Host factors important for bacterial invasion and persistence in the urinary tract

Urinary tract infections (UTI) are one of the most prevalent human diseases, with uropathogenic Escherichia coli (UPEC) being the primary cause of Gram-negative UTIs. Left untreated, bacteria can ascend from the bladder to the kidneys and eventually escape into the blood stream, causing a potentially lethal case of sepsis. With the shocking increase of antibiotic resistance, new treatments are necessary to combat these infections. UPEC are invasive bacteria that reside within host cells and can remain in a quiescent, metabolically inactive state that renders most current generation antibiotics useless. The primary aims of this dissertation are: 1. to identify host factors important for bacterial invasion and 2. determine how sensing of the bacteria by the innate immune system contributes to persistence or clearance of uropathogens. First, I investigated how plant-based natural products could be used to prevent UPEC invasion into host bladders. These natural products are often used as a part of traditional medicinal practices and may hold promising new methods of treatment for UTIs. The natural products tested successfully inhibited invasion into host cells in an in vitro model of UTI through inhibition of the focal adhesion kinase host factor. When tested in an in vivo model of UTI, invasion into bladder cells was also inhibited. Next, I investigate the role of the host protein histone deacetylase 6 (HDAC6) in an in vitro and in vivo model of UTI in both the somatic and hematopoietic compartments. I discovered that although HDAC6 is a vital factor for host cell invasion in an in vitro model, in an in vivo HDAC6 knockout model bacteria were better able to invade the bladder mucosa at an early time point. Although there are elevated titers in HDAC6 knockouts very early, it is rapidly brought under control and remains near wild type levels through the examined time points. Investigation of neutrophils revealed both genotypes recruit similar numbers, though HDAC6 knockout neutrophils contain higher numbers of viable bacteria. Finally, I investigated the interplay between a bacterial motility organelle and its corresponding receptor in the host, Toll-Like Receptor 5, to elaborate how pro-inflammatory signaling through the receptor altered the host's ability to resolve a UTI. Overall, the sum of the research presented in this dissertation aims to identify the host factors important for bacterial invasion and persistence with the goal to eventually develop ways to manipulate the host to better treat UTIs and prevent recurrent UTIs.

HOST FACTORS IMPORTANT FOR BACTERIAL INVASION AND PERSISTENCE IN THE URINARY TRACT by Adam John Lewis A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Department of Pathology The University of Utah December 2016 Copyright © Adam John Lewis 2016 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Adam John Lewis has been approved by the following supervisory committee members: Matthew A. Mulvey , Chair 09/02/2016 Date Approved Janis J. Weis , Member 09/02/2016 Date Approved Diane M. Ward , Member 09/02/2016 Date Approved Matthew A. Williams , Member 09/02/2016 Date Approved Markus Babst , Member Date Approved and by the Department/College/School of Peter E. Jensen and by David B. Kieda, Dean of The Graduate School. , Chair/Dean of Pathology ABSTRACT Urinary tract infections (UTI) are one of the most prevalent human diseases, with uropathogenic Escherichia coli (UPEC) being the primary cause of Gram-negative UTIs. Left untreated, bacteria can ascend from the bladder to the kidneys and eventually escape into the blood stream, causing a potentially lethal case of sepsis. With the shocking increase of antibiotic resistance, new treatments are necessary to combat these infections. UPEC are invasive bacteria that reside within host cells and can remain in a quiescent, metabolically inactive state that renders most current generation antibiotics useless. The primary aims of this dissertation are: 1. to identify host factors important for bacterial invasion and 2. determine how sensing of the bacteria by the innate immune system contributes to persistence or clearance of uropathogens. First, I investigated how plant-based natural products could be used to prevent UPEC invasion into host bladders. These natural products are often used as a part of traditional medicinal practices and may hold promising new methods of treatment for UTIs. The natural products tested successfully inhibited invasion into host cells in an in vitro model of UTI through inhibition of the focal adhesion kinase host factor. When tested in an in vivo model of UTI, invasion into bladder cells was also inhibited. Next, I investigate the role of the host protein histone deacetylase 6 (HDAC6) in an in vitro and in vivo model of UTI in both the somatic and hematopoietic compartments. I discovered that although HDAC6 is a vital factor for host cell invasion in an in vitro model, in an in vivo HDAC6 knockout model bacteria were better able to invade the bladder mucosa at an early time point. Although there are elevated titers in HDAC6 knockouts very early, it is rapidly brought under control and remains near wild type levels through the examined time points. Investigation of neutrophils revealed both genotypes recruit similar numbers, though HDAC6 knockout neutrophils contain higher numbers of viable bacteria. Finally, I investigated the interplay between a bacterial motility organelle and its corresponding receptor in the host, Toll-Like Receptor 5, to elaborate how proinflammatory signaling through the receptor altered the host’s ability to resolve a UTI. Overall, the sum of the research presented in this dissertation aims to identify the host factors important for bacterial invasion and persistence with the goal to eventually develop ways to manipulate the host to better treat UTIs and prevent recurrent UTIs. iv In memory of William F. Morgan TABLE OF CONTENTS ABSTRACT .......................................................................................................... iii LIST OF FIGURES ............................................................................................. viii LIST OF ABBREVIATIONS .................................................................................. x ACKNOWLEDGEMENTS .................................................................................... xi Chapters 1. INTRODUCTION .............................................................................................. 1 Medical Consequences and Complications of Urinary Tract Infections ...... 2 Important Factors of Bladder Cell Invasion ................................................ 4 Host Immune Response to Bacteria in the Bladder .................................... 7 References ............................................................................................... 10 2. PLANT PHENOLICS ABLATE ACTIVATION OF FOCAL ADHESION KINASE AND INHIBIT BLADDER CELL INVASION BY UROPATHOGENIC ESCHERICHIA COLI ............................................................................... 17 Introduction .............................................................................................. 18 Materials and Methods ............................................................................. 20 Results ..................................................................................................... 24 Discussion ................................................................................................ 36 References ............................................................................................... 40 3. HISTONE DEACETYLASE 6 REGULATES BLADDER ARCHITECTURE AND HOST SUSCEPTIBILITY TO UROPATHOGENIC ESCHERICHIA COLI ......................................................................................................... 47 Abstract .................................................................................................... 48 Introduction .............................................................................................. 48 Results and Discussion ............................................................................ 49 Experimental Section ............................................................................... 53 Conclusions.............................................................................................. 56 Acknowledgements .................................................................................. 56 Author Contributions ................................................................................ 56 Conflicts of Interest .................................................................................. 56 References ............................................................................................... 57 4. THE FLAGELLIN SENSOR TOLL-LIKE RECEPTOR 5 PROMOTES BACTERIAL PERSISTANCE IN THE BLADDER .................................... 59 Introduction .............................................................................................. 60 Materials and Methods ............................................................................. 62 Results ..................................................................................................... 64 Discussion ................................................................................................ 71 References ............................................................................................... 77 5. DISCUSSION.................................................................................................. 80 APPENDIX: HOST CELL INVASION BY PATHOGENIC ESCHERICHIA COLI ................................................................................................................... 85 vii LIST OF FIGURES Figure Page 2.1 Plant phenolics used in this study ................................................................. 25 2.2 CAPE, resveratrol, and EGCG inhibit UTI89 invasion into a bladder cell line. ..................................................................................................................... 27 2.3 Catechin reduces growth of Salmonella while EGCG reduces growth of Shigella flexneri................................................................................................... 28 2.4 Invasion into host cells is not dependent on RNA transcription or protein synthesis ............................................................................................................. 30 2.5 FAK 576 phosphorylation is greatly reduced when bladder cells are treated with CAPE and resveratrol .................................................................................. 32 2.6 Plant-based compounds show altered morphologies of filopodia and lamellipodia revealed by staining for actin .......................................................... 34 2.7 Plant-based secondary metabolites reduce invasion by other pathogens in an in vitro model ...................................................................................................... 35 2.8 Resveratrol inhibits bacterial invasion into bladders in an in vivo model of UTI ...................................................................................................................... 37 3.1 HDAC6 is critical for UPEC entry into cultured MEFs ................................... 50 3.2 HDAC6 affects initial colonization of the bladder by UPEC, but not the levels of persistent bacteria........................................................................................... 50 3.3 HDAC6 deletion alters bladder architecture and volume capacity ................ 51 3.4 HDAC6 deletion results in increased levels of acetylated tubulin within superficial umbrella cell and smooth muscle layers of the bladder ..................... 52 3.5 Neutrophils recovered from the bladders of infected HDAC6-/- contain higher numbers of viable bacteria .................................................................................. 53 4.1 Bladder and kidney TLR5 increases susceptibility to infection by two different strains of UPEC .................................................................................................. 65 4.2 CFT073 lacking flagella does bring unity in titers between WT and TLR5 -/mice .................................................................................................................... 67 A.1 Types of invasive E. coli strains with associated virulence factors (VFs) used to enter target host cells ................................................................... 86 A.2 UPEC invasion of the urothelium ........................................................ 88 A.3 Transcytosis of NMEC across the blood brain barrier ........................ 97 A.4 EIEC/Shigella invasion of the colonic epithelium ................................ 99 ix LIST OF ABBREVIATIONS ΔFBS ............................................................ Heat Inactivated Fetal Bovine Serum BEC ..................................................................................... Bladder Epithelial Cell BSA.....................................................................................Bovine Serum Albumin CAPE ......................................................................... Caffeic Acid Phenethyl Ester CFU ....................................................................................... Colony Forming Unit DC...................................................................................................... Dendritic Cell DMEM ............................................................. Dulbecco’s Modified Eagle Medium EGCG ...............................................................................Epigallocatechin Gallate ExPEC ................................................. Extraintestinal Pathogenic Escherichia coli H&E ................................................................................... Hematoxylin and Eosin HDAC6 ................................................................................ Histone Deacetylase 6 HlyA .....................................................................................................α-hemolysin LB .................................................................................................. Lysogeny Broth MEF .......................................................................... Murine Embryonic Fibroblast MPO............................................................................................. Myeloperoxidase NFκB ................................................................................. Nuclear Factor Kappa B PBS.............................................................................. Phosphate Buffered Saline PRR ......................................................................... Pattern Recognition Receptor RT-PCR ................................. Reverse Transcriptase Polymerase Chain Reaction SEM .............................................................................Standard Error of the Mean TLR ............................................................................................ Toll-Like Receptor TNFα ........................................................................ Tumor Necrosis Factor Alpha UPEC ..................................................................... Uropathogenic Escherichia coli UTI ....................................................................................... Urinary Tract Infection WT .......................................................................................................... Wild Type ACKNOWLEDGEMENTS I will forever be grateful to Dr. Matthew Mulvey, who is an incredible mentor. His steady, guiding hand in research, sense of humor, enthusiasm, and supportive attitude made coming to the lab a privilege. My thanks to the committee members that have provided valuable advice and guidance. I also thank the members of the Mulvey lab who shared in the victories and greeted frustrations with the type of gallows humor that makes it easy to persist. My thanks to the members of the department who always made time to help this wayward graduate student. To my parents, my brother, my in-laws, and my friends: thank you for helping me keep my sanity. I’m sure whenever I told you what I was doing in the lab, it sounded like I had lost it. Most importantly, my wife, Whitney, and son, Jack: I am humbled by your unwavering and amazing patience and support. Without it, and the supply drops during nights spent in the lab, this journey would not have been as enjoyable. My love to you always. CHAPTER 1 INTRODUCTION 2 Medical Consequences and Complications of Urinary Tract Infections Urinary tract infections (UTI) are one of the most prevalent bacterial diseases. Females are at a higher risk of contracting a UTI likely due to their shorter urethra [1]. In their lifetimes, 50% of women will contract at least one UTI and are at a 25% risk of contracting a second UTI within 6 months of the first [2, 3]. In the United States alone, UTIs are annually responsible for 8 million infections, resulting in 8.1 million doctor’s visits and $2.14 billion in health care costs [4, 5]. In some cases, UTIs can ascend to the kidneys and may even escape into the blood stream leading to urosepsis, which carries a lethality rate of up to 40% [6]. The primary pathogen responsible for Gram-negative UTIs is uropathogenic Escherichia coli (UPEC), a subset of extraintestinal pathogenic Escherichia coli (ExPEC) [7]. These strains act as commensals in the intestinal flora without any apparent pathogenesis, but can cause diseases like meningitis and UTI once they escape the intestinal niche. Currently, the standard treatment for UPEC-caused UTIs is administration of oral antibiotics, but the effectiveness of this regimen is compromised by the intracellular lifestyle of UPEC, which protects the bacteria from antibiotics that cannot cross the host cell membrane, and the increasing prevalence of antibiotic resistance. The intracellular lifestyle offers many advantages to UPEC including access to sequestered nutrients [8-10] and protection from antibiotics [11], immune cells, antibodies, and antimicrobial peptides. UPEC strains are 3 particularly well adapted for pathogenesis as they are able to invade and survive within a wide variety of host cells, both somatic and hematopoietic [12, 13]. Additionally, invasion of host cells can provide a niche that facilitates long-term survival and may contribute to recurrent infections [14, 15]. This concept becomes evident in mouse models where, despite sterilization of the urine through sustained administration of antibiotics, there is a failure to clear bladderresident bacteria [11]. Recurrent UTIs are often due to reinfection with the same strain that caused the primary infection, and are likely contributing to the increase of antibiotic resistance due to the often repeat administration of the same antibiotics for recurrent infections [16-19]. ST131, a strain type of UPEC that carries multiple drug resistance genes, has recently seen global dissemination [20-22]. As these resistance genes are carried on plasmids, spread of antibiotic resistance to other bacterial strains is made disturbingly easy [23]. This trend emphasizes the need for new preventative measures as well as novel therapeutics. One possibility for new therapeutics is to target host factors that UPEC requires for invasion. By blocking invasion at the earliest stages of infection, the pathogen fails to gain entry to the host cell and may be stopped before any meaningful disease afflicts the patient. Another possibility is to modify the host immune response in such a way that promotes complete resolution of the infection and minimizes recurrence. 4 Important Factors of Bladder Cell Invasion Within the bladder, invasion can occur in any of the three distinct cell layers that comprise the urothelium: basal cells, intermediate cells, and superficial cells [24]. Basal and intermediate cells are undifferentiated cells that undergo rapid replication, differentiation, and repair of the luminal barrier upon damage [25]. In contrast, superficial cells are terminally differentiated and form a barrier that protects the underlying cells through a network of tight junctions and membrane-bound uroplakin plaques [26-28]. Exposure to bacteria can induce a basic antimicrobial defense in which the superficial cells are shed from the urothelium and expelled in the urine [29, 30]. While this can remove bacteria from the bladder, it opens up breaches in the urothelial barrier and gives rise to the possibility of invasion into the underlying cells [31]. Bacteria able to invade the underlying cells may contribute to recurrent infections. Unique to superficial cells are uroplakin plaques comprised of four major proteins including uroplakin Ia (UPIa), UPIb, UPII, and UPIIIa [32, 33]. These proteins form heterodimers consisting of UPIa/UPII and UPIb/UPIIIa, then into heterotetramers, and are finally assembled into hexameric complexes [34]. Uroplakin hexamers are stored in mature fusiform vesicles characterized by the presence of two uroplakin plaques [34, 35]. These large hexagonal structures can be as large as 1 µm in diameter and comprise up to 90% of the luminal surface [36]. As an integral part of the superficial cell membrane, uroplakins play important roles in normal bladder physiology, preventing urine reflux into the kidneys and assisting in maintenance of barrier function [28, 37, 38]. They are 5 also thought to play an integral role in bladder expansion and contraction, whereby additional uroplakin plaques are trafficked to the luminal membrane during expansion or endocytosed and trafficked to lysosomes for degradation during contraction of the bladder [39-41]. One of the earliest steps of a UTI is adhesion between UPEC and the uroplakin-expressing superficial cells. This is accomplished through interaction between Type 1 pili on the bacteria and various host receptors [13, 30]. Type 1 pili are adhesive organelles consisting of repeating structural units tipped by the FimH mannose-binding adhesin [42, 43]. FimH allows UPEC to bind mannosylated residues on the cell surface, and thereby resist removal by emptying of the bladder. This is accomplished through a catch-bond mechanism that enhances adhesion under shear flow conditions [44, 45]. UPEC lacking FimH are impaired at cell attachment and are quickly cleared by the host [46, 47]. Several specific membrane proteins on the superficial cell have been identified as targets of FimH including UPIa, α3 and β1 integrins, and Toll-Like Receptor (TLR) 4. Of the uroplakins, only UPIIIa has any appreciable intracellular region [48]. Therefore, it is likely that binding between UPIa and FimH does not directly signal to downstream factors to initiate invasion. However, binding of FimH to uroplakin plaques induced phosphorylation of the intracellular domain of UPIIIa and subsequently increased intracellular calcium concentration [49]. One hypothesis is that this cascade activates casein kinase II and promotes bacterial entry into host cells in vitro. It is also possible that signaling through UP complexes is not 6 required, and instead bacteria are endocytosed through natural cycling of uroplakin plaques. This scenario is problematic in that bacteria are significantly larger than uroplakin plaques, requiring the simultaneous uptake of multiple plaques. Another issue is that FimH-expressing bacteria are able to invade a wide variety of cells, while uroplakins are only expressed in the bladder and eye. A more ubiquitous receptor found in the bladder is α3 and β1 integrins [5052]. Previous work found that although FimH binds integrins α2, α6, and β4, only blocking the α3 and β1 subunits showed any significant reduction of invasion into bladder cells [53]. Further, the importance of signaling mediated by the intracellular tail of β1 integrin was indicated by the reduced invasion into host cells harboring mutations at critical residues that prevent phosphorylation. Interestingly, FimH did not associate with the binding pocket created by integrin, but rather bound to glycosylated residues on the integrins. FimH binding to integrins promoted clustering, which in turn led to focal adhesion kinase (FAK) autophosphorylation at tyrosine 397 (Y397). Phosphorylation of Y397 promotes binding by Src family kinases, which leads to phosphorylation of two additional residues and subsequent activation of FAK [54]. Activated FAK promotes actin nucleation and invasion by UPEC. Inhibition of either FAK or Src family kinases significantly attenuates the ability of UPEC to invade host cells [53]. Integrin receptors and downstream signaling events that lead to actin rearrangements are attractive targets for therapy as multiple pathogens, like Shigella, Salmonella, and Yersinia, are also dependent on actin cytoskeletal rearrangements for entry into host cells. 7 Another cytoskeletal component important for UPEC invasion are microtubules. Disruption of microtubule dynamics negatively impacts the ability of UPEC to invade host cells even though microtubules do not appear to associate with invading bacteria in the same way actin does [31, 55]. Current data suggest that microtubules do not directly interact with invading bacteria. Instead, kinesin, the motor protein that travels along the microtubules towards the plasma membrane, has been shown to be important for invasion. It is possible that kinesin is delivering factors important for invasion at the site of bacterial association [55]. Host Immune Response to Bacteria in the Bladder Detection of bacteria within the bladder lumen is accomplished through a number of pattern recognition receptors (PRR) including TLR4 (lipopolysaccharide), TLR5 (extracellular flagellin), and TLR11 (extracellular flagellin). Binding between FimH and TLR4 has been shown to induce nuclear factor kappa B (NFκB) signaling, which initiates critical immune signaling [56]. Mice that lack TLR4 have greater bacterial burdens within the bladder when compared to their wild type counterparts [56, 57]. Previous experimentation has also shown that mice lacking TLR5 or TLR11 also contain greater bacterial burdens in comparison to wild type mice [58, 59]. It would be easy to assume inflammation induced by these PRRs during bladder infections yields positive outcomes for the host, but elevated inflammation at early time points during UTI are associated with increased bacterial persistence, suggesting that not all inflammation is beneficial to the host [60]. Therefore, it is more appropriate to 8 consider inflammation in terms of quality rather than quantity. This distinction is evident in humans where polymorphisms in the TLR1, 2, 4, and 5 genes have been associated with either resistance or susceptibility to UTIs. These polymorphisms have also been associated with asymptomatic bacteriuria, which has been shown to be protective against pathogens that would otherwise cause symptomatic bacteriuria [61-65]. Recruitment of immune cells to the bladder is initiated through bladder resident macrophages. Subsequent immune cells are recruited through a licensing mechanism in which circulating macrophages infiltrate the bladder and secrete tumor necrosis factor α (TNFα) [66]. TNFα induces resident macrophages to release CXCL2 and thereby recruit matrix metalloproteinase-9 (MMP9)-expressing neutrophils to the sites of infection [66]. Without proper licensing, neutrophils fail to express MMP9 and are unable to enter the bladder. Neutrophils are the primary phagocyte during the early phases of infection, though they cede this role to macrophages as the immune response progresses [66]. One notable feature of UTIs is how commonly they recur shortly after the primary infection, suggesting that an adaptive immune response is not fully developing to provide long-term protection to the host. RAG -/- mice, which lack mature B and T cells, demonstrate similar bacterial burdens on primary challenge, but increased (approximately 100 fold) bacterial burden on secondary challenge when compared to wild type mice [67, 68], indicating that it is possible to induce an adaptive response in experimental systems. The limited adaptive 9 response is developed between antigen presenting dendritic cells (DCs) and T cells, depletion of which negatively impacts the immune response to the secondary challenge, but not the primary [67]. Depletion of macrophages improves the adaptive immune response to secondary challenge, suggesting that macrophages are stymying adaptive immunity through phagocytosis of bacteria and subsequent antigen sequestration [67]. With antibiotic resistance rapidly spreading among UPEC strains coupled with the high risk of recurrence, the challenge facing modern medicine lies in identifying new therapeutics that can act either synergistically with, or independently of, currently prescribed antibiotics. Specifically, these therapies would target host factors and thereby reduce the development of resistance mechanisms by pathogens. Theoretical bipartite therapeutics would target both invasion and the immune response. These types of treatments could be taken prophylactically by high risk patients or simultaneously with another treatment to minimize reinfection of the bladder. The research presented in the following chapters attempts to identify host factors that can be targeted similarly to how natural products derived from plants used in traditional medicines prevent invasion into the host cell. 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Mora-Bau G, Platt AM, van Rooijen N, Randolph GJ, Albert ML, Ingersoll MA. Macrophages subvert adaptive immunity to urinary tract infection. PLoS Pathog. 2015;11(7):e1005044. doi: 10.1371/journal.ppat.1005044. PubMed PMID: 26182347; PubMed Central PMCID: PMCPMC4504509. 68. Frendéus B, Godaly G, Hang L, Karpman D, Svanborg C. Interleukin-8 receptor deficiency confers susceptibility to acute pyelonephritis. J Infect Dis. 2001;183(Suppl 1):S56-S60. CHAPTER 2 PLANT PHENOLICS ABLATE ACTIVATION OF FOCAL ADHESION KINASE AND INHIBIT BLADDER CELL INVASION BY UROPATHOGENIC ESCHERICHIA COLI 18 Introduction Urinary tract infections (UTIs) are one of the most abundant human bacterial infections worldwide [1, 2]. Women are more susceptible to UTIs than men with nearly one third experiencing an acute UTI by the age of 24 and at least 25% of these individuals suffering a recurrent infection [2, 3]. The high rate of recurrence and the spread of antibiotic resistant uropathogens [4-6] are jeopardizing effective treatment options and pose an imminent global health risk [2]. Plants have long had a role in medicine, with herbs being components of traditional medicines used to treat pathogenic diseases like malaria [7], diarrhea [8], and respiratory diseases [9]. Modern medicine has recognized the usefulness of plant-based natural products for the basis of numerous therapeutics such as morphine as an analgesic [10] and Taxol as an anti-cancer compound [11]. Looking to plant-based traditional medicines as a basis for new treatments has revealed potential compounds for treating for heart disease [12, 13], cancer [14, 15], and pathogens [16-20]. In popular culture, plant products are recommended as folk remedies, such as cranberry juice to treat UTIs [21]. Chemically, cranberry extracts contain a number of phenolic compounds that are biologically active in humans [22]. Recent studies have demonstrated that consumption of cranberry extracts is associated with a decreased incidence of recurrent urinary tract infections [23], likely by inhibiting adhesion to host cells through type 1 and P pili [24-28]. Cranberry extracts have also been associated with reduction in the risk of UTIs in postoperative patients [29, 30] and high-risk elderly populations 19 [31, 32]. In another study, investigators used compounds isolated from traditional seagrass-based medicines used to treat UTIs and found that many inhibited bacterial growth [33]. Along the lines of this study, traditional medicinal preparations from mandarin orange seeds were found to inhibit invasion into bladder epithelial cells, potentially by down regulation of 1 integrin [34]. Though the studies outlined are important steps in identifying new natural product-based treatments, many are very broad and generally do not investigate mechanism. Additionally, they identify interference with association between host and pathogen, but few investigate effects on invasion. They also tend to focus on a single pathogen as opposed to identifying broadly applicable invasive pathways used by multiple pathogens. This is a major oversight as invaded bacteria are able to remain hidden from the immune system and sequestered from antibiotics [35-37]. Specifically with UTIs, comparisons between primary and secondary infection strains has revealed an overwhelming incidence of reinfection with the same strains [38]. By using compounds that inhibit bacterial invasion, either prophylactically or as treatment for a UTI, the risk of reinfections could be reduced by preventing the creation of latent bacterial populations housed in host cells [39]. Therefore, we designed this study to investigate the anti-invasive mechanisms of multiple plant-based natural products that are commonly found in a variety of foods and drinks and tested them in the context of multiple pathogens. 20 Materials and Methods Bacterial strains, cell lines and pharmacological inhibitors The clinical cystitis isolate UTI89 was grown statically in modified M9 minimal medium (6 g/l Na2HPO4, 3 g/l KH2PO4, 0.5 g/l NaCl, 0.1 mM CaCl2, 1 g/l NH4Cl, 1 mM MgSO4, 0.1% glucose, 0.0025% nicotinic acid, 0.2% casein amino acids, and 16.5 μg/ml thiamine) or Lysogeny Broth (LB; Difco) at 37C. AAEC185/pRI203, encoding the Yersinia invasin protein, was grown shaking in LB at 37C to stationary phage. Bacteria were diluted in M9 or LB to an OD600 of approximately 0.5 prior to use. Shigella flexneri (ATCC 12022) and Salmonella enterica serovar Typhimurium (SL1344) were grown shaking at 37C in LB overnight, then diluted 1:33 and grown for an additional 3.5 hours as previously described [40]. 5637 cells (ATCC HTB-9) were grown and maintained in RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum (Hyclone) in a 37C humidified incubator with 5% CO2. CAPE, resveratrol, EGCG, and catechin were purchased from Sigma-Aldrich, Biomol, or Cayman Chemical and dissolved in DMSO. Actinomycin D and cycloheximide were purchased from Sigma-Aldrich and dissolved in ethanol. Bacterial cell-association and invasion assays Bacterial invasion and cell-association assays were performed as described previously [41]. Briefly, confluent 5637 monolayers in 24-well tissue culture plates were pretreated with CAPE (25 µg/ml), resveratrol (100 µM), EGCG (25 µg/ml), or catechin (25 µg/ml) for 1 hour prior to infection. Controls 21 were treated with the carrier DMSO alone. BECs were infected with UTI89 or AAEC185/pRI203 at a multiplicity of infection (MOI) of approximately 15-25, or with S. flexneri or S. enterica at an MOI of 100. Tissue culture plates were centrifuged at 600 x g for 5 minutes to expedite and synchronize bacterial contact with the host cells. After 2 hours in the continued presence of the plant-based compound or carrier, sets of triplicate wells were washed 4 times with PBSMg+2/Ca+2 and lysed in PBS containing 0.4% Triton-X 100. These lysates were plated on LB agar plates to determine numbers of cell-associated bacteria. Alternatively, sets of triplicate wells were first treated with 100 µg/ml of gentamicin for 2 hours to kill extracellular bacteria prior to 4 washes PBSMg+2/Ca+2 and lysis in 0.4% Triton-X 100. These lysates were plated to quantify the numbers of surviving intracellular bacteria. In experiments using S. enterica, host cells were infected for 30 min followed by 1-hour incubations in the presence of gentamicin prior to washes and host cell lysis. To assess the effects of host cell translation and transcription in the cell association and invasion assays, BECs were treated continuously with 26 µM cycloheximide, 5 µg/ml actinomycin D, or an equal volume of ethanol. Bactericidal activity of each compound was tested by treating bacteria in RPMI1640 + 10% heat inactivated FBS for 2 hours in the presence of DMSO or the compounds at the previously indicated concentrations. All assays were repeated at least three times in triplicate. 22 Western blot analysis Confluent BEC monolayers in 6-well plates were starved for serum overnight and then treated with CAPE (25 µg/ml), resveratrol (100 µM), EGCG (25 µg/ml), catechin (25 µg/ml), or carrier (DMSO) alone for 1 hour prior to infection with UTI89 (MOI~15). Plates were spun at 600 x g for 5 minutes and incubated 15 minutes. Wells were then washed with cold PBS-Mg+2/Ca+2, lysed in 50 mM Tris pH 7.4, 1 mM NaCl, 1% NP-40, 1X complete protease inhibitor (Roche), 1 mM PMSF, 1 mM NaF, and 0.4 mM orthovanidate, and processed for Western blot analysis. In other experiments, serum-starved BECs were infected with 100 µL of diluted bacterial suspension and incubated for up to 60 minutes before lysing the host cells in RIPA lysis buffer containing1X complete protease inhibitor, 1 mM PMSF, 1 mM NaF, and 0.4 mM orthovanidate at indicated time points. The primary antibodies used include mouse anti-FAK, mouse anti-p397 (BD), and rabbit anti-p576 (Upstate). Kinexus “Kinetworks” multi-immunoblotting service 5637 cells were seeded into 6 well plates and confluent in 24 hours. CAPE (25 µg/ml) or DMSO (Sigma-Aldrich) was added to each well. The drug treatment was allowed to proceed for 1 hour. UTI89 with an OD600 of approximately 0.5 was added to each well for 15 minutes. The cells were washed 3 times with PBS2+ (PBS supplemented with Mg2+/Ca2+). The cells were then lysed with a buffer containing 50 mM Tris pH 7.4, 1 mM NaCl, 1% NP-40, 1X Complete Protease Inhibitor (Roche Applied Sciences, Indianapolis, IN), 0.1 µg PMSF (Elastin Products, Owensville, MO), 1 mM NaF (Sigma-Aldrich), 0.4 23 mM orthovanidate (Sigma-Aldrich), 5 µM leupeptin (Sigma- Aldrich), and 1 mM aprotinin (Roche Applied Sciences). A BCA protein assay was used to determine protein content. The protein concentration was diluted to 0.8 µg/µl and Kinexus 4X sample buffer was added. The samples were shipped to Kinexus (Vancouver, Canada) for multi-immunoblotting analysis. Mouse infections Eight- to nine-week-old female CBA/JCrHsd mice (Harlan Laboratories) were inoculated via transurethral catheterization with 50 µl of a bacterial suspension (~107 CFU in PBS from a 24-hour static culture of UTI89) containing resveratrol (300 µM) or DMSO as previously described. Mice were killed 1-hour post infection, bladders were harvested aseptically, and quadrisected. Bladder pieces were treated with gentamicin (100 µg/ml in 1 mL PBS) for 30 minutes, washed twice in 1 ml PBS and lysed in 1 ml PBS containing 0.025% Triton X100. Total number of intracellular bacterial titers within the lysates was determined by plating serial dilutions on LB agar plates. A total of eleven mice from two independent experiments were tested for each treatment. Microscopy 5637 cells were seeded into 24-well plates containing sterile 12 mm diameter coverslips for 24 hours until confluent. DMSO, CAPE, EGCG, catechin, and resveratrol were added at the concentrations described previously and the cells were incubated for 3 hours. After 3 hours, the cells were washed 3x with sterile PBS2+ and fixed with 2.5% paraformaldehyde. The cells were stained with 24 Alexa568-conjugated phalloidin (1:40; Molecular Probes, Eugene, OR) to visualize F-actin. The slides were mounted with FluorSave (Calbiochem) and observed with an Olympus Fluoview series confocal microcope equipped with argon and krypton lasers providing excitation energy at 568 nm. Images were captured using Olympus Fluorview software. Statistics Prism 6.07 (GraphPad Software) was used for all statistical tests. p values less than 0.05 were considered significant. Results Plant-based compounds inhibit host cell invasion by uropathogenic Escherichia coli We were interested in testing a variety of plant-based compounds in the context of a bladder infection model that allows us to quantify the ability of bacteria to associate with and invade host cells. To this end, we investigated the phenolic natural products caffeic acid phenethyl ester (CAPE), resveratrol, epigallocatechin gallate (EGCG), and catechin (Figure 2.1) in an in vitro model of urinary tract infections. These are phenolic compounds, much like many of the compounds found in cranberry extracts [22]. The bladder epithelial cell (BEC) line 5637 was treated with the indicated compounds, and then infected with the cystitis strain UTI89. After 2 hours of incubation, the wells were washed to remove bacteria that remained unassociated with the host cells. Each well was then titered to enumerate the number of UTI89 cells associated with host cells, 25 Figure 2.1 Plant phenolics used in this study. Structure and plant source for (A) caffeic acid phenethyl ester (CAPE), (B) resveratrol, (C) catechin, and (D) (-) epigallocatechin gallate (EGCG). 26 either intracellularly or extracellularly. Of the compounds tested, only resveratrol significantly reduced the number of bacteria associated with the host cells (Figure 2.2A). Wells were further treated with gentamicin to eliminate extracellular bacteria, and the number of invaded bacteria were normalized to association. All of the compounds significantly inhibited the ability of UTI89 to invade host cells when compared to the DMSO control (Figure 2.2B). None of the compounds tested negatively impacted survival of UTI89 (Figure 2.3). These results suggest that the plant-based compounds are most likely inhibiting host factors required for invasion. Inhibition of invasion is independent of translation and transcription One potential explanation for how the plant-based compounds inhibit bacterial invasion includes the observation that CAPE can be an inhibitor of nuclear factor kappa B (NFκB), a transcription factor important for the immune response during infection [42]. In the context of UTIs, NFκB has not been investigated and warranted further pursuit as a possible mechanism of our observed phenotype. To determine if downstream NFκB-related factors were important for pathogen entry into host cells, we treated 5637 cells with actinomycin D (ActD), a transcription inhibitor, or cycloheximide (CHX), a protein synthesis inhibitor, and infected cells with UTI89 in an in vitro gentamicin protection assay. We hypothesized that if NFκB was involved in invasion through production of a host factor that facilitates invasion, either ActD or CHX would inhibit invasion. Instead we found that inhibiting either transcription or protein 27 Figure 2.2 CAPE, resveratrol, and EGCG inhibit UTI89 invasion into a bladder cell line. (A) Cells were pretreated with the compounds for 2 hours, then infected with UTI89 in the presence of the compounds. Only resveratrol significantly inhibited association between host and pathogen. (B) Bacteria invaded into host cells normalized to DMSO control. CAPE, resveratrol, and EGCG significantly inhibited invasion. Bars indicate mean values ± standard error of the means (SEM) with n ≥ 3 independent experiments with 3 technical replicates per condition. p-values calculated by student’s t-tests. 28 Figure 2.3 Catechin reduces growth of Salmonella while EGCG reduces growth of Shigella flexneri. UTI89, Salmonella Typhimurium, Shigella flexneri, and AAEC185/pRI203 were treated with DMSO or phenolic compounds for 2 hours, then titered. Colony forming units were enumerated and all values normalized to DMSO. Significant reductions in survival of Salmonella (a: p = 0.0393) and Shigella (b: p = 0.0368) were observed. Bars indicate mean values ± SEM with n = 3 independent experiments. p-values calculated by a one-sample t-test against a theoretical mean of 100. 29 synthesis did not significantly affect the ability of bacteria to associate with (Figure 2.4A) or invade (Figure 2.4B) host cells. This result demonstrates that CAPE’s ability to inhibit invasion is independent of NFκB and related transcribed immune factors, but rather inhibits an already present host factor necessary for invasion. Plant-derived phenolic compounds reduce phosphorylation of an activating FAK residue Since the plant-based compounds were acting on a factor already present in the cell, we hypothesized that the natural products were inhibiting posttranslational modifications. Previously published work found subapoptotic doses of CAPE can inhibit activation of focal adhesion kinase (FAK) [43]. FAK is a protein kinase that associates with integrins and is autophosphorylated at Y397 by integrin clustering [44]. Activation of FAK by phosphorylation signals through many different pathways that can influence cell migration, survival, adhesion, and invasion [45-47]. Previously, we discovered that knockdown or knockout of FAK reduced bacterial invasion into 5637 bladder cells [48]. Similarly, inhibition of Src family kinases, which phosphorylate FAK at Y576 and Y577, reduces bacterial invasion into host cells [48]. To assess the level of phosphorylation of both FAK and other possible downstream targets, we used a phosphoprotein array from Kinexus to investigate phosphorylation status during infection and treatment with either DMSO or CAPE. Though phosphorylation levels in several proteins were different in CAPEtreated cells, the most dramatic difference was the reduction in phosphorylation 30 Figure 2.4 Invasion into host cells is not dependent on RNA transcription or protein synthesis. (A) Bladder cells treated with actinomycin D (ACD) or cycloheximide (CHX), and then infected with UTI89, showed no significant differences in association 1-hour postinoculation. (B) Bladder cells treated with actinomycin D or cycloheximide showed no significant differences in invasion. Bars indicate mean values ± SEM with n = 3 independent experiments, each with 3 technical replicates. p-values calculated by student’s t-tests. 31 of FAK Y576 (Figure 2.5A). Since the other natural products demonstrated similar phenotypes to CAPE, we were interested if they also inhibited phosphorylation of FAK Y576. We treated 5637 cells with DMSO, CAPE, EGCG, catechin, or resveratrol and infected them with UTI89. Probing infected cell lysates for phosphorylation of FAK Y397, the tyrosine autophosphorylated by integrin clustering [49], revealed some minor fluctuation between samples when compared to DMSO-treated controls (Figure 2.5B). However, probing for phospho-Y576 confirmed the dramatic reduction in phosphorylation observed by Kinexus when cells were treated with CAPE or resveratrol, and more modest reduction when treated with EGCG or catechin (Figure 2.5C). Interestingly, comparing intensities in Figure 2.5C with invasion in Figure 2.2B reveals a correlation between inhibition of phosphorylation of Y576 and invasion. These results suggest that inhibiting phosphorylation of FAK Y576 has downstream effects resulting in reduced invasion into the host cells. Plant-derived phenolic compounds alter the structure and morphology of filopodia and lamellipodia FAK has previously been shown to control cell motility and spreading through actin polymerization [45]. To investigate if the plant-based compounds altered cell morphologies, cells were incubated with DMSO, CAPE, catechin, EGCG, or resveratrol, fixed after 3 hours, and then visualized for actin. The most notable phenotype of treated cells was the morphological differences in lamellipodia, the actin skirt on the leading edge of a cell, and filopodia, actin filaments that form projections past the lamellipodia [50]. Cells treated with 32 Figure 2.5 FAK 576 phosphorylation is greatly reduced when bladder cells are treated with CAPE and resveratrol. (A) Phosphorylation of specific residues expressed as a percent of control. FAK phospho-Y576 (black bar) is the most downregulated of any of the residues when cells are treated with FAK. (B) FAK phospho-Y397 levels remain mostly unchanged under treatment with natural products. Overall FAK levels remain similar between treatments. (C) FAK phospho-Y576 is reduced when bladder cells are treated with CAPE, EGCG, catechin, or resveratrol. Overall FAK levels remain similar between treatments. 33 DMSO (Figure 2.6A) were characterized by the presence of lamellipodia and a large number of regular filopodia. In comparison, cells treated with CAPE (Figure 2.6B) showed no lamellipodia and very few, small filopodia. Catechin (Figure 2.6C) and EGCG-treated cells (Figure 2.6D) still had lamellipodia, but filopodia were reduced in number and had a crooked, irregular appearance. Resveratrol (Figure 2.6E) greatly reduced both lamellipodia and filopodia. As normal actin dynamics are critical for host cell invasion by UPEC strains, this finding points to a potential role for inhibition of FAK Y576 resulting in altered actin nucleation that ultimately manifests as changes in the morphology of lamellipodia and filopodia. Plant-derived phenolic compounds can also inhibit invasion by a variety of intracellular pathogens We wanted to further investigate other invasive pathogens to assess the ability of the plant-based compounds to inhibit association or invasion. 5637 cells were challenged with Salmonella enterica serovar Typhimurium, Shigella flexneri, or AAEC185/pRI203, a K12 strain encoding the Yersinia invasin, in an in vitro model of invasion. With S. Typhimurium (Figure 2.7A), there was no notable change in association and only resveratrol significantly inhibited invasion (Figure 2.7B). In the case of S. flexneri (Figure 2.7C) and AAEC185/pRI203 (Figure 2.7E), EGCG and catechin increased association with host cells. When normalized to association, we found that all but catechin reduced S. flexneri (Figure 2.7D) invasion while all plant-based compounds reduced invasion by AAEC185/pRI203 (Figure 2.7F). Only treatment of Salmonella with catechin (8.4%) and S. flexneri with EGCG showed any significant impact on survival at 34 Figure 2.6 Plant-based compounds show altered morphologies of filopodia and lamellipodia revealed by staining for actin (white). Bladder cells treated with (A) DMSO show wild type morphologies of both filopodia and lamellipodia. Bladder cells treated with (B) CAPE show reduced numbers of small filopodia and no lamellipodia. (C) Catechin and (D) EGCG induce reduced numbers of filopodia and remaining filopodia have altered morphologies. (E) Resveratrol treatment reduces filopodia and clusters them in small patches of lamellipodia. 35 Figure 2.7 Plant-based secondary metabolites reduce invasion by other pathogens in an in vitro model. (A) Salmonella association is not affected by any of the plant-based compounds, but (B) resveratrol does significantly reduce invasion into host cells. (C) Shigella association is higher with treatment of EGCG and catechin, but is not significant. (D) However, all compounds except catechin significantly reduce invasion. (E) AAEC185 expressing the Yersinia invasin associate at a significantly higher rate upon treatment with EGCG and catechin, but (F) all compounds significantly reduce host cell invasion. Bars indicate mean values ± SEM with n ≥ 3 independent experiments. p-values calculated by student’s t-test. 36 8.3% and 18.4% mean reduction, respectively (Figure 2.3). However, catechin did not reduce Salmonella invasion and EGCG treatment of Shigella actually increased association. These data demonstrate the robust ability of these compounds to inhibit invasion by a variety of pathogens. Resveratrol inhibits invasion of UTI89 into murine bladders Ultimately, the goal of this work was to identify if plant-based compounds could potentially be used to prevent or reduce bacterial burden in UTIs. Using an in vivo murine model of UTIs, we catheterized mice with UTI89 and either DMSO or resveratrol. Bladders were extracted, treated with gentamicin to kill extracellular bacteria, and the surviving intracellular bacteria were counted. Similar to what we had observed in vitro, bacterial numbers were significantly reduced in bladders treated with resveratrol (Figure 2.8). This result validates the in vivo effectiveness of resveratrol when directly instilled into bladders at the time of infection. Discussion The desperate need for new therapeutics to treat common bacterial infections gave rise to this study which looks at phenolic compounds found in food and drink. The activity of the chosen compounds was investigated in terms of the host instead of the bacteria, a strategy to reduce the opportunity for bacteria to develop resistance. This manuscript demonstrates that CAPE, resveratrol, and EGCG have anti-invasive properties in an in vitro model of UTI. This effect is not due to inhibition of NFκB, which is inhibited by CAPE, as 37 Figure 2.8 Resveratrol inhibits bacterial invasion into bladders in an in vivo model of UTI. Bladders were catheterized with approximately 10 7 CFU of UTI89 and either DMSO or resveratrol, extracted 1 hour later, and treated with gentamicin. Treatment with resveratrol significantly reduces invasion into bladder cells. Bars indicate median values with p-values calculated by Mann-Whitney Utest. n = 10-11 mice in two independent experiments. 38 treatment with a transcription inhibitor or protein synthesis inhibitor did not reduce invasion. Rather, inhibition of invasion is correlated with a reduction in phosphorylation of FAK Y576. Phosphorylation of FAK Y397, the residue that allows for binding of Src family kinases and leads to phosphorylation of FAK Y576, remained largely consistent during treatment. Treatment of BECs with CAPE, resveratrol, EGCG, or catechin altered the number and morphology of filopodia and lamellipodia, both actin-dependent structures. These findings suggest that the plant based compounds are inhibiting activation of FAK by blocking phosphorylation of key residues and altering actin nucleation. This finding agrees with previous findings that normal actin dynamics are important for host cell invasion by UPEC [51, 52]. Previous work using human breast cancer cell lines found that treatment with resveratrol actually induced extension of filopodia and a reduction in FAK Y397 phosphorylation, but only in cells expressing estrogen receptors [53]. This indicates that effects of these plant-based compounds are likely cell-specific. Expanding the scope of our pathogen to other invasive bacteria, we found that these compounds reduced invasion into host cells by Salmonella, Shigella, and a nonpathogenic K12 E. coli strain expressing the Yersinia invasin. The importance of actin dynamics during invasion has been previously implicated with each of these pathogens, agreeing with the inhibitory mechanism proposed in this manuscript [54-57]. When considering the role of filopodia in pathogenesis, previous work has shown that interaction with filopodia are important for invasion by Shigella and bacteria expressing the Yersinia invasin [58, 59]. Therefore, 39 disruption of filopodia would be expected to reduce invasion, which is what our results demonstrate. Importantly, we found that treatment of mouse bladders with resveratrol significantly reduced bacterial invasion in an in vivo model of UTI, providing valuable translational evidence of effectiveness. Overall, these results point to a role for the plant-based compounds to inhibit invasive pathogens by reducing phosphorylation of FAK Y576. This has the effect of reducing FAK activity, altering actin nucleation, and placing invasive pathogens at a disadvantage. In a host, these pathogens would be left exposed to antibiotics, antibodies, and immune cells when they otherwise would be protected in the intracellular niche. This is also an exciting finding as it could help reduce the number recurrent UTIs, a major issue that is likely contributing to the rise of antibiotic resistance [60, 61]. By targeting the host cell and reducing the quantity of antibiotics used during treatment, this strategy could slow the development of resistance. We demonstrated that co-catheterization with resveratrol reduced the number of invaded bacteria in mouse bladders, giving promising evidence that natural products can be developed for prophylactic and combination treatments. Future experiments should focus on the pharmacology of these compounds with the goal of adapting them into a form that would be present and active at relevant concentrations in target tissues. Ideally, the natural products would be in a stable form to make transport, ingestion, and adherence to a treatment plan by a patient as easy as possible. These results further validate traditional medicines in a time where 40 antibiotic resistance is spreading rapidly. As we look for new treatments, traditional medicines may hold critical therapeutic tools to combat invasive bacterial pathogens. References 1. 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CHAPTER 3 HISTONE DEACETYLASE 6 REGULATES BLADDER ARCHITECTURE AND HOST SUSCEPTIBILITY TO UROPATHOGENIC ESCHERICHIA COLI Copyright © MDPI, Pathogens, 5, 20, 2016, 1-11, doi: 10.3390/pathogens5010020 Reprinted with permission 48 49 50 51 52 53 54 55 56 57 58 CHAPTER 4 TOLL-LIKE RECEPTOR 5 SIGNALLING PROMOTES PERSISTANCE BY UROPATHOGENIC ESCHERICHIA COLI IN THE BLADDER AND KIDNEY 60 Introduction Urinary tract infections (UTIs) are one of the most prevalent bacterial infections to afflict humans [1]. Uropathogenic Escherichia coli (UPEC) are the primary causative agent. Even when the patient is cleared of UTI symptoms through the use of antibiotics, there is a 25% chance of reinfection within 6 months of the primary infection [2]. This recurrent infection is disproportionately due to infection with the same strain as the primary infection, suggesting that the bacteria responsible for the primary infection are often not sufficiently cleared by the combination of antibiotics and the host immune system [3, 4]. Detection of UPEC within the bladder is mediated by pattern recognition receptors including Toll-Like Receptor (TLR) 4 and TLR5 [5, 6]. Mice lacking TLR4 were previously shown to have higher bladder titers during UTI, suggesting that clearance of UTIs is in part reliant on TLR4-mediated proinflammatory signaling [5, 7]. Inflammatory signaling mediated through TLR5 is likely more nuanced, as demonstrated in published research showing that flagella can induce strain-dependent inflammation in both zebrafish and mice [8]. Flagella are a bacterial motility organelle that have been shown to play a role in kidney colonization during UTI [9, 10]. In mice, flagella are detected by TLR5 and TLR11, though TLR11 is a pseudogene in humans that is not expressed [11]. In epithelial cells, binding between TLR5 and flagella activates nuclear factor kappa B (NFκB) and induces pro-inflammatory cytokines like IL-1β and IL-8 [12, 13]. Generally, detection of bacteria within the urinary tract triggers activation of the host innate immune system, which includes activation of bladder 61 resident macrophages leading to the recruitment of circulating macrophages [14]. In turn, recruited macrophages license the recruitment of neutrophils, which are the primary phagocyte at early time points. As the infection progresses, macrophages take a more prominent role in combating UPEC as neutrophils numbers are reduced within the bladder. Despite the influx of innate immune cells, there does not appear to be an appreciable role for the adaptive immune system during the primary infection as mice lacking T cells and B cells have similar bacterial burdens during UTI [15, 16]. Investigation into the development of adaptive immunity during UTI has suggested that this may be due to antigen sequestration by macrophages, which prevent proper presentation to adaptive immune cells [16]. Proper signaling by pattern recognition receptors has been shown to be important for clearance of UTIs. Blocking recruitment of neutrophils, ablation of macrophages or improper TLR4 signaling has been demonstrated to increase bacterial burdens in the bladder, likely due to a reduced proinflammatory response [5, 14, 17]. However, the presence of high levels of proinflammatory cytokines has been shown to be a risk factor for bacterial persistence in the bladder [17]. The overall bladder immune response appears to be inadequate under normal conditions. Blocking recruitment of neutrophils or macrophages to the bladder has either no or only modest increases in titers and, in the case of macrophages, may improve outcomes during a secondary infection. Adaptive immune cells also seem to have a modest effect on the secondary infection. 62 Reducing proinflammatory cytokines increases acute titers, but increasing proinflammatory cytokines increases the likelihood of persistence within the bladder. These seemingly contradictory findings can be reconciled if the quality of inflammation is considered instead of the quantity. With this framing of the host-pathogen interaction during UTIs, inflammation is not a matter of too much or too little, but rather the proper tuning of the immune system for complete clearance of the pathogen. This chapter investigates the role of TLR5-mediated signaling during UTIs and presents evidence that signaling through TLR5 induces a quality of inflammation that promotes bacterial persistence. Materials and Methods Bacterial and mouse strains The pyelonephritis strain CFT073 and cystitis strain F11 were obtained from the lab of Harry Mobley at the University of Michigan. CFT073ΔfliC::kanR and F11ΔfliC::kanR were generated and described previously [8]. Bacterial cultures for bladder catheterizations were grown from frozen stocks in 20 mL Lysogeny Broth (LB; Fisher Scientific) or M9 minimal media static cultures at 37°C. C57BL/6J and TLR5 knockout mice (TLR5 -/-) in the C57BL/6J background were obtained from The Jackson Laboratories. All mice used were between 7-10 weeks old. Mice were genotyped using primers and thermocycler protocols described by The Jackson Laboratories. Animals were used in accordance to protocols approved by the Animal Studies Committee at the University of Utah. 63 Bladder catheterization Mice were anesthetized by isoflurane inhalation and catheterized with approximately 107 CFU of bacteria in a volume of 50 µL PBS. Mice were then returned to their cages to recover. At the indicated time points, mice were killed by cervical dislocation under anesthesia. Bladders and kidneys were aseptically removed, quartered, and placed in microcentrifuge tubes with PBS. Bladders and kidneys were homogenized using 3.2 mm stainless steel balls and a BulletBlender Storm 24 tissue homogenizer (NextAdvance). Serial dilutions of tissue homogenates were plated on LB agar and counted to enumerate bacteria. Data was collected from 2 or more independent assays. RNA sequencing and analysis Wild type and TLR5 -/- mice were infected with F11 and the infection was allowed to proceed until the indicated time points. Mice were killed by cervical dislocation under anesthesia and bladders extracted. Bladders were processed for RNA using either the Norgen Total RNA Isolation Kit (Norgen Biotech) or the GenElute Total RNA Isolation Kit (Sigma) according to the manufacturer’s protocol. RNA was submitted to the University of Utah High Throughput Genomics Core and libraries were prepared using RiboZero treatment. Sequencing was performed with Illumina TruSeq Stranded RNA-seq in 50 singleend cycles. Sequences were analyzed with assistance from the University of Utah Bioinformatics Core. Pathways were investigated using Qiagen Ingenuity Pathway Analysis (IPA) with a false discovery rate cutoff of 0.05. 64 Statistics Prism 6.07 (GraphPad Software) was used for statistical testing in all experiments other than RNA-seq. p values of less than 0.05 were considered significant. Results TLR5 -/- mice are more resistant to UTIs Though TLR5 -/- mice lack one of their extracellular flagellin receptors, they maintain the presence of TLR11 (another extracellular flagellin receptor) and NLRC4 (an intracellular flagellin receptor). We hypothesized that without a full complement of flagellin receptors, mice would be less able to contain a urinary tract infection. To test this, we catheterized wild type (WT) and TLR5 -/- mice with the F11 cystitis strain and extracted bladders and kidneys 1 or 5 days later. At 1day postinoculation, WT and TLR5 -/- bladder titers were similar between the genotypes while kidneys showed significantly reduced titers in TLR5 -/- mice (Figure 4.1A). At 5-days postinoculation, we found that TLR5 -/- mice had significantly reduced titers in both the bladder and kidney when compared to wild type mice (Figure 4.1B). To confirm that this phenotype was not strain-specific, we repeated the experiment with the CFT073 pyelonephritis strain and enumerated titers at 5-days postinoculation. Though the magnitude of the difference was more modest, we found that TLR5 -/- bladder and kidney titers were significantly reduced in comparison to wild type (Figure 4.1C). This demonstrates that TLR5 -/- mice are better able to combat bacterial persistence within the urinary tract and that the phenotype does not appear to be strain 65 Figure 4.1 Bladder and kidney TLR5 increases susceptibility to infection by two different strains of UPEC. WT and TLR5 -/- mice were catheterized with F11 and bladders and kidneys titered (A) 1-day or (B) 5-days postinoculation. Bladders showed similar titers at 1 day, though kidneys had significantly reduced colonization in TLR5 -/- mice. (C) WT and TLR5 -/- mice were catheterized with CFT073 and bladders and kidneys titered at 5-days postinoculation. Titers were significantly lower in both bladder and kidneys of TLR5 -/- mice. n ≥ 8 mice catheterized in at least 2 independent experiments. Bars indicate median values. Significance calculated using a Mann-Whitney nonparametric U-test. p-values ≤ 0.05 were considered significant. 66 specific. Reduced bacterial titers in TLR5 -/- is independent of flagellin Since TLR5 is a flagellin receptor, we hypothesized that TLR5 -/- mice infected with CFT073 lacking flagellin would no longer show lower titers than wild type mice. To test this, we infected WT and TLR5 -/- mice with the CFT073ΔfliC pyelonephritis strain lacking the fliC component of flagellin, and extracted their bladders and kidneys 5 days later. To our surprise, TLR5 -/- mice maintained lower titers in both bladder and kidneys when compared to wild type mice (Figure 4.2A). Further, comparing CFT073 and CFT073ΔfliC infections in either wild type (Figure 4.2B) or TLR5 -/- (Figure 4.2C) mice revealed no significant difference, suggesting that flagella may not play an important role in bladder pathogenesis, as has been previously published [9, 10], but rather TLR5-mediated signaling is promoting bacterial persistence within the urinary tract. RNA-seq results suggest differential involvement of the immune system in TLR5 -/- mice Our previous experiments demonstrated that lack of the TLR5 receptor was beneficial to the host in the context of bladder and kidney infections, regardless of whether the bacteria actually produced flagella. One possibility to explain this difference is that knockout of TLR5 results in differential regulation of other TLRs. This phenotype has been observed with TLR7 and 8, in which knockout of TLR8 results in upregulation of TLR7 [18]. Another possibility is that knockout of TLR5 does not affect the TLR complement on the cell surface, but 67 Figure 4.2 CFT073 lacking flagella does not bring unity in titers between WT and TLR5 -/- mice. (A) WT and TLR5 -/- mice catheterized with CFT073ΔfliC showed significant differences in both bladder and kidney titers 5-days postinoculation with TLR5 -/- maintaining lower titers. When CFT073 and CFT073ΔfliC titers were compared between (B) WT or (C) TLR5 -/- mice, no significant difference was observed in either bladder or kidney titers. n ≥ 8 mice catheterized in at least 2 independent experiments. Bars indicate median values. Significance calculated using a Mann-Whitney nonparametric U-test. p-values ≤ 0.05 were considered significant. 68 rather signaling occurs preferentially through existing TLRs. To gain a global perspective, we infected WT and TLR5 -/- mice with F11, isolated RNA from bladders at 1- and 5-days postinoculation, and performed RNA-seq. At 1-day postinoculation, 36 genes were significantly downregulated while 37 genes were significantly upregulated in TLR5 -/- when compared to WT. Many of these genes are involved in innate immunity and may hold clues to the mechanism behind TLR5 -/- phenotype (Table 4.1). For example, emr1 (F4/80), a marker of macrophages in mice, and lyz2 (lysozyme 2), an antimicrobial enzyme, were both upregulated in TLR5 -/- mice. Also observed is the upregulation of csf1r (macrophage colony-stimulating factor 1 receptor), which regulates macrophage differentiation and function. Notably missing from the list of differentially regulated genes was the presence of any cytokines normally associated with UTIs. In fact, the only cytokine with any significant differential regulation was IL-33. IL-33 has been found to be upregulated during UTIs through TLR4-signaling, though its role in the bladder has not been determined [19]. Additionally, with the exception of TLR5, no TLR demonstrated significantly different expression levels in TLR5 -/- mice. To gain better insight of the pathways that were differentially regulated between WT and TLR5 -/- mice, we used IPA software to analyze our RNA-seq data. Interestingly, pathways involving increased proliferation and differentiation of immune cells, inflammation, and immune cell trafficking were predicted to be significantly upregulated. Further, pathways representing myeloid and lymphoid cell death were predicted to be downregulated. These results suggest that in 69 Table 4.1 Differentially regulated genes at day 1 postinfection Gene Name log2 Fold Change Tlr5 -1.500 Pydc3 -1.030 Per1 -0.803 Mmrn1 -0.744 Plk5 -0.740 Gm4956 -0.709 Per2 -0.666 Usp2 -0.666 Cys1 -0.661 Ide -0.660 Fgfbp3 -0.635 Olfr1033 -0.610 Hlf -0.596 Ttr -0.590 Lonrf3 -0.584 Kcnq5 -0.570 Reln -0.559 Dock3 -0.540 Csmd3 -0.525 Pcdh9 -0.520 Cdh19 -0.486 Fgl2 -0.474 Tsc22d3 -0.458 Snhg11 -0.457 Abca8a -0.450 Zbtb16 -0.447 Igf2 -0.428 Exph5 -0.428 Abca8b -0.425 Trpm8 -0.422 Hcn1 -0.405 Unc5c -0.392 Shank2 -0.381 Plagl1 -0.375 Tacc2 -0.329 Ssfa2 -0.286 KO1 KO2 KO3 KO4 KO5 WT1 WT2 WT3 WT4 WT5 70 Table 4.1 Continued Gene Name log2 Fold Change Itga6 0.252 Btg1 0.260 Prdx1 0.284 Tmsb4x 0.318 Csf1r 0.336 Cadm3 0.346 Maged2 0.361 Pcolce 0.387 Ctsc 0.387 Mgst3 0.387 St6gal1 0.390 Pdia6 0.394 H2-Aa 0.413 Cfp 0.420 Mcm7 0.425 Arsi 0.431 Krt15 0.432 Cotl1 0.438 Gm20390 0.439 C1qa 0.460 Nme2 0.468 Ifi27l2a 0.475 Lgmn 0.487 Pf4 0.502 Krt5 0.510 Hcar2 0.517 NA 0.524 Adamts15 0.529 Fbn1 0.538 Il33 0.543 Clec11a 0.546 Lyz2 0.547 Tyrobp 0.561 Hist1h1b 0.597 Hist1h1d 0.613 Diras2 0.627 Emr1 0.669 KO1 KO2 KO3 KO4 KO5 WT1 WT2 WT3 WT4 WT5 71 TLR5 -/- bladders there may be a larger and more sustained immune response than in WT bladders. At 5-days postinoculation, 11 genes were significantly downregulated and 34 genes were significantly upregulated (Table 4.2). Though some immunerelated genes were identified as being differentially regulated, no clear trend emerged. Of note, nos1, a nitric oxide synthase gene, was significantly downregulated in TLR5 -/- while dll1, a gene expressed in maturing T-cells, was significantly upregulated. The use of IPA software also failed to identify any clearly relevant pathways that were differentially regulated between WT and TLR5 -/- bladders. These results suggest that by day 5, the expression profiles between WT and TLR5 -/- mice are similar despite the significantly lower bacterial titers in TLR5 -/- mice. Discussion Flagella are important for bacterial motility and, in the context of UTIs, has been previously shown to promote bacterial ascension to the kidneys [9, 10]. Previous work using TLR5 -/- mice and CFT073 in a UTI model demonstrated that although titers in both the bladder and kidney were similar at early time points, at day 5 there was a significant resurgence of bacteria in both organs of knockout mice [6]. In our hands, repeating previously published experiments using the same strains of mice and bacteria (CFT073) demonstrated that bladders and kidneys of TLR5 -/- mice had significantly lower titers at 5 days postcatheterization (Figure 4.1C). We confirmed this finding using a second strain of bacteria (F11; Figure 4.1A and B) showing that our findings were not 72 Table 4.2 Differentially regulated genes at day 5 postinfection Gene Name log2 Fold Change Pydc3 -1.277 Tlr5 -1.108 Ide -0.927 Fgfbp3 1500011K16Rik -0.710 Nos1 -0.649 Rps24-ps3 -0.648 Itpa -0.639 Rps24 -0.577 Clec2d -0.461 Btaf1 -0.340 -0.671 Sqstm1 0.208 Itfg3 0.233 Dcaf7 0.259 Kpna6 0.261 Copa 0.284 Uba1 0.290 Supt5 0.291 App 0.299 Tnpo2 0.302 Flii 0.303 Snx33 0.303 Dcaf5 0.308 Tmem63a 0.339 Os9 0.347 Zfp652 0.348 Trim28 0.371 Lmna 0.382 Actb 0.403 Ldlr 0.410 Lamb1 0.412 Tesk1 0.414 Ggcx 0.427 Wdr6 0.440 Loxl1 0.471 Lamb2 0.471 KO1 KO2 KO3 KO4 KO5 KO6 WT1 WT2 WT3 WT4 WT5 73 Table 4.2 Continued Gene Name Cbx4 log2 Fold Change 0.484 Gpc3 0.486 Fbln2 0.491 Zfp574 0.527 Pigt 0.561 Flnc 0.607 Dll1 0.618 Cybrd1 0.623 Itgbl1 0.695 KO1 KO2 KO3 KO4 KO5 KO6 WT1 WT2 WT3 WT4 WT5 74 strain specific, but are instead due to the specific genotypes of the mice. This suggests that our phenotype is valid and that the previously published research may be due to procedural differences. Most notable is the catheterization procedure in which our mice were catheterized and returned to their cage for recovery while the previous authors catheterized their mice and sealed the urethra using collodion, eventually unsealing it after 6 hours. Also, comparisons with their data findings on 5 day titers in WT mice in which bacteria are cleared from the bladder are not repeated in the literature with C57BL6/J and CFT073. Regardless, the ability to repeat the results found in TLR5 -/- mice with two strains of bacteria suggests a role for TLR5-mediated signaling that results in bacterial persistence. Our findings show that TLR5 detrimentally affects the ability of the host to clear bacteria from the urinary tract. Particularly surprising in these findings is the discovery that the lack of flagellin expression by bacteria did not equalize organ titers between WT and TLR5 -/- mice, contrary to our hypothesis. This suggests that TLR5-mediated sensing of flagellin is not important, but rather other TLR5-mediated functions in the presence of bacteria is important. This could be through freeing of TLR4 in the TLR4/5 heterodimer and altering the overall immune response [20]. RNA-seq results from bladders isolated at 1-day post-inoculation suggest that TLR5 is altering the recruitment of immune cells to the bladder as well as the quality of inflammation in a manner that prevents resolution of the infection. The only cytokine significantly upregulated in the TLR5 -/- bladder is IL-33, whose 75 role in UTIs has not been defined, but may be shaping the immune response to promote bacterial clearance. Pathway analysis of the results predicts that immune cell recruitment and survival is increased as well as inflammation, suggesting that the quality of the immune response in TLR5 -/- bladders is improved. At 5-days postinoculation, there are fewer differentially regulated genes and analysis fails to identify any pathways that could be contributing to our observed phenotype. An interpretation of this finding is that by day 5, WT and TLR5 -/- bladders are responding similarly to the infection despite WT bladders having significantly greater bacteria. More widely, similar expression profiles in the presence of significantly different levels of bacteria within the urinary tract could be reflective of the high recurrence of UTIs in human patients and the failure of the immune system to fully resolve the infection. Future experimentation will focus on validating and further exploring the RNA-seq data to elaborate the immune response at early time points. It is not surprising that an event occurring early in the infection does not impact bacterial titers until later, so elaborating that event is critical in identifying the mechanism behind the phenotype of lower titers in TLR5 -/-. Preliminarily, bladders will be infected, extracted at early time points, and fixed for sectioning. Hematoxylin and eosin staining will be used to assess gross inflammation as well as general localization of infiltrating immune cells. This infiltrate will be characterized using previously established methods in which bladders will be infected and extracted at different time points for analysis by flow cytometry and immunofluorescence 76 [21]. Specifically, antibodies against both myeloid and lymphoid populations will be used to characterize recruitment to the bladder during UTI. Macrophage-related genes were notably upregulated in the TLR5 -/bladders at 1-day post infection, suggesting a role for macrophages. TLR5 -/mice will be treated with clodrolonate liposomes to deplete macrophages and bladders will be catheterized. If macrophages are the key to bacterial clearance, the expectation is that macrophage-depleted TLR5 -/- mice will have elevated levels of bacteria in the bladders at day 5. It is also possible that the critical cell type to bacterial clearance is actually somatic rather than hematopoietic. By creating bone marrow chimeras (TLR5 -/- with WT bone marrow and vice versa), the critical cell compartment could be identified. IL-33 was the only cytokine whose expression was significantly altered in TLR5 -/- mice. The role of IL-33 in bladder infection will be further described by treating WT mice with IL-33 or treating TLR5 -/- mice with an IL-33 neutralizing antibody. If IL-33 is playing a critical role in bacterial clearance, the expectation is that IL-33-treated mice would show bladder titers more similar to TLR5 -/- mice. Likewise, neutralizing IL-33 in TLR5 -/- mice would cause bladder titers to resemble WT mice. Findings from these experiments could suggest new clinical treatments with the goal to fully resolve UTIs in patients. Incomplete resolution of UTIs is likely leading to recurrence and, therefore, spread of antibiotic resistance through repeated use [3, 22]. Development of new therapies could give new life to existing treatments and slow the development of resistance mechanisms within 77 UPEC by manipulating the host immune response. References 1. Foxman B, Barlow R, D'Arcy H, Gillespie B, Sobel JD. Urinary tract infection: Self-reported incidence and associated costs. Ann Epidemiol. 2000;10(8):509-15. 2. Foxman B, Brown P. Epidemiology of urinary tract infections: Transmission and risk factors, incidence, and costs. Infect Dis Clin North Am. 2003;17(2):227-41. doi: 10.1016/s0891-5520(03)00005-9. 3. Russo TA, Stapleton A, Wenderoth S, Hooton TM, Stamm WE. 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Pulsed-field gel electrophoresis typing of Escherichia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women. J Clin Microbiol. 2006;44(5):1776-81. doi: 10.1128/JCM.44.5.1776-1781.2006. PubMed PMID: 16672406; PubMed Central PMCID: PMCPMC1479185. CHAPTER 5 DISCUSSION 81 The prevalence of UTIs, high rates of recurrence, and spread of antibiotic resistance amongst UPEC creates a dire global health condition. Failure to properly treat UTIs raises the risk of renal scaring and urosepsis, particularly in pediatric and elderly patients. Therefore, new treatment methods must be rapidly identified and verified to avoid total therapeutic failure. The findings presented within this body of work demonstrate that targeting different stages of UTI may lead to positive patient outcomes. In conjunction with standard antibiotic treatments, these combination therapies may give physicians the edge. Since invasion by UPEC is necessary for initiation and continuation of pathogenesis, the host-based mechanisms required for invasion provide therapeutic targets. To this end, Chapters 2 and 3 investigated how manipulation of the cytoskeleton in host cells could impact invasion during a UTI. Chapter 2 investigated the effect of common plant phenolic compounds on the ability of UPEC to invade host cells. In our in vitro model of UTI we found that CAPE and resveratrol significantly inhibited invasion into host cells. When we tested these findings in an in vivo model using resveratrol, we were successfully able to recapitulate our in vitro results. We pursued these findings and discovered that treatment with the plant phenolic compounds were correlated with reducing activating phosphorylation event of FAK. This was observed morphologically as the BECs qualitatively demonstrated altered filopodia and lamellipodia, both actin-dependent structures. Likewise, we found invasion by Shigella flexneri, Salmonella Typhimurium, and a K12 strain expressing the Yersinia invasin could be inhibited by our plant phenolic compounds. This finding demonstrates the 82 usefulness of plant phenolic compounds in the prevention of disease, especially as the plant compounds are commonly consumed by humans. Further work would characterize other pathogens that may be susceptible to these compounds as well as similar plant-derived compounds. Ideally, diets could be “prescribed” for at-risk populations to minimize disease. Chapter 3 focused on another part of the cytoskeleton: the microtubules. Previous work found that inhibiting HDAC6, which deacetylates microtubules, significantly reduced invasion in an in vitro model of UTI. We extended these findings into an HDAC6 -/- mouse line and found that while MEFs derived from the knockout mice were resistant to invasion, the mice themselves were actually more susceptible to invasion in an in vivo model of UTI, though titers normalized between WT and HDAC6 -/- as early as 6 hours postinoculation. We identified that HDAC6 -/- bladders had altered physiologies with greater maximum capacities and also had thicker muscle layers. These results suggest that HDAC6 is involved in the development of normal bladder architecture and that the abnormal bladder physiology contributes in some part to the higher invasion rates. Neutrophils recruited to bladder at early time points contained greater numbers of bacteria. The phenotype observed in HDAC6 -/- neutrophils may be due to slower recruitment to the bladder, giving less time for clearance when we assayed their bacterial content. Higher titers within the neutrophils may also be due to slower killing. It is also possible that the elevated titers found in HDAC6 -/bladders are due to increased phagocytosis of bacteria and the urothelium does 83 not play a major role in the phenotype. These findings better describe how HDAC6 is involved in the bladder, though it does not provide a clear path for treatment. Further investigation of posttranslational modifications of microtubules and their associated motors may provide clearer target to combat bacterial invasion into host cells. Finally, Chapter 5 looked at how persistence within the bladder was impacted by TLR5-mediated detection of bacterial flagellin. We had hypothesized that TLR5 -/- mice would be more susceptible to bacteria, but instead discovered that bacterial titers were lower in both the bladder and kidney at 5 days. This was not strain-specific, as another UPEC strain showed a similar phenotype in the bladder. TLR5 is canonically identified as a receptor of bacterial flagella, though using UPEC strains lacking flagella recapitulated bacterial quantities when wild type UPEC strains were used in either WT or TLR5 -/- mice. This may reflect TLR5 detecting an uncharacterized bacterial antigen. RNA-seq of samples 1 day postinoculation revealed that that there were strong associations with pathways related to immune cell infiltration, differentiation, and survival in TLR5 -/bladders. We also observed a number of macrophage-associated genes upregulated in TLR5 -/- bladders. At 5 days, no clearly differentially regulated pathways were identified, suggesting that WT and TLR5 -/- bladders are acting similarly, despite WT bladders harboring significantly greater numbers of bacteria. This suggests that TLR5-induced inflammation places the host at a disadvantage for bacterial clearance and presents a tantalizing target. By blocking signaling through TLR5, bacterial persistence could be reduced. The 84 immune signaling revealed by the RNA-seq data suggests that potentially administering IL-33 could promote a better quality of immune recruitment and promote bacterial clearance. The goal of this work was to identify host factors that could be targeted to prevent or treat UTIs. Targeting host factors is potentially less likely to generate resistance mechanisms in pathogens and, in combination with traditional antibiotics, could reduce the recurrence rate in patients. Through preventing invasion into the bladder through targeting of cytoskeletal components, bacteria can be stymied at one of the earliest stages of infection. Later stages of infection could be better resolved through manipulation of the host immune response by ensuring a better quality of inflammation. Together, these therapies could bring the startling rise of antibiotic resistant UPEC under control and reduce disease burden, cost, and suffering of patients. APPENDIX HOST CELL INVASION BY PATHOGENIC ESCHERICHIA COLI Lewis, A. J., et al. (2014). Host Cell Invasion by Pathogenic Escherichia coli. Pathogenic Escherichia coli. S. Morabito. Great Britain, Caister Academic Press: 231-254. Reprinted with permission 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109