW129 ssrB::cm mutant

4-6 2 hour Infection of oyster hemocytes with gfp labeled S. enterica 14028 wild type and MJW129 ssrB::cm mutant

5-1 Activity of the lsrG-tnpR-lacZY reporter in response to DPD.

5-2 Resolution of AHL related RIVET reporters during 24 hour incubation in live oysters at 22 ˚C

5-3 Resolution of AI-2 related RIVET reporters during 24 hour incubation in live oysters at 22 ˚C

5-4 Resolution of two-component regulator RIVET promoters during 24-hour incubation in live oysters at 22 ˚C

5-5 Growth curves of QS mutants in LB.

5-6 Competitive co-infections of defined mutants vs. wild type S. enterica 14028 in live oysters

5-7 Percent resolution of an sdiA-tnpR reporter in response to LB containing varying NaCl concentrations at 22 ˚C and 37 ˚C.

5-8 AI-2 activity in CFS

6-1 Resolution of the lsrG-tnpR RIVET reporter in the 14028 wild type (CEC0015) or isogenic luxS::FRT-kanR-FRT (CEC0018) backgrounds alone or in co-culture with Pectobacterium

6-2 AI-2 activity of Salmonella and Pectobacterium CFS

6-3 Resolution of RIVET reporters in normal and soft-rotted green tomatoes........ 187 6-4 Competitive co-infections of defined Salmonella mutants versus 14028 wild type in normal and soft-rotted tomatoes

6-5 JS246 vs. 14028 in normal tomatoes

–  –  –

ABr Antibiotic Resistance AHL N-Acyl Homoserine Lactone AI-2 Auto-Inducer 2 AI-3 Auto-Inducer 3 AMC Activated Methyl Cycle

–  –  –

ASW Artificial Seawater BSA Bovine Serum Albumin BSLII Biosafety Level 2 CFA Colony Forming Antigen CFU Colony Forming Unit CFS Cell Free Supernatant

–  –  –

DI H2O De-Ionized Water DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid dNTPS Deoxynucleotide Triphosphates DPD (S)-4,5-Dihydroxy-2,3-Pentandione EBU Evans Blue Uranine Agar EGTA Ethylene Glycol Tetraacetic Acid FACS Flourescence Activated Cell Sorting

–  –  –

gfp Green Flourescent Protein HBOI Harbor Branch Oceanographic Institute HDPE High Density Polyethylene ICBR Interdisciplinary Center for Biotechnology Research kan Kanamycin

–  –  –

OD600 Optical Density at 600 nm ONPG Ortho-Nitrophenyl-β-galactoside PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PEL Pectate Lyase ppt Parts per Thousand

–  –  –

RIVET Recombinase based In Vivo Expression Technology RNA Ribonucleic Acid ROS Reactive Oxygen Species SAH S-Adenosylhomocysteine SAM S-Adenosyl Methionine SDS Sodium Dodecyl Sulfate

–  –  –

TAE Tris-Acetate-EDTA Buffer tet Tetracycline TTSS Type Three Secretion System UF University of Florida US United States of America USPS United States Postal Service v/v Volume to Volume

–  –  –

w/v Weight / Volume, Mass Concentration of Solution X-gal 5-Bromo-4-Chloro-3-Indolyl- beta-D-Dalactopyranoside XLD Xylose Lysine Deoxycholate Agar

–  –  –

Chair: Max Teplitski Major: Interdisciplinary Ecology Salmonella caused foodborne gastroenteritis is a tremendous public health burden, accounting for 1.4 million infections, 400-500 deaths and several billion dollars in costs each year in the US alone. Contaminated fresh produce and seafood now account for more Salmonella outbreaks than beef, poultry or eggs. Relatively little is known about how Salmonella successfully colonizes these “non-traditional” hosts.

Because they are typically consumed raw, oysters and tomatoes represent important vectors for salmonellosis. Understanding the mechanisms Salmonella employs to invade and persist in these hosts will help devise preventative strategies which would benefit public health as well as the state of Florida which is a major producer of both commodities.

As antibiotic resistant bacteria become more common new strategies to control pathogens are needed. Non-essential targets which contribute to environmental persistence may be one strategy to avoid evolved resistance. Because these strategies are not be lethal there would be less selective pressure for individuals which are resistant to a potential small molecule inhibitor of the targeted system. The GacS/GacA two-component system is a non-essential regulator which controls expression of virulence genes, biofilm formation, motility and surface colonization. In an attempt to identify the unknown GacS signal or a signal agonist, I developed a screening methodology based on luminescent reporters. I screened two chemical libraries for activity but was unable to identify any potential candidates.



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