DEFINING THE INTERPLAY BETWEEN BACTERIAL PORE-FORMING

TOXINS AND HOST PROTEASES

by

Elizabeth M Enrico

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 2019 Copyright © Elizabeth M Enrico 2019

All Rights Reserved The University of Utah Graduate School

STATEMENT OF DISSERTATION APPROVAL

The dissertation of Elizabeth M Enrico has been approved by the following supervisory committee members:

Matthew A. Mulvey , Chair 4-25-2019 Date Approved

Markus Babst , Member 4-25-2019 Date Approved

Diane M. Ward , Member 4-25-2019 Date Approved

Jessica Brown , Member 4-25-2019 Date Approved

Ryan O’ Connell , Member Date Approved

and by Peter E. Jensen , Chair/Dean of the Department/College/School of Pathology and by David B. Kieda, Dean of The Graduate School.

ABSTRACT

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), gram-negative bacteremia, and urosepsis. More than half

of UPEC isolates secrete a pore-forming called alpha- (HlyA). At the molecular level, HlyA intoxication of cells activates proteolytic and signaling cascades such as MAP kinase, caspases, and more recently discovered serine proteases. Previous work by our lab demonstrated that serine proteases, in particular Trypsinogen 4 (Try4), were activated in response to HlyA intoxication and trafficked into the nucleus. Here I present work that begins to determine how

HlyA promotes the activation of the host serine protease Try4 and also how Try4 traffics into the nucleus. Using confocal microscopy and Try4 truncations

mutants, I discovered that the mesotrypsin core domain was necessary and

sufficient for Try4 translocation into the nucleus. We identified that Try4 trafficked

into the nucleolus, a subnuclear organelle. Inhibitor and drug studies in bladder

epithelial cells showed that Try4 trafficking into the nucleolus required potassium

efflux. Using mass spectrometry, we discovered potential Try4 interacting

partners during HlyA intoxication. One interacting partner was identified as

caspase-14, a relatively unknown caspase. Using caspase-14 over-expression

constructs and confocal microscopy, I observed that Try4 trafficking into the

nucleolus was significantly increased by caspase-14 over-expression. To ascertain the relevance of Try4 activation in vivo, we used two models of infection a UTI and sepsis. We found that inhibiting activation of Try4 was beneficial to the host, resulting in increased survival during a sepsis challenge and decreased inflammation during a UTI. At the cellular level, these studies begin to elucidate the intracellular mechanism of PFT-mediated activation and recruitment of Try4 into the nucleolus. On the organismal level, my work reveals that HlyA-mediated activation of Try4 is harmful to the host.

iv

TABLE OF CONTENTS

ABSTRACT ...... iii

LIST OF FIGURES ...... vii

LIST OF ABBREVIATIONS ...... viii

ACKNOWLEDGEMENTS ...... ix

Chapters

1-INTRODUCTION ...... 1

References ...... 19

2-PRODDING YOUR INNER DEMONS: PORE-FORMING ACTIVATE HOST PROTEASES ...... 27

Introduction ...... 28 Calpains: More Than Just Dynamics ...... 30 Cathepsins: Suicide Bags or Friends? ...... 34 Furin: The Traitor Within ...... 41 Trypsins and Mysterious Proteases ...... 42 Future Perspectives ...... 49 References ...... 50

3-BACTERIAL PORE-FORMING TOXINS PROMOTE THE NUCLEOLAR DETENTION OF HOST SERINE PROTEASE TRYPSINOGEN 4 ...... 65

Introduction ...... 66 Results ...... 68 Alpha-hemolysin Is Necessary for Nucleolar Localization of Try4 . 68 The Mesotrypsin Core Is Required for HlyA-Mediated Try4 Nucleolar Trafficking ...... 69 PFT-Mediated Nucleolar Localization of Try4 Does Not Require Its Activation or Enzymatic Activity ...... 71 Potassium Fluxes Are Necessary and Sufficient for Nucleolar Localization of Try4 During Intoxication ...... 73 The Karyopherin Importin-β Mediates the Nuclear Import of Try4 . 74 Caspase-14 Facilitates Try4 Trafficking to the Nucleolus ...... 75 Inhibition of Try4 is Beneficial to the Host in Models of UTI and Sepsis ...... 76 Discussion ...... 78 Materials and Methods ...... 83 , Cell Culture, and Drugs ...... 83 Cell Culture and Western Blotting ...... 84 Trypsinogen4-EGFP Cloning ...... 86 Immunofluorescence/Microscopy ...... 86 Nucleolar Puncta Quantification ...... 87 Immunoprecipitation ...... 88 Mouse UTI Model ...... 89 Histology ...... 90 Mouse Sepsis Model ...... 91 References ...... 91

4-DISCUSSION ...... 120

The Interplay Between Host Proteases and Pore Forming Toxins ...... 121 Trypsinogen 4: An Unusual Serine Protease Living a Double Life ...... 123 Nucleolar Detention: Helpful or Harmful? ...... 129 References ...... 132

vi LIST OF FIGURES

1.1-Trypsinogen 4 Is Encoded by the PRSS3 Gene ...... 18

3.1-Alpha-hemolysin Intoxication of BECs Promotes the Re-localization of Trypsinogen 4 into Nucleolar Puncta ...... 98

3.2-The Mesocore Is Necessary and Sufficient for Targeting of Try4 into the Nucleolus ...... 100

3.3-Try4 Trafficking into the Nucleolus Is Independent of Its Activation or Catalytic Function ...... 102

3.4-Potassium Efflux Is Necessary and Sufficient for Mediating Try4 Localization into the Nucleolus ...... 106

3.5-Try4 Trafficking into the Nucleus Is Mediated by Importin β ...... 108

3.6-Caspase-14 Facilitates Try4 Trafficking into the Nucleolus ...... 110

3.7-Inhibition of Try4 During UTI or Sepsis Protects the Host from Inflammation and Death ...... 112

3.8-Working Model of the Intracellular Trafficking of Try4 in Response to HlyA Intoxication ...... 115

S.3.9-UTI89 Is More Sensitive to Diminazene than F11 ...... 117

LIST OF ABBREVIATIONS

ExPEC ...... extraintestinal pathogenic E. coli HlyA ...... α-hemolysin LF/EF ...... lethal factor/ edema factor LLO ...... LMP ...... lysosomal membrane permeabilization MAPK ...... mitogen-activated kinase NLRP3 ...... NLR family pyrin domain containing 3 NLS ...... nuclear localization signal PCD ...... programmed cell death PFT ...... pore-forming toxin PFP ...... pore-forming protein PLY ...... pneumolysin RTX ...... repeat-in-toxin Try4 ...... Trypsinogen 4 UPEC ...... uropathogenic E. coli UTI ...... urinary tract infection WT ...... wild type

ACKNOWLEDGEMENTS

I want to thank Dr. Matthew Mulvey for his mentorship and support during my graduate career. He helped me through some difficult times both scientifically as well as personally, and for that I will always be grateful. I also want to acknowledge past and present members of the Mulvey lab for their support and friendship throughout the years. Without my fellow lab members science is so much harder to do. To all the graduate students who entered the MB/BC program around when I did, your friendship and support made graduate school so much fun. I am incredibly grateful to my friends from college who patiently waited for me to return to Utah. Their understanding, friendship, love, and children were big reasons that I made it through my PhD. Finally, I want to thank my mother and my father for instilling in me a deep love of science and for always believing in me even when I did not.

CHAPTER 1

INTRODUCTION 2

Every day we interact directly with many different types of bacteria; some are harmless commensals, and others are pathogens. The first step in causing an infection is to colonize the host and gain access to restricted tissues. Our most fundamental defense mechanism against bacterial colonization is the skin and mucosal surfaces. If the pathogen can penetrate and colonize host tissues, the next defensive step is activation of the host . The human immune system is made up of two arms: the innate and adaptive. They are crucial in mounting an effective response to invading pathogens.

The innate immune system is the generic first response to invading pathogens. These signals can come from molecules released by injured tissue, toll-like receptor (TLR) detection of pathogen-associated molecular patterns

(PAMPs), and immune signaling molecules (chemokines and cytokines) secreted by tissue-resident immune cells and epithelial cells. These signals serve to recruit leukocytes, i.e., neutrophils, macrophages, and dendritic cells, into the site of infection to begin bacterial clearance.

On the other hand, the adaptive immune response is highly specific to a particular pathogen. Cytokines and chemokines secreted by the innate immune cells prime and signal to lymphocytes (T cells and B cells) what type of pathogen, i.e., intracellular, extracellular, or parasitic, has infected the host. These signals are crucial for the recruitment, activation, and maturation of the appropriate T and

B cell types. Importantly, after the infection has been cleared, a subset of T and

B cells will enter into a memory state which can protect against the same pathogen in the future. 3

The human immune system is very efficient at detecting, isolating, and clearing bacterial infections. As such, pathogens have devoted a tremendous amount of their genomes to encoding factors that promote the destruction of tissue barriers and subversion of the immune response. Bacterial pathogens have impressive suites of virulence factors that are often necessary for causing infection. These factors facilitate various aspects of the infection, such as adherence and invasion, metabolite acquisition, preventing , and toxins. The most common type of toxins used by bacterial pathogens to promote dissemination and modulate the immune response is a type of pore-forming protein (PFPs) called pore-forming toxins (PFTs)1,2.

There are examples of PFPs in all five kingdoms of life, but their functions can vary significantly3. Interestingly, studies have identified conserved structural domains of PFTs, such as the those from Cholesterol-Dependent Cytolysins, that are present in immune system C8 and C9 from the complement cascade and perforin4,5. Moreover, in other bacterial species, , and eukaryotes conserved structures from the PFT aerolysin from Aeromonas species have been identified6-8.

PFTs are the most common type of PFPs, and they segregate into two classes based on the secondary protein structure that insert into the plasma membrane, including α-helices (α-PFT) and β-barrels (β-PFT). PFTs classification is further broken down by common protein domains or motifs.

These motifs, for example, the aerolysin domain or the RTX domain, are found in the founding members of their particular PFT families. There are six PFT 4 families, three α-PFTs and three β-PFTs. The colicins, actinoporins, and cytolysin A belong to the α-PFTs, while the , aerolysins, and cholesterol-dependent cytolysins (CDCs) are β-PFTs9. There is one other family of PFTs called the repeat in toxin (RTX) family10 that has been troublesome to classify by the conventional designations and therefore often remains unclassified. The RTX domain is a protein motif of repeating arrays of glycine and aspartate-rich nonapeptide amino acid repeats that binds calcium11.

As their namesake suggests, PFTs form pores in plasma membranes.

Damaging the host’s cell wall causes the immediate fluxes of cations Ca2+ and K+ in particular. Loss of ionic homeostasis immediately results in the activation of phosphorylation cascades such as mitogen-activated kinase pathways (MAPKs) of p38, JNK, and ERK12-14. Cells also initiate Ca2+-dependent membrane repair to mitigate the damage caused by PFTs4,15,16. Potassium efflux mediates the assembly and activation of the intracellular multiprotein signaling platform known as the nucleotide-binding oligomerization domain (NOD) leucine-rich repeat

(LRR) pyrin domain containing (PYD) 3 (NLRP3) inflammasome17. The NLRP3 inflammasome is comprised of three proteins NOD, ASC, and caspase-1. NOD serves as the sensor domain and is activated in response to changes in K+ levels, RNA-DNA hybrids, extracellular ATP, uric acid crystals, and PFTs18. The

NOD protein forms a complex with the adaptor protein called apoptosis- associated speck-like protein containing a caspase recruitment domain (ASC) which initiates the binding, oligomerization, and self-cleavage of caspase-1.

Active caspase-1 cleaves proinflammatory cytokines prointerleukin IL1B (IL-1β) 5

and interleukin-18 (IL-18) and also promotes the inflammatory cell death known

as pyroptosis19-21.

The human pathogens Bordetella pertussis, Vibrio cholerae, Kingella

kingae, and Extra-intestinal pathogenic Escherichia coli (ExPEC) all express RTX

toxins11. The most well studied and founding member of the RTX family is alpha- hemolysin (HlyA) from ExPEC. ExPEC is the leading cause of gram-negative

sepsis and neonatal meningitis. They have a wide range of virulence factors that

aid in promoting infection such as adhesins, nutrient acquisition genes, and the

PFT HlyA22,23.

Uropathogenic E. coli (UPEC) is a pathotype of ExPEC and is the number

one cause of urinary tract infections (UTI)24. HlyA is expressed by ~40% of UTI

causing strains, up to 78% of pyelonephritis causing strains, and 80% of

bloodstream isolates25-27. Retrospective studies of human data, in vitro tissue

culture assays, and experiments using mouse models of UTIs have shown that

HlyA mediates increased tissue damage and inflammation28,29. Furthermore,

studies in the urinary tract or in sepsis models have demonstrated that HlyA is

not essential in the colonization of those tissues29,30.

Further research has shown that HlyA can target specific cell types during

infection. Within the urinary tract, HlyA can promote cell death in renal cells and

bladder epithelial cells28,31-33. HlyA can also kill innate immune cell types such as

natural killer cells (NK), neutrophils, and macrophages through necrosis or

apoptosis34-38. Recently, the relevance of HlyA was further demonstrated using

an in vivo zebrafish model of infection. Wiles and co-workers established a 6

zebrafish model of disease that served as a surrogate for a local infection. They

injected into the zebrafish pericardial cavity either WT UPEC strain UTI89 or a

UTI89∆hlyA mutant. The WT UTI89 strain killed zebrafish efficiently, but the

isogenic hlyA mutant was no longer lethal, suggesting that HlyA was a significant

mediator of zebrafish death in their model. Plasmid complementation of HlyA

restored lethality in the mutant, demonstrating that zebrafish killing was reliant on

the presence of HlyA. Furthermore, in zebrafish depleted of phagocytes, the HlyA

mutant regained the ability to cause fatality in the pericardium. These data

establish that phagocytes are a significant target of HlyA during infection39.

Interestingly, not all UPEC isolates share the requirement of HlyA to

cause lethality. Experiments with another UPEC isolate CFT073 demonstrated

that HlyA is not necessary for death in the zebrafish pericardial cavity. These data are consistent with the idea that there are functionally redundant genes within CFT073’s genome that can compensate for the loss of HlyA. As such, the

Mulvey lab is trying to identify those genetic factors. We hope that these factors

may serve as potential vaccine candidates or possible therapeutic targets for

treatment in diseases such as sepsis or UTIs.

HlyA is the prototypic member of the RTX family and was one of the first

bacterial virulence factors that fulfilled molecular Koch's postulates30,40,41. RTX

toxins must meet two criteria to fall into the RTX family. First, they must contain

an iteration of the RTX domain. Secondly, secretion must occur by the type I

secretion system (TISS). For proteins to get out of Gram-negative bacteria, they

need to pass through a cytoplasmic membrane, the periplasm, and the outer 7 membrane. Gram-negative bacteria have devised different secretion systems to allow transport of proteins from inside the bacterium, through the cell envelope, and into the extracellular milieu. The TISS is made up of three different proteins: an ATP-Binding Cassette (ABC) transporter, a trimeric membrane

(MFP), and the outer membrane protein (OMP) (reviewed in42 and11).

RTX toxins all share a similar genetic organization which is exemplified by the hlyCABD operon. The hlyCABD operon is composed of 4 genes that are transcriptionally ordered and encode the following proteins: hlyC the activating protein, hlyA the RTX toxin, hlyB the ABC transporter, and hlyD the MFP.

Another gene tolC encodes the OMP but is outside of the operon at a distant chromosomal locus. Together HlyB, HlyD, and TolC make up the constituents of the TISS11.

Genes in the hlyCABD operon play specific roles in mediating the maturation and secretion of HlyA. HlyA is produced as an inactive ~107 kDa protoxin. It is made up of two domains: an N-terminal series of 10 alpha-helices and the C-terminus contains the RTX repeats followed by a secretion signal. In the bacterial cytosol, the acyltransferase HlyC post-translationally modifies HlyA.

HlyC acylates two internal lysine residues on HlyA, converting the protoxin into an active toxin43. Acylation of the lysine residues is necessary for the cytotoxic function of HlyA, but the modifications are thought to be dispensable for secretion44. HlyA is then recognized by HlyB the ABC transporter, and HlyD the

MFP, by its C-terminal secretion signal. HlyB utilizes ATP to pump HlyA through the trimeric channel of HlyD and TolC into the extracellular milieu10,11,45. 8

Once secreted from the bacteria, the RTX domains of HlyA bind extracellular calcium, which induces conformational changes that enable absorption into host membranes. The exact structure of the pore is not known as researchers have yet to determine the crystal structure or visualize the channel by electron microscopy. HlyA can target many cell types, including epithelial, endothelial, immune cells types, and erythrocytes. Based on the promiscuity of

HlyA’s interactions with multiple cell types, some have argued that interactions with host cells are receptor-independent46. In contrast, other reports have identified at least two different receptors that appear to be crucial in mediating cytotoxicity: glycophorin A an erythrocyte specific receptor, and lymphocyte function-associated 1 (LFA-1) that is present on leukocytes of the immune system47,48. After binding to the host membrane HlyA monomers oligomerize, and form pores in the host membrane.

In the field of PFT biology, there are generally two paradigms about how

PFTs influence host cells. There are “generic” responses, and there are those responses that fall under a newer archetype called host cell “tampering or manipulation.” Intoxication by PFTs results in a set of “generic” host cell responses that include pathways that are immediately responsive to ion fluxes and occur during intoxication with many different PFTs12,14,49. The influx of Ca2+ ions signals to initiate endocytic and exocytic membrane damage repair pathways4,16,50. Potassium efflux activates MAPK signaling pathways as well as the assembly of the NLRP3 inflammasome, both of which result in the production of proinflammatory cytokines13,17,51-53. In agreement with our lab's observations, 9 there is considerable evidence in the literature that PFTs can “tamper” with other host cell pathways, including the activation of proteases. In Chapter 2, I present a literature review of the interplay between PFTs and host proteases. The review highlights how PFTs can either activate or usurp protease functions and what impact those actions have on the outcome of infection. Similarly, research in the

Mulvey lab has also discovered instances of host cell “tampering” involving the inactivation of critical signaling cascades, as well as aberrant activation of a host serine protease.

Wiles and co-workers discovered that HlyA intoxication of bladder epithelial cells resulted in the inactivation of the serine/threonine kinase Akt54. Akt is a central signaling molecule that regulates cell survival, metabolism, cytoskeletal dynamics, and immune responses55. Furthermore, they also showed that two other PFTs, alpha-toxin from Staphylococcus aureus and aerolysin from the Aeromonas hydrophilia, also potently inactivated Akt in bladder cells. Of note, earlier studies from other groups had shown that HlyA intoxication inhibited the production of the proinflammatory cytokines interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNFα)56,57. Taken together, the inactivation of Akt may serve as one molecular mechanism employed by UPEC to mute the initial inflammatory response or facilitate particular types of cell death.

During infection, the cell has to regulate multiple signaling cascades in response to many stimuli. Input from TLR engagement, cytokine and chemokine receptor signaling, and other danger signals (i.e., extracellular ATP and others) all converge in the cell and mediate survival and immunity. Mounting the 10

appropriate immune response versus a dysregulated one can be the difference between resolution of the infection or result in host death.

Apoptosis, necroptosis, and pyroptosis are three major programmed cell death (PCD) pathways that can be manipulated by microbes to promote infection

as well as utilized by the host to aid in survival58. Apoptosis is a silent non-

inflammatory regulated multistep process while necroptosis and pyroptosis are

forms of inflammatory cell death. At the center of two of these PCD pathways are

a family of cysteine protease, the caspases. Caspases are the gatekeepers for

initiating and executing all three types of PCD. The caspase family is made up of

13 different members, and they are divided into the initiator and executioner

subtypes. The initiator caspases become activated in response to various stimuli

such as the binding and activation of death receptors such as TNF-R and FAS or

the leakage of mitochondrial components such as cytochrome c58. Initiator

caspases are activated by recruitment to the death induced signaling complexes

(DISC) where they oligomerize and self-cleave. Executioner caspases are the

business end of the pathway and are kept in the cytosol in an inactive state

through their short inhibitory prodomain. The initiator caspases activate

executioners by proteolytic cleavage of their inhibitory domain. Once activated,

the executioner caspases are free to target their down-stream effectors and

initiate the apoptotic program.

Pyroptotic and necroptotic pathways are mediated by caspase-dependent

and independent mechanisms, respectively. For example, necroptosis occurs

when the ligation of death receptors fails to initiate caspase activation and 11

instead, the protein kinases receptor-interacting serine/threonine protein kinase

1/3 (RIPK1 and RIPK3) are recruited to the DISC. The RIPKs initiate pathways

that result in necrosis through lysosomal membrane permeabilization (LMP),

activation of the calcium-dependent calpain proteases, and the generation of

reactive oxygen species58. In contrast, activation of the supramolecular complex known as the inflammasome initiates pyroptosis. Different types of

inflammasomes are differentiated by their sensor domain (NOD), but the

downstream repercussions of activation are the same. The inflammasome

facilitates the interaction between the NOD domain, the adaptor protein, and

procaspase-1. Recruitment and oligomerization of caspase-1 promote its auto-

activation, the processing of proinflammatory cytokines IL-1β and IL-18, and

pyroptotic cell death.

The intoxication of host cells by PFTs can also promote cell death that is

caspase-independent and mediated by other proteases such as lysosomal

cathepsins or serine proteases. PFTs can facilitate lysosomal membrane

permeabilization (LMP), a type of PCD where lysosomal components such as

cathepsins leak out into the cytosol59,60. Once released, the hydrolases can

degrade proteins and activate other proteolytic cascades. (For specific examples

and details see Chapter 2). While there have been numerous reports of LMP

caused by PFTs61-63, there have been fewer reports involving the activation of

serine proteases in response to PFTs.

Research into the multifaceted effects of HlyA on host cells has ranged

from manipulation of the immune response, inactivation of Akt, activation of 12 calpains and caspases, and more recently the aberrant activation of host serine proteases. Dhakal and Mulvey discovered that HlyA intoxication of bladder epithelial cells resulted in the proteolysis of host proteins33. In contrast to the shared phenomenon of the inactivation of Akt54, the degradation of host proteins appears to be specific to HlyA intoxication and not shared by alpha-toxin or aerolysin. One of the proteins they identified as being degraded during HlyA intoxication was the factor RelA, a member of the proinflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) family.

The NF-kB signaling pathway is responsible for modulating the inflammatory response to infection, specifically the production of inflammatory cytokines and chemokines64. Intriguingly, they were able to link the degradation of RelA, and others, to the activation of a host serine protease called mesotrypsin, also referred to as Trypsinogen 4 (Try4). siRNA knockdown or chemical inhibition of

Try4 significantly protected host proteins from proteolysis. Indeed, preventing

Try4 activation increased the production of proinflammatory cytokine IL-6 in bladder epithelial cells. These data suggest that activation of Try4 is perhaps another mechanism utilized by HlyA to suppress the host immune response. In addition to the proteolysis of host proteins, microscopic analysis of intoxicated bladder cells, as well as RAW macrophages, demonstrated that many serine proteases were trafficked along microtubules and localized within the nucleus.

How Try4 is activated and what governs its intracellular trafficking into the nucleus is not known. In Chapter 3, I present my work that elucidates some of the intracellular trafficking requirements for Try4 and investigates the 13 consequences of Try4 activation for the host during infection.

Trypsinogen 4 is a member of the trypsins, a family of serine proteases.

Trypsins are digestive , secreted by the pancreas and small intestines where they proteolyze proteins at specific amino-acid sequences aiding in the digestion of food. There are three trypsins encoded genetically by PRSS1

(cationic), PRSS2 (anionic), and PRSS3 (mesotrypsinogen). Trypsins regulation shares some similarities with the apoptotic caspases. They are translated as an inactive zymogen (trypsinogens) and like the executioner caspases, require cleavage of the prodomain to become active. Furthermore, trypsins can also self- activate through autocleavage, thereby amplifying the catalytic cascade. In contrast to the caspases, trypsins function primarily extracellularly, and the serine protease enterokinase cleaves the trypsinogens at the trypsinogen activation peptide (DDDDK)65,66.

Cationic and anionic trypsins are known for their roles in the digestion of food in the digestive tract. In addition to the requirement of proteolytic processing, there are other safeguards in place to ensure that digestive enzymes do not activate prematurely. Pancreatic cells secrete potent endogenous trypsin inhibitors, which are crucial for keeping PRSS1 and PRSS2 repressed.

Interestingly, mesotrypsin (PRSS3) is resistant to most endogenous trypsin inhibitors and can degrade them67-69. Trypsin inhibitors resistance is one way that mesotrypsin is thought to regulate the function of PRSS1 and PRSS2 in the digestive tract.

Mesotrypsin regulation, tissue expression, and even physiological 14 functions are quite different from those of cationic and anionic trypsins. For instance, PRSS3 encodes two distinct mRNAs due to alternative splicing and the use of different start codons. The two mRNA species generate three splice forms and are designated isoforms A, B, and C (Figure 1.1). Isoforms A and B, also known as Trypsinogen 4 (Try4) or brain trypsin, use an alternative upstream exon that is outside of the PRSS3 locus70. The mRNA species are different from each other concerning the first exon used, but exons 2-5 are the same between the two70. Isoforms A and B differ only by their start codon usage, AUG vs. CUG, which changes the length of the N-terminal leader sequence71. Isoform C is referred to as mesotrypsinogen and is encoded by exons 1-5 within the PRSS3 locus. Unlike isoforms A and B, isoform C has a typical signal sequence and is secreted into the extracellular milieu by the pancreas and small intestines69. Of note, all three isoforms are indistinguishable at the amino acid level starting from the trypsinogen activation peptide (DDDDK) through the carboxy terminus (see

Figure 1.1 for graphical representation of the PRSS3 loci).

As previously noted, isoforms A and B do not have the typical signal sequence characteristic of secreted proteins; thus, how these isoforms are secreted is currently unknown. Try4 is a unique trypsin as evidenced by the fact that it is expressed in extrapancreatic tissues such as the brain, immune cells, and many different epithelial cell types72-74. Previous work in understanding the regulation and function of Try4 and mesotrypsin have primarily centered on their reported extracellular roles in various , neurodegenerative diseases, pancreatitis68,75-77, and its intracellular role in the skin differentiation78. In light of 15

resistance of mesotrypsin to trypsin inhibitors, researchers have attributed the

onset of acute pancreatitis to the degradation of endogenous inhibitors which

unleashes the other trypsins. Mesotrypsin activity is also associated with

increased metastasis and the progression of pancreatic, breast, and prostate

cancers76,79,80. For example, in models of breast , mesotrypsin cleaved the cell marker CD109, which corresponded to increased malignant growth and alterations in gene expression80.

Try4 and mesotrypsin are also associated with the pathological

mechanisms of multiple sclerosis and neuroinflammation. Several reports have

shown that both proteases can cleave protease-activated receptors (PARs),

myelin basic protein, and amyloid precursor protein81-87. Along with our lab’s

finding that HlyA intoxication activates Try4, another report suggested that Try4 is responsive to other stimuli. Experiments in mouse astrocytes expressing

recombinant human Try4 have illuminated some of the requirements for Try4

(isoform A) intracellular trafficking. Tarnok and coworkers discovered that when

astrocytes were treated with anoxia inducing conditions, Try4 trafficked from the

cytosol to the plasma membrane, and subsequently the protease was activated

extracellularly.

Further investigation uncovered what parts of the N-terminal leader

sequence of Try4 mediated plasma membrane localization. Using N-terminal truncation mutants of fluorescently tagged Try4, they showed that the first 44 amino acids of the 72 amino-acid leader sequence did not target Try4 to the plasma membrane in astrocytes88. These results are suggestive in light of our 16 lab’s previous observation in bladder cells that in response to HlyA intoxication

Try4 co-localized to the plasma membrane and along microtubules. Perhaps similar domains of the N-terminal leader sequence are responsive to both anoxia as well as PFT intoxication.

In Chapter 3, I present the work I’ve done to understand how bladder cells regulate Try4 trafficking in response to HlyA intoxication. My work has discovered what features of the Try4 protein mediate trafficking into the nucleus and also what cellular signals initiate relocalization. Furthermore, I categorize host factors that facilitate nuclear trafficking and identify the subnuclear compartment that

Try4 is sequestered within. Finally, I show the consequence of Try4 activation to the host using mouse models of UTIs and sepsis. This work supports a small but growing body of evidence that Try4 has essential roles in mediating inflammation in infection. Future work is needed to start understanding if Try4 mediates host responses to other pathogens.

17

Figure 1.1 Trypsinogen 4 Is Encoded by the PRSS3 Gene. The PRSS3

gene is located on chromosome 9 and encodes two different mRNA species.

Isoforms A and B utilize an upstream exon that encodes the N-terminal leader sequence, which lacks the secretion signal. Isoforms A and B differ only in their use of start codon (Met for isoform A and Leu for isoform B). Those isoforms are expressed in extrapancreatic tissues. Isoform C encodes mesotrypsin and utilizes all five exons within the PRSS3 locus. Mesotrypsin is expressed in the pancreas and contains a known secretion signal within the first exon of the

PRSS3 locus. Importantly, exons 2-5 encode the “mesotrypsin core” domain, which is identical between all three isoforms. Adapted from “Regional Distribution of Human Trypsinogen 4 in Human Brain at mRNA and Protein Level” by J. Toth et al., 2007 Neurochem Res, 32, p. 1424. Copyright 2007 by Springer+Business

Media, LLC.

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CHAPTER 2

PRODDING YOUR INNER DEMONS: PORE-FORMING TOXINS

ACTIVATE HOST CELL PROTEASES 28

Elizabeth M Enrico1, Amanda C Richards1, and Matthew A Mulvey1*

1Division of Microbiology and Immunology, Pathology Department, University of

Utah School of Medicine, Salt Lake City, Utah, USA

Introduction

Pore-forming proteins (PFP) are a vast family of proteins that are present

in every kingdom of life. The function of the PFP varies between the kingdoms. In

Metazoans the PFPs Bax and Bak initiate apoptosis by punching holes in the

mitochondrial membrane releasing cytochrome c which activates caspase 31.

Another example of a PFP is the complement cascade which is essential for

innate and adaptive immune responses in mammals. The complement cascade

functions to remove pathogens by -mediated opsonization, recruitment of phagocytes, or cell lysis. After initial binding of complement protein to the bacterial membrane, the proteolytic cascade is activated and can culminate in the deposition of C5-C9 (the membrane-attack complex). C9 is the terminal PFP in the membrane attack complex and it oligomerizes and forms the pore that lyse

the bacteria (reviewed in2).

Pore-forming toxins (PFTs) are the most abundant proteinaceous

virulence factor employed by bacterial pathogens and are often crucial factors

that promote infection and worsen disease outcomes. PFTs are categorized

based on protein domain characteristics and the structures that penetrate the cell

membrane. They are broadly classified into alpha and beta toxins named for the 29

protein domains alpha helices and beta barrels which they form upon insertion

into plasma membranes3. The toxins are further subclassified into six different

families: colicin, cytolysin A (ClyA), actinoporins, hemolysins, aerolysin,

cholesterol-dependent cytolysins (CDC) and the unclassified repeat-in-toxin family (RTX)4.

As their namesake suggests, PFTs form pores in membranes which can

lead to outright lysis and cell death. Cell lysis is thought to promote infection

through the release of essential nutrients like iron and depletion of immune cells5-

7. PFT/host cell interactions begin with the secretion of the toxin into the

extracellular space typically as a water-soluble molecule. There is growing

support for the idea that PFTs act in a short-range manner and rarely reach

concentrations that are lytic, but rather intoxicate the cell. Once inserted into host

membranes, the most immediate effects are the transient but rapid fluxes of ions.

Potassium efflux has been shown to activate the innate immune signaling

receptor NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome8.

This multiprotein signaling platform mediates the activation of caspase-1.

Caspase-1 is known to mediate cleavage and secretion of Interleukin 1-β (IL-

1β)9 , and it is necessary for the initiation of a highly inflammatory type of cell death called pyroptosis10,11. There are 11 members of the IL-1 family of cytokines

that promote inflammation and can mediate the adaptive immune response12.

Similarly, PFT’s damage to host membranes has been shown to cause

transient calcium oscillations which can activate Mitogen activated protein kinase

(MAPK) pathways (ERK, p38, and JNK)13-18. MAPK cascades are central hubs 30

that control processes such as cellular differentiation, proliferation, inflammatory

responses, and apoptosis19,20. Indeed, it appears that signaling through MAPKs,

in particular, p38 is a conserved response that aids the host in recovery17,18,21.

Furthermore, other intracellular components like ATP can also be released and

can serve as danger signals to neighboring cells through binding to their

receptors22. This review will focus on the phenomenon of protease activation in

response to PFTs and their role in the outcome of infection.

Calpains: More Than Just Cell Membrane Dynamics

Given the nature of PFTs to induce membrane damage and ion fluxes, it is

no surprise that numerous PFTs activate calpains. Calpains are intracellular

cysteine proteases that require high concentrations of Ca2+ for autoactivation and

proteolytic activity. They have described roles in regulating gene expression,

protein turnover, cell migration and plasma membrane plasticity, cell cycle

progression, inflammation, and inducing apoptosis23,24. Calpain activation is crucial for mounting an effective host immune response by regulating the innate

immune effector interleukin 1- α (IL-1α)24,25. Recent research has shed light on

how PFTs utilize the activation of calpains to promote invasion into tissues, and

how calpain activation mediates an effective immune response.

Streptococcus pneumoniae is a gram-positive bacterium that can cause pneumonia, septicemia, and meningitis. One major virulence factor that promotes disease progression and severity in S. pneumoniae infection is Pneumolysin

(PLY). PLY is a pore-forming toxin that belongs to the cholesterol-dependent 31 cytolysin (CDC) family and is expressed by nearly all clinical isolates26. How, or even if, PLY is secreted into the surrounding milieu is controversial. However, recent research has identified two potential pathways for PLY release. One mechanism involves autolysis27, and the other is nonautolytic28. In the stationary phase, S. pneumoniae produces an autolysin called N-acetyl-muramoyl-l-alanine amidase (LytA). LytA and other autolysins degrade the bacterial cell wall peptidoglycan, thereby lysing the bacterium and releasing the toxin27.

PLY is a crucial virulence factor for S. pneumoniae that initiates tissue damage and aids in effective colonization of the host. PLY mutants are attenuated in mouse models of infection29,30, and PLY deficient strains generate less tissue damage compared to their wild type (WT) counterparts30-32.

Furthermore, several reports have highlighted PLY involvement in the activation of host innate immune responses. Infections with PLY positive strains result in the upregulation of the proinflammatory cytokines IL-1β, IL-1α, tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and interleukin-8 (IL-8)33-35.

The production of IL-1 family cytokines (IL-1β and IL-1α) during models of pneumococcal meningitis and pneumoniae is essential in promoting survival and bacterial clearance35-38. Shoma et al. discovered that PLY strongly stimulated the secretion of IL-1α, IL-1β, and IL-18 in peritoneal macrophages, and their production was dependent on the activation of caspase-1. However, the mechanism for IL-1α secretion remained elusive35.

Calpains have previously been shown to process and promote the secretion of IL-1α25,39. Other groups have discovered that intracellular bacterial 32

pathogens Mycobacterium tuberculosis and Listeria monocytogenes40,41 induce

the secretion of IL-1α by calpain activation. Recent work by Fang et al. has

uncovered how PLY mediates the secretion of mature IL-1α in macrophages. To

test if calpains were involved in this process, the authors used calpain inhibitors,

genetic mutants, and gene knockdown experiments. They showed that host

calpains activation was dependent on PLY and calpain activation relied on

calcium fluxes, which in turn mediated the maturation and secretion of IL-1α.

Calpain activation was beneficial to S. pneumoniae infection due to IL-

1α mediated tissue damage and access to deeper tissues42.

Another pathogen that benefits from the aberrant activation of calpains is

Staphylococcus aureus. S. aureus is a significant cause of skin infections, pneumonia, and bacteremia43. The pathogen produces a PFT called α-hemolysin

(Hla) or alpha toxin that belongs to the beta-barrel PFT family. Hla has reported

roles in mediating immune cell death, destruction of tissue barriers, and can

activate the NLRP3 inflammasome44-47. Soong et al. reported that Hla potently

stimulated the activation of caspase-1 in keratinocytes. In turn, caspase-1

promoted pyroptosis, a form of inflammatory cell death that is mediated by IL-

1β10. In parallel, they found that caspase-1 degraded the endogenous calpain

inhibitor, calpastatin that facilitated keratinocyte necrosis. Also, inhibiting

caspase-1 or calpain activation rescued keratinocytes from both inflammatory

types of cell death and prevented staphylococcal invasion across the skin cells.

These studies illustrate that activation of calpains, either through Ca2+ fluxes or

by altering levels of the endogenous inhibitor, facilitated tissue invasion, damage, 33

and cell death.

In contrast, not all PFT-mediated calpain activation is detrimental to the

host. Research by Velasquez et al. focused on the prototypic pore-forming toxin

for Uropathogenic Escherichia coli, alpha-hemolysin (HlyA). Using sublytic levels of HlyA on erythrocytes, they observed that Ca2+ fluxes activated calpain I (µ-

calpain), and that resulted in the cleavage of known cytoskeletal protein targets

of calpain (spectrin). Cytoskeletal destabilization perturbed plasma membrane

dynamics, and this increases the activity of sphingomyelinases, enzymes that

catalyze the production of ceramides. Ceramides are lipid molecules that are

found in high concentrations of plasma membranes and can serve as signaling

molecules. They are also potent initiators of apoptosis and an erythrocyte

specific form of apoptosis called eryptosis48,49. Ceramides facilitate the exposure

of phosphatidylserine (PS) on the cell membrane that targets apoptotic cells for

macrophage engulfment50. The authors speculated that the calpain-mediated

degradation of cytoskeletal components and increased ceramide formation

facilitated eryptosis51. There are thought-provoking implications in undergoing

this type of regulated cell death versus a more inflammatory death. For instance,

the iron sequestered within heme is not released into the extracellular milieu but

recycled through macrophage engulfment. Iron is tightly regulated in host cells,

and eryptosis is possibly an effective way to deprive extracellular pathogens of

vital metabolic co-factors52.

The canonical functions of calpains are to degrade intracellular proteins

that facilitate cellular homeostatic processes to proceed. It is fascinating that the 34 very nature of PFTs, to damage membranes and allow ion fluxes can exploit this function to further infection and disease severity. On the other hand, it is interesting how the host has evolved proteins that are delicately sensitive to ion concentrations. And, those proteins serve as a first-line defense against pathogens by initiating immune signaling pathways.

Cathepsins: Suicide Bags or Friends?

The ability of cells to degrade and recycle proteins is vital for maintaining cellular homeostasis. Cells can bring in extracellular proteins by the endosomal/lysosomal pathway or recycle intracellular proteins by autophagy.

Lysosomes are vesicles of the endocytic pathway that contain different hydrolases that are crucial for regulating protein degradation and turnover.

Lysosomes were initially described as “suicide bags” for their roles in the degradation of intracellular proteins. However, it has become clear that lysosomal hydrolases, like the cathepsins, participate in many more cellular processes than initially thought.

The cathepsins are a family of proteases classified as papain-like cysteine proteases. Interestingly, not all of their members utilize the same amino acid in their catalytic site. There are 11 cysteine proteases (B, C, F, H, K, L, O, S, V, X, and W), two serine proteases (A and G), and two aspartic proteases (D and E).

Cathepsins have demonstrated functions in , bone remodeling, keratinocyte differentiation, and others53-56. Furthermore, genetic mutations and misregulation of cathepsins are implicated in the metastasis of 35 cancer, cardiovascular pathologies, and genetic disorders57.

Cathepsins are optimally active in acidic environments such as those found in lysosomes. Interestingly, there are reports that cathepsins S, D, and B can function at neutral pH58-63. Cathepsin activation is initiated by autocleavage of the zymogen form followed by dimerization. However, the N-terminal pro- peptide can remain associated with the protein, thereby inhibiting its activity until dissociation that is driven by acidification within the endosome.

A more recently appreciated theme in the fields of PFT biology and cell death is the phenomenon of lysosomal membrane permeabilization (LMP)64.

LMP is the destabilization of the lysosome where the luminal contents leak out into the cytosol. This biological process can occur in response to several stimuli that are found during an infection such as reactive oxygen species, tumor necrosis factor, interferon gamma, and intoxication by bacterial PFTs58,64-70.

Uncontrolled LMP can lead to necrosis, but partial or selective LMP results in apoptosis. Lysosomes contain many different hydrolases, but studies have shown that cathepsin B and D appear to be significant players in driving LMP mediated cell death71,72.

Listeria monocytogenes is a Gram-positive bacterium which causes , a leading cause of food-borne related deaths. L. monocytogenes is particularly adept at invading into cells and across tissue barriers. It can cross the intestinal epithelium, the blood-brain barrier, and the maternal-placental barrier.

Upon ingestion of contaminated food, Listeria invades into the host intestinal cells through a zipper-like mechanism that is mediated by clathrin and others73,74. 36

As the Listeria internalization vesicle traffics through the phagolysosomal pathway, the pH of the vesicle drops. The acidic environment drives the activation of Listeria’s cholesterol-dependent cytolysin PFT, listeriolysin O

(LLO)75,76.

LLO is a significant determinant of virulence and is required in mouse models of listeriosis to cause infection77-79. LLO has been shown to promote apoptosis and necrosis of immune cells, activate host signaling cascades, and mediate invasion into the intestinal epithelium74,80-83. Arguably the most important and well-studied role of LLO is its role in destabilizing the internalization vacuole that allows L. monocytogenes access to the host’s cytosol. Invasion into the cell and escape into the cytosol are crucial in facilitating the cell to cell spread of

Listeria and avoiding extracellular detection by the immune system78,79,84.

A recent report by Mallet et al. has uncovered a new intracellular ramification of LLO intoxication, the initiation of LMP85. Using HeLa cells, the authors showed that LMP was dependent on LLO but was independent of calcium fluxes. Interestingly, while epithelial cells lines were susceptible to LLO induced LMP, macrophage cell lines appeared resistant. LLO-LMP released the lysosomal protease cathepsin D into the cytosol, and the protease remained active for some time. They also showed that two other PFTs, Perfringolysin O from and Pneumolysin from Streptococcus pneumoniae, also induced LMP in HeLa cells.

However, what the consequence(s) are of having active cathepsin D in the cytosol remains unclear. Perhaps free cathepsins or other lysosomal hydrolases 37

could degrade cellular proteins and promote different kinds of cell death

(apoptosis or necrosis) and further stimulate the host’s immune response.

In another study, LLO intoxication of peripheral blood mononuclear cells

(PBMCs) ruptured the and released the cysteine protease, cathepsin

B, into the cytosol60. The cytosolic release and activity of cathepsin B are sensed

by the NLRP3 inflammasome60,86, which results in the potent production of the

proinflammatory cytokine IL-1β. The induction of IL-1β relied on the NLRP3

inflammasome and LLO, but LLO was crucial for the release of cathepsin B.

However, PBMCs treated with purified LLO illuminated a cathepsin B-

independent mechanism for NLRP3 inflammasome activation and IL-1β release.

These results demonstrate that PFTs-induced LMP has many different outcomes

for host cells, but this pathway plays an essential role in the type and magnitude

of the inflammatory response during infection.

Bacillus anthracis is a Gram-positive spore-forming bacterium that is found

in the environment and makes the well-known toxin, . Most cases of

anthrax are associated with herbivores that accidentally encounter B. anthracis

spores in the environment that can result in death. There are three routes of

anthrax infection, including inhalation, ingestion, and cutaneous. Spore

introduction into a nutrient-rich environment of the host signals the transition into

the vegetative form, which initiates programs to express virulence factors

including anthrax toxin87.

Anthrax toxin belongs to the AB toxin family and is made of three

components: the receptor-binding protective antigen (PA), and two catalytic 38 subunits lethal and edema factor (LF and EF). The toxin is secreted as an inactive form that requires cleavage of PA by the host protease furin or furin-like proteases88,89. PA cleavage mediates the heptamerization of the receptor binding subunit, binding of LF and EF, and receptor-mediated endocytosis87,90. The toxin- containing endosome traffics along the endocytic pathway and endosomal acidification causes PA to form pores in the endosomal membrane. Pore formation allows the translocation of endosomal contents as well as LF and EF into the host-cytosol87,91.

In the cytosol, LF and EF exert their cytotoxic effects. LF is a metalloprotease that cleaves mitogen-activated protein kinases (MAPKs) and potently inactivates the p38, ERK, and JNK MAPK pathways92-94. MAPK signaling is crucial for regulating cellular stress responses, proinflammatory cytokines, and growth factors20. This is interesting considering many PFTs activate MAPK pathways14,18. EF converts ATP to cyclic AMP (cAMP), thereby depleting intracellular ATP levels. cAMP acts as a secondary messenger by binding to proteins like protein kinase A (PKA), which initiate signaling cascades.

Activation of PKA leads to dysregulation of ion gradients in the intestinal lumen and other tissues and results in massive edema95. In spite of the efficient manner in which anthrax toxin kills the host, recent reports have demonstrated a new player in mediating anthrax toxicity, cathepsin B58,61,65.

Anthrax lethal toxin (LF+PA) can cause LMP and the release of active cathepsin B into the cytosol58,96. Proteolytically active cathepsin B promotes the lethal toxin-mediated inflammatory cell death pathways of caspase-1 dependent 39

pyroptosis, and caspase-1 independent pyronecrosis58,97. Two reports have

recently shown that inhibiting cathepsin B activation during lethal toxin challenge, either with the small molecule inhibitor CA-074 or the cell permeable version CA-

074Me, promoted survival of macrophage cell lines58,65. Of note, it appears that

these works have discovered two different mechanisms of action for cathepsin B

during lethal toxin challenge. One mechanism described cathepsin B mediated

fusion of the endosome with the lysosome thereby inhibiting LF release into the

cytosol65. In a lethal rat model of anthrax, researchers co-injected anthrax toxin

and an antimalarial drug, amodiaquine (which shares similar chemical structures

to CA-074). All the anthrax challenged rats survived and had no overt signs of

toxin-associated symptoms61. Further experiments demonstrated that

amodiaquine directly inhibited cathepsin B, which in light of the previous research

supports the biological importance of cathepsin B in mediating anthrax toxicity

and death. These studies highlight the crucial role that cathepsin B plays in

mediating LF and EF host cell death.

Sometimes cathepsin B activity and escape from the lysosome can be

protective for the host. While bacterial PFTs make up a significant portion of all

pore-forming proteins (PFP), there are PFPs represented in all domains of life.

Interestingly, one family of PFP has emerged as playing a vital role in host

defense against bacterial infection. The aerolysin family of proteins is named for

containing the “aerolysin domain” that is comprised of 2 beta strands β1 and β2,

a membrane insertion domain β-hairpin (for aerolysin), and two more beta

strands β3 and β498. 40

Recent work in the frog species Bombina maxima has shown that they

possess a unique aerolysin-like PFP called βγ-CAT99. The PFP is comprised of

an alpha and beta subunit: βγ -crystallin fused aerolysin-like protein and a trefoil

factor, respectively. βγ-CAT is present on mucosal surfaces like the skin and

gastrointestinal tract as well as the blood. Upon bacterial challenge, researchers

observed the upregulation of βγ-CAT protein. Earlier research showed that βγ-

CAT oligomerization and endocytosis were necessary for its full biological

activity100. Once endocytosed, βγ-CAT destabilized the lysosome and released cathepsin B into the cytosol. The authors went on to show that cathepsin B activity facilitated the assembly of the NLRP3 inflammasome, activation of caspase-1, and the secretion of IL-1β. βγ-CAT -dependent induction of IL-1β was

crucial to promoting survival in both a frog model of peritonitis as well as a mouse

model of sepsis99.

How the trefoil proteins mediate protective inflammation is mechanistically

similar to how PFTs mediate pathologic inflammation. They induce LMP,

resulting in the leakage of cathepsins (B and others) into the cytosol, and the

subsequent activation of the inflammatory response. What is so striking is the

host is purposefully inducing LMP to mediate protection during infection. In

contrast, it can be argued that PFT-induced LMP could be either harmful or

helpful to the host’s survival. It is clear there are other factors during infection that

play significant roles in deciding what the outcome of LMP will be for the host.

Further research into understanding what those factors are and how they are

regulated will shed light on the issue of LMP during PFT challenge. 41

Furin: The Traitor Within

Another common theme in PFT-biology is the activation of toxins by host proteases such as furin. Furin is a Type-1 transmembrane endopeptidase (pro-

convertase) that traffics between the trans-Golgi network, the plasma membrane,

and the endosomal pathway. It is responsible for converting inactive proprotein

substrates into their active forms, and some bacterial toxins have taken

advantage of this function90,101. As previously mentioned, furin cleaves the N- terminal tail of PA of anthrax toxin, which is essential for anthrax toxin cytotoxicity87-89. For instance, the PFTs aerolysin from Aeromonas hydrophilia102

and alpha-toxin from Clostridium septicum103 require furin processing to convert

their protoxin form into their active form. In addition to furin’s activities at the

plasma membrane, it can also process different substrates within the endocytic

pathway.

Another class of toxins, the A/B class, subvert furin’s endosomal functions

to promote disease and pathogenesis. Upon receptor binding, the toxin is

endocytosed and traffics along the endosomal pathway. The toxin’s A domain

and B domain are separated by cleavage of the furin site that links them. Upon

separation, the A domain translocates into the cytosol and promotes host-cell

dysfunction. The A/B toxins , , and Pseudomonas

exotoxin all utilize this pathway to transition from protoxin to active toxin104,105.

Additionally, there is in vivo evidence that inhibiting furin in a mouse model of

Pseudomonas exotoxin significantly increased overall survival106. The roles of

other related proconvertases and cellular proteases most certainly overlap with 42

furin; however, furin’s role in activating toxins is undisputed. Current research is ongoing to find better furin inhibitors to mitigate the cytotoxic effects of PFTs on

host cells106,107.

Trypsins and Mysterious Proteases

ExPEC are a diverse group of pathogens that cause a variety of diseases

like skin infections, urinary tract infections, meningitis, and sepsis. Uropathogenic

E. coli (UPEC) is a pathotype of ExPEC that is responsible for the majority of

UTIs and pyelonephritis cases in humans108,109. UPECs have an extensive suite

of virulence factors that allow them to colonize, replicate, hijack nutrients, and

even disarm the host immune system. As previously discussed, PFTs are a

widely used tool in many pathogen’s arsenals, and UPEC is no exception. More

than 80% of ExPEC isolates from the bloodstream and ~50% of UTI isolates

express the prototypic repeat-in-toxin (RTX) α-hemolysin (HlyA)110-112. The RTX

toxins are named for protein domain comprised of repeating nonapeptide calcium

binding sequence. All RTX toxins also have a C-terminal secretion signal that is

recognized by the type I secretion system which facilitates toxin secretion113.

Research into HlyA’s effects on host cells has demonstrated some

similarities with other PFTs. Similarly to aerolysin and alpha-toxin, HlyA can

activate MAPK pathways14, caspases, and the NLRP3 inflammasome114-116. The

intoxication of bladder cells with either alpha-toxin, aerolysin, or HlyA all resulted

in the inactivation of protein kinase B (Akt)117. Akt is a central signaling hub in the

cell and regulates cellular processes such as growth, metabolism, glucose 43

uptake, angiogenesis, and survival118. Of note, other pathogens also target the

Akt signaling axis. Salmonella enterica, Shigella flexneri, and Neisseria

gonorrhoeae all activate Akt signaling119-121. However, those pathogens affect Akt

signaling by directly injecting effector molecules into the host cells instead of

through PFTs.

In addition to the previously discussed classical families of proteases,

there are limited reports of proteases, such as trypsins, that are modulated by

PFTs intoxication. Research in our lab, using the prototypic RTX toxin HlyA from

Extraintestinal pathogenic Escherichia coli (ExPEC), has uncovered a role for

host trypsin during intoxication114.

Trypsins are a super-family of serine proteases found in vertebrates that

hydrolyze proteins. Three genes encode trypsins: PRSS1 (cationic), PRSS2

(anionic), and PRSS3 (mesotrypsinogen). Trypsins are secreted into the

extracellular intestinal lumen as inactive zymogens or trypsinogens. They are

activated by cleavage of the trypsin motif (DDDDK) by another protease,

enterokinase122. Active trypsins can also self-activate through autocleavage,

thereby amplifying the catalytic cascade. The cationic and anionic trypsins cleave

peptide bonds and aid in digesting food. In the pancreas, mesotrypsin is thought to regulate trypsin activation by degrading endogenous inhibitor of PRSS1 and

PRSS2. However, mesotrypsin is unlike the other trypsins because of its reported intracellular functions and broad tissue distribution.

The first description of mesotrypsin was that it was a minor component of pancreatic juice. It is resistant to endogenous trypsin inhibitors and can cleave 44

them 123. The gene PRSS3 is unique because it encodes two distinct mRNAs

due to alternative splicing and uses different start codons. The mRNAs are

different from each other with respect to the first exon used. However exons 2-5 conserved between the two124. The two mRNA species generate three splice

forms and are designated isoforms A, B, and C. Isoforms A and B are also

known as Trypsinogen 4 (Try4) or brain trypsin. These isoforms use an

alternative upstream exon that is outside of the PRSS3 locus. Isoforms A and B

differ only by their start codon usage, AUG vs. CUG, which changes the length of

the N-terminal leader sequence. Extrapancreatic tissues such as the brain,

immune cells, and many different epithelial cell types express isoforms A and

B125-127. Also, these isoforms do not have the typical signal sequence

characteristic of secreted proteins. Isoform C, referred to in most of the literature

as mesotrypsinogen, uses the first exon in the PRSS3 locus. The isoform has a

typical signal sequence and is expressed within the pancreas and small

intestines123. Importantly, all three isoforms are identical in sequence starting

from the propeptide (DDDDK) through the carboxy terminus, thereby making it

difficult to determine the exact isoform in some cases.

Previous work in understanding the regulation and function of Try4 and

mesotrypsin has primarily centered on their reported roles in various cancers,

neurodegenerative diseases, and in pancreatitis128-131. Because mesotrypsin is

resistant to endogenous inhibitors, researchers have attributed the initiation of

acute pancreatitis to the degradation of trypsin inhibitors and the unleashing of

the other trypsins. Isoforms of PRSS3 are implicated in the pathological 45

mechanisms of multiple sclerosis and neuroinflammation132-138. As Try4 is

associated with various models of disease, it is thought that its functions are

almost exclusively extracellular.

Work in our lab has discovered an intracellular role for Try4 in a UTI model

of infection. HlyA intoxication of bladder epithelial cells resulted in activation of

host caspases as well as serine proteases. Serine protease activity promoted the

degradation of many host proteins, including those associated with cell survival,

membrane dynamics, and inflammation. Try4 facilitated the targeted degradation

of members of the nuclear factor kappa-light-chain-enhancer of activated B cells

(NF-κB) immune mediators IKKB and RelA. Using inhibitors, siRNA, and co- immunoprecipitation studies, we identified Try4 as a significant moderator of

these processes114. The degradation of NF-kB signaling components may be one

mechanism by which HlyA can inhibit early cytokine signaling139,140.

Surprisingly, HlyA intoxication of bladder cells and RAW macrophages

resulted in the translocation of activated serine proteases into the nucleus. In

follow up studies we have discovered that Try4, as well as other serine

proteases, localize within the nucleolus, the site of ribosomal biogenesis. We

identified the molecular mechanisms of Try4 nucleolar trafficking, and we also

observed that two other PFTs, aerolysin and alpha-toxin, promoted the nucleolar

translocation of Try4 independent of its activation.

Using mouse models of UTIs and sepsis, we have also uncovered a

physiological ramification of Try4 activation. We inhibited Try4 activation, using

the putative Try4 inhibitor diminazene141, and were able to reduce inflammation 46

and tissue damage in our UTI model. Furthermore, inhibition of Try4 during sepsis dramatically improved survival as compared to the vehicle-treated mice. In light of Try4’s roles in regulating inflammation, our results support the hypothesis that HlyA-mediated activation of Try4 is detrimental to the host.

Even though aerolysin and alpha-toxin do not activate Try4, we speculate

that nucleolar localization may be a way to sequester or detain “misbehaving”

proteins142. For instance, during hypoxia, members of the ubiquitin pathway are

detained in the nucleolus until the restoration of normoxic conditions143.

Alternatively, one report demonstrates that caspase-2 is recruited to and

activated within the nucleolus in response to DNA damage144.

Another possible explanation is that Try4 has an unknown function within the nucleolus and may regulate the cell's response to intoxication stress. We

propose that Try4 could be part of a cellular network that is responsive to PFTs.

Why Try4 is being recruited or detained in the nucleolus is subject to much

speculation. Further studies are needed to begin delineating the function(s) of

Try4 within the nucleolus.

Another report has suggested that an unknown host serine protease is

responsible for mediating cell death in a model of intoxication with Clostridium

perfringens (CPE). Clostridium perfringens is a Gram-positive spore-

forming anaerobic bacteria that causes food poisoning in livestock and humans.

C. perfringens isolates been reported to produce 17 different toxins which allow

for classification into five different types of C. perfringens (A-E). C. perfringens

type A causes food poisoning in humans as well as gastrointestinal diseases like 47 -resistant diarrhea145. CPE is most often associated with type A although other types can also produce the toxin. While about 5% of all C. perfringens produce CPE, the toxin is required for C. perfringens type A food poisoning in a rabbit model of enteritis146,147.

CPE is an unusual toxin because it shares structural similarities to the aerolysin pore-forming toxin family148,149, but its primary sequence lacks homology to other toxins150. Upon ingestion of contaminated food, C. perfringens undergoes sporulation, and the mother cell undergoes lysis releasing CPE into the intestinal milieu. Of note host trypsins and chymotrypsins cleave the N- terminal leader sequence, thereby activating the toxin151. Previous work has identified the receptor for CPE as the family of claudins, a mammalian protein necessary for maintaining tight junctions. After receptor binding, the toxin forms small CPE complexes which are rapidly followed by oligomerization into a large prepore and then pore-formation. The intoxication of enterocytes with a high amount of CPE caused rapid and large calcium influxes. The calcium fluxes have been shown to activate calpains and drive oncosis, a form of necrotic death152,153.

However, at lower concentrations, CPE initiates a smaller influx of calcium and cells undergo classical apoptosis mediated by caspases 3/7. The claudin receptors are vital mediators of CPE intoxication as cells that do not express the receptors are highly resistant to physiologically relevant CPE concentrations154.

The gastrointestinal tract is made up of at least six different cell types155, and not all of those cell types are equally responsive to CPE156,157. Work by

Shrestha et al. investigated whether there were essential interactions between 48

CPE sensitive and insensitive cells in a co-culture model of the gastrointestinal environment158. They modeled CPE intoxication in rat fibroblasts expressing the human claudin-4 receptor (Cldn Trans) or naturally sensitive Caco-2 cells. They treated CPE-sensitive cells and CPE-resistant cells with sublytic doses of the toxin and then measured cell death using viability dyes. CPE-resistant cells were unaffected by CPE treatment. In contrast, co-culturing the resistant cells with the

CPE-sensitive cells increased the resistant cell death by approximately 10%.

The authors hypothesized that the CPE-sensitive cells were releasing factor(s) that could mediate CPE-resistant cell death. To test this idea, they collected supernatants from CPE-sensitive cells after intoxication and then treated CPE-insensitive cells and measured viability. Again, they observed a decrease in overall viability in the CPE-resistant cells. They went on to show that decreased cell viability was not due to free or extracellular membrane-associated

CPE but rather an unidentified heat labile proteinaceous factor. Further experiments demonstrated that the cytotoxic factors were sensitive to trypsin inhibitors and had a molecular size between 10-30 kDa.

Finally, Shrestha et al. examined how the cytotoxic factors promoted cell death in CPE-resistant cells. Previous work had identified that sublytic doses of

CPE caused Caco-2 cells to undergo caspase 3/7 mediated classical apoptosis153. Indeed, inhibiting CPE activation caspase 3/7 in Caco-2 cells or

Cldn Trans cells had two interesting impacts in their model. First, it prevented cytotoxicity in both Caco-2 cells or the Cldn Trans fibroblasts. But more interestingly, caspase 3/7 activity was required for the release of the cytotoxic 49 factors from CPE-sensitive cells. Importantly, inhibiting caspase3/7 did not reduce the amount of CPE-induced membrane vesicles secreted, further supporting the importance of caspases in mediating CPE toxicity.

In conclusion, this study has demonstrated that CPE intoxication of sensitive cells causes the release of cytotoxic factors from host cells. These factors act in a paracrine manner and mediate classical apoptosis in CPE- insensitive cells. Furthermore, the cytotoxic factors are likely serine proteases which have been shown to function in apoptosis even in caspase inhibiting conditions159,160. It is tempting to speculate if this mechanism of cell death is similar in cells exposed to other PFTs.

Future Perspectives

The effects of PFTs on the host has been dutifully studied for the past century. Many of those studies focused on the immediate ramifications of PFT intoxication of host cells such as outright lysis and cell death. More recently, research has shown that the interactions of PFTs and host cells can be more nuanced and typically do not result in outright lysis but rather host cell

“tampering.” Studies have shown that PFTs activate a myriad of host signaling cascades that all feed into the outcome for the host cell. Many of those pathways that were initially characterized were MAPK pathways and cell death pathways.

But in the past decade or so investigators have observed that PFTs also perturb other pathways like proteolytic cascades. There appear to be some common themes that can be categorized based on the mechanism of activation, such as 50

calcium fluxes, LMP, and aberrant activation of serine proteases. These

mechanisms appear in part to be programmed responses to changes in cellular

homeostasis, but interestingly sometimes the pathogen benefits from the

response and other times the host benefits. Undoubtedly, much more research is

needed to begin to understand what impact these proteases have in different

models of PFT-mediated infections. It is very likely that with further investigation, perhaps currently approved drugs could be repurposed to treat the effects of PFT

intoxication during infection, such as the antimalarial drug amodiaquine used in

the anthrax studies61.

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CHAPTER 3

BACTERIAL PORE-FORMING TOXINS PROMOTE THE NUCLEOLAR

DETENTION OF HOST SERINE PROTEASE TRYPSINOGEN 4

66

Elizabeth M Enrico1, Alex Tran, Brittany A Fleming1, Amanda C Richards1, Ting

Liu, and Matthew A. Mulvey1*.

1Division of Microbiology and Immunology, Pathology Department, University of

Utah School of Medicine, Salt Lake City, UT, USA 84112-0565

Introduction

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of gram-negative sepsis and neonatal meningitis, and the number one cause of urinary tract infections (UTI)1. ExPEC has many virulence factors that aid in promoting infection such as adhesins, nutrient acquisition, and a pore-forming repeat-in-toxin called alpha-hemolysin (HlyA)2,3. HlyA is expressed by ~40% of

UTI causing strains, and 80% of bloodstream isolates 4. Pore-forming toxins

(PFTs) are the most abundant proteinaceous bacterial virulence factors and are often necessary to promote infection. They can facilitate bacterial colonization by breaking down tissue barriers, inactivation or killing of professional phagocytes, and outright lysis of host cells5. However, research using sublytic levels of intoxication has demonstrated that there can be more nuanced effects on the host.

Several studies have shown that HlyA is important in the dissemination of

ExPEC into surrounding tissues, promoting tissue damage, suppressing cytokine production, and killing of host phagocytes6-11. We have previously shown that sublytic levels of intoxication by HlyA or two other PFTs, aerolysin and alpha- 67

toxin, result in the inactivation of AKT (protein kinase B), the master regulator of cellular homeostasis12. We have also shown that HlyA potently promotes the

activation of many host serine proteases. One serine protease that we identified

as activated in a mouse model of UTI was Trypsinogen 4 (Try4)6.

Serine proteases are proteolytic enzymes that are ubiquitously expressed

and necessary for life functions in mammalian cells. Trypsins and trypsin-like

enzymes are a subset of serine proteases that have well described regulatory

roles in digestion, coagulation, and the immune response13,14. The digestion

related trypsins (cationic trypsin, anionic trypsin, and mesotrypsin PRSS3) are

expressed in the digestive tract. In contrast, Try4 is unique in that it is expressed

15 outside of the pancreas and small intestines

We have previously shown that Try4 becomes activated in a HlyA dependent manner in bladder epithelial cells. Activation resulted in the

degradation of many host proteins, some of which are involved in cytoskeletal

regulation as well as immune signaling. In vitro microscopy studies in bladder

epithelial cells also demonstrated Try4 re-localizing along microtubules and

trafficking into the nucleus during HlyA intoxication6. In this study, we identify

what domains mediate Try4 relocalization into the nucleus and show that Try4

trafficks into the nucleolus, the cite of ribosomal biogenesis. We also discovered

that caspase-14 promotes Try4 trafficking into the nucleolus. And finally, we

determine the consequence(s) of Try4 activation using in vivo mouse models of

UTIs and sepsis. Our results show that inhibiting Try4 activation decreased

disease severity and promoted survival in a mouse model of UTI and sepsis, 68 respectively.

Results

Alpha-hemolysin Is Necessary for Nucleolar Localization of Try4

To address whether HlyA intoxication regulates Try4 trafficking, we used recombinant Try4 fused to EGFP to monitor its intracellular movements. We infected Try4-EGFP expressing bladder epithelial cells (BECs) with ExPEC isolate UTI89 or the isogenic UTI89∆hlyA mutant and used confocal microscopy to visualize the cellular localization of Try4. In uninfected cells or cells infected with the isogenic hlyA mutant, Try4 remained localized broadly throughout the cell. However, in BECs infected with UTI89 Try4 was trafficked into distinct nuclear puncta, demonstrating that HlyA is necessary for Try4-EGFP re- localization in BECs (Figure 3.1A).

We hypothesized that the puncta where Try4 localized was the nucleolus.

The nucleolus is the site of ribosomal biogenesis and is composed of proteins,

DNA, and RNA organized around regions in the genome encoding ribosomal genes16. It is easily visible due to the lack of DNA staining (blue) within the subnuclear compartment. To confirm this, we utilized fluorescently labeled nucleolar proteins, td-tomato-fibrillarin, and DS-red-B23/nucleophosmin (data not shown), and transfected BECs to mark the nucleolus. Cells were infected with either UTI89 or UTI89∆hlyA and stained for endogenous Try4 using an antibody that recognizes the trypsin motif DDDDK. Importantly, 5637 BECs express no other trypsins6; therefore staining of DDDDK serves as a surrogate for Try4. HlyA 69 intoxication resulted in the co-localization of endogenous Try4 with td-tomato- fibrillarin (Figure 3.1B). In contrast, Try4 did not colocalize with td-tomato- fibrillarin in BECs infected with the hlyA mutant (Figure 3.1B). These results show that HlyA promotes the trafficking of Try4 into the nucleolus.

To test the requirement of HlyA in Try4 nucleolar localization, we used a nonpathogenic strain of E. coli (AAEC185) transformed with plasmids encoding either WT HlyA or an inactive HlyA. BECs were treated with culture supernatants for 4 hours from WAM582 (WT HlyA) or supernatants from WAM783 (inactive toxin). As can be seen in Figure 3.1C, Try4 localized into discreet punctae within the nucleolus after intoxication with active HlyA but not with the inactive toxin.

These results demonstrate that active HlyA, but not other ExPEC associated virulence factors, is sufficient for Try4 re-localization into the nucleolus.

The Mesotrypsin Core Is Required for HlyA-Mediated Try4

Nucleolar Trafficking

The PRSS3 gene encodes Try4 which has three isoforms17,18. Two of the isoforms use an alternative exon and therefore have longer N-terminal leader sequences in contrast to the pancreatic form of mesotrypsinogen. The Try4 protein is an inactive zymogen containing a 72 amino acid leader sequence (pro- domain) followed by a mesotrypsin “core domain,” which is common between all three isoforms. All isoforms also share the canonical trypsin activation motif

(DDDDK). In pancreatic acinar cells, mesotrypsinogen is secreted into the intestinal lumen and is activated by cleavage of the DDDDK motif by 70 enteropeptidase19,20

A series of N-terminal truncation mutants were tested to identify what protein domains of Try4 mediated intracellular trafficking during HlyA intoxication

(see Figure 3.2A for constructs). Experiments with different N-terminal truncation mutants revealed that the first 44 amino acids of theTry4 pro-domain are not required for HlyA mediated nucleolar localization (Figure 3.3B). BECs transfected with constructs lacking the mesotrypsin “core domain” were no longer capable of trafficking into the nucleolus (p72M and p28M). We performed reciprocal experiments using a mesotrypsin core-EGFP fusion and restored nucleolar punctae formation during intoxication (Figure 2.2B). Interestingly, the N-terminal leader sequence EGFP fusions by themselves strongly co-localized along microtubules and the plasma membrane in response to HlyA intoxication. These results suggest that the N-terminal leader sequence may have a role in the initial trafficking of Try4 to the plasma membrane.

We next went on to quantify instances of nucleolar localization across all the Try4 constructs. Figure 2.2C shows that the N-terminal leader sequence is dispensable for nucleolar trafficking during HlyA intoxication. To account for intoxication effects on nonspecific protein or aggregation, we performed the same experiments with the empty vector (EGFP). We did not observe any significant localization of EGFP within the nucleolus. These demonstrate that intoxication mediated relocalization of proteins into the nucleolus is a regulated process and not simply an artifact of EGFP over-expression. Furthermore, the mesotrypsin-core domain is necessary and sufficient for HlyA mediated 71

trafficking of Try4 into the nucleolus.

PFT-Mediated Nucleolar Localization of Try4 Does Not Require

Its Activation or Enzymatic Activity

We next tested the importance of Try4 enzymatic activity in mediating

nucleolar localization. To answer this question, we created two different mutants a catalytically dead mutant (S257A) and a trypsin activation motif mutant

(EEEEE) (see Figure 3.3A for constructs). We performed the same experiment

as previously described and observed that the catalytically dead protease

(S257A) as well as the EEEEE mutant translocated with similar efficiencies as

compared to the WT Try4 (Figure 3.3B). To further test if Try4 enzymatic activity

was needed for trafficking into the nucleolus, we used a pharmacological

inhibitor, Tosyl-L-lysyl-chloromethane hydrochloride (TLCK), a trypsin and

trypsin-like serine protease inhibitor. In agreement with the previous genetic

mutant data, we observed the TLCK treatment did not significantly change Try4

trafficking into the nucleolus as compared to vehicle control (Figure 3.3C). While

the trafficking efficiency of the Try4-EEEEE trended lower than the WT, these

results clearly show that Try4 localization into the nucleolus is independent of its

catalytic function as well as its activation.

Previous work in our lab has shown that other PFTs share some common

mechanisms of actions on host cells12. These observations led us to test whether

another PFT, aerolysin, could activate Try4 or mediate its nucleolar localization.

In Figure 3.3D we intoxicated Try4-EGFP expressing BECs with HlyA, 10ng/mL 72

aerolysin, or 10 ng/mL heat-killed aerolysin for 4 hours and then marked active

serine protease using SR-SLCK, which is a cell-permeable TLCK conjugated to

Texas red (Figure 3.3D). As shown in Figure 3.3E, active aerolysin can stimulate

Try4 trafficking into the nucleolus. Furthermore, the colocalization of activated

Try4 (yellow) in HlyA treated cells appears more intense than aerolysin treated

cells suggesting that aerolysin intoxication does not significantly activate Try4. Of

note, the population of activated serine proteases within these nucleoli appears

distinct in their segregation within the subnuclear structure (Figure 2.3D zoom).

To further test if aerolysin intoxication activated Try4, we analyzed the

population of activated serine proteases by western blot. We intoxicated BECs with either HlyA or 5 ng/mL aerolysin for 4 hours in the presence of

Carboxyfluorescein-spacer-leucine chloromethyl ketone (FSLCK). FSLCK covalently binds, and fluorescently tags activated serine proteases and can be detected in a western blot using anti-FITC. Whole-cell lysates were probed with anti-FITC, and a representative blot is shown in Figure 3.3F. The serine proteases that become activated in response to each toxin are unique though some also appear to be shared. Our western blot analysis shows that HlyA

intoxication alone activated Try4 as indicated by the * in the blot. In addition, we

previously published that activation of Try4 resulted in the degradation of many

host proteins, including paxillin6. To further demonstrate that aerolysin

intoxication does not activate Try4, we examined protein levels of paxillin by

western blot. Indeed, the intoxication of BECs with increasing amounts of

aerolysin did not induce the degradation of paxillin, further supporting the 73 observation that Try4 is not activated (Figure 3.3G). In sum, these data suggest to us that the nucleolar localization of Try4 does not require its activation and that aerolysin does not activate Try4.

Potassium Fluxes Are Necessary and Sufficient for Nucleolar

Localization of Try4 During Intoxication

The most well-studied consequence of PFTs interactions with host membranes is to cause damage5,21. When PFTs insert into host membranes, there are immediate and sporadic influxes of calcium and effluxes of potassium ions22-25. To ascertain the effect of calcium homeostasis onTry4 trafficking, we utilized the intracellular calcium chelator BAPTA-AM. We pretreated Try4-EGFP expressing BECs for 1 hour, and then performed the microscopy experiments as previously described. The chelation of intracellular calcium did not have a significant impact on the translocation of Try4 into the nucleolus when compared to vehicle control (Figure 3.4A).

To test if potassium efflux was necessary for the nucleolar trafficking of

Try4, we incubated Try4-EGFP expressing BECs with excess potassium chloride and then performed the experiments as previously noted. Of note, we observed that preventing potassium efflux significantly reduced the efficacy of Try4-EGFP translocation when compared to vehicle control (Figure 3.4B). We then wanted to test if potassium fluxes were sufficient on their own in driving Try4 nucleolar localization. To do this, we utilized a pore-forming protein (PFP) valinomycin, which is produced by strains of Streptomyces. The PFP forms potassium specific 74 channels in the lipid bilayer and is known to cause apoptosis in some cells26. We treated BECs with either valinomycin or DMSO control for 4 hours and quantified nucleolar puncta. Treatment of BECs with valinomycin alone was able to promote significant nucleolar localization of Try4 when compared to DMSO (Figure 2.4C).

These results reveal that the fluxes of potassium are necessary and sufficient to drive Try4 relocalization into nucleoli.

The Karyopherin Importin-β Mediates the Nuclear Import of Try4

We next tested what nuclear import mechanisms mediated Try4 trafficking during HlyA intoxication. First, we tested whether microtubules mediated Hlya induced nuclear import of Try4. We pretreated BECs with the microtubule depolymerizing drug nocodazole (Figure 3.5A) and performed microscopy experiments as discussed above. Quantification of nucleolar punctae revealed that transport along microtubules did not significantly contribute to Try4 nucleolar localization during HlyA intoxication. Next, we tested specifically what nuclear import mechanism was important for Try4 trafficking into the nucleolus. We identified two inhibitors, ivermectin and importazole, that targeted importin α/β and importin β mediated nuclear import, respectively. We pretreated BECs either ivermectin, importazole or vehicle control and then performed the experiment as previously described. As can be seen in Figure 3.5B inhibiting nuclear import that is mediated by α/β heterodimers did not affect the translocation of Try4 into the nucleolus during intoxication as compared to the vehicle control.

In contrast, inhibition of importin β mediated nuclear import resulted in 75 significantly reduced nucleolar localization of Try4 compared to vehicle control or ivermectin treated cells (Figure 3.5B). These results show that Try4 trafficking into the nucleolus is mediated in part by importin β but not by importin

α/β heterodimers. Furthermore, as the import of Try4 was not absolutely abolished, these results also suggest that there are likely other importins or nuclear import pathways that are facilitating Try4 trafficking into the nucleolus.

Caspase-14 Facilitates Try4 Trafficking to the Nucleolus

To further investigate the intracellular mechanisms of Try4 trafficking, we decided to identify potential Try4 interacting partners in BECs. To accomplish this, we performed co-immunoprecipitation experiments using a FLAG-tagged

Try4 (see Figure 3.1A for construct) and infected with UTI89 or the isogenic hlyA mutant for 3 hours. We pulled down Try4-FLAG, visualized the interacting proteins by silver staining, and selected bands for further analysis by mass spectrometry. One band of particular interest to us (marked by the *) increased significantly in the presence of HlyA. Mass spectrometry analysis identified that band as caspase-14 (Figure 3.6A).

Having identified that one caspase interacted with Try4, we wanted to test if other caspases were involved in Try4 trafficking into the nucleolus. We inhibited caspase activity using a caspase-1 inhibitor (Ac-YVAD-CMK) or inhibited all the caspases with the pan-caspase-inhibitor (Z-VAD-FMK). As shown in Figure 3.6B, caspase-1 does not mediate trafficking of Try4 into the nucleolus during intoxication. However, inhibiting all caspase activity with Z-VAD-FMK significantly 76

increased Try4 trafficking into the nucleolus as compared to vehicle control or

Ac-YVAD-CMK. We decided to follow up on our mass spectrometry findings and

test if caspase-14 affected Try4 localization during intoxication. We co-

transfected BECs with Try4-EGFP and caspase-14-FLAG-cMyc or an empty- vector and performed the experiments as previously described. Over-expression

of caspase-14 during HlyA intoxication significantly increased nucleolar Try4

when compared to the empty vector control. Importantly, the over-expression of

caspase-14 alone did not promote Try4 trafficking into the nucleolus. These

results suggest that activation of caspases can inhibit Try4 trafficking into the

nucleolus. Also, our over-expression experiments demonstrated that caspase-14

could mediate Try4 trafficking into the nucleolus in a HlyA dependent manner.

Inhibition of Try4 Is Beneficial to the Host in Models of UTI and Sepsis

Try4 has been associated with other disease and inflammatory

processes27-29; we wanted to test if Try4 activation was significant in models of

UTI or sepsis. To test this hypothesis, we used a previously identified inhibitor of

mesotrypsin called diminazene30. Diminazene has been used since the 1950s to

treat animal trypanosomiasis, although recent work has shown that diminazene

may have anti-inflammatory effects on the host31,32. The drug also has

antimicrobial properties against enterohemorrhagic E. coli O157:H733 as such we

first evaluated the impact of diminazene on two Urinary pathogenic E. coli

(UPEC) isolates F11 and UTI89. Bacterial growth curve analysis and disk

diffusion assays all demonstrated that UTI89 was more sensitive to the drug than 77

F11 (supplemental Figure 3.9). Therefore, we carried forward with our in vivo studies using UPEC isolate F11.

Next, we tested whether treatment of diminazene affected the ability of

F11 to colonize the bladder. Female CBA/J mice were transurethrally catheterized with ~1x10^8 CFU of F11, and then 1 hour later, we intraperitoneally

(i.p.) administered diminazene or vehicle control. Twenty-four hours after infection, the mice were sacrificed, bladders removed, and titers enumerated.

Figure 3.7A shows that diminazene treatment did not significantly affect F11 colonization in the bladder compared to vehicle control. Next, we wanted to evaluate the repercussions of Try4 activation in our mouse model of UTI. Mice were infected as described above, and the bladders were sectioned and stained with Haemotoxylin and Eosin (H&E) to allow for histological evaluation of cystitis severity. Bladder sections were scored blindly by a pathologist for signs of edema, mucosal immune infiltrates, and submucosal immune infiltrates. Mice treated with diminazene had significantly reduced numbers of infiltrating immune cells but interestingly, increased signs of edema compared to the vehicle control treated mice (Figure 3.7B and 3.7C for representative H&E images). These results are noteworthy as overall bacterial titers were similar between the drug and control treated mice (Figure 3.7A), suggesting that diminazene’s effect is host centric and not due to antibacterial functions.

We then wanted to test what role Try4 may play in promoting sepsis.

Before we could answer those questions, we first established a mouse model of sepsis that was primarily mediated by HlyA. We infected female Swiss Webster 78

mice i.p. with ~5x10^7 CFU of ExPEC cystitis isolate F11 or F11∆hlyA and then

monitored the survival of the mice over 48 hours. The survival graphs clearly show that HlyA is a potent mediator of mortality in an F11 model of sepsis

(Figure 3.7D).

We next sought to test whether inhibition of Try4 was beneficial or harmful

to the host during sepsis. First, we examined the effects of diminazene using

F11∆hlyA to ascertain if there were effects that were independent of HlyA. We

infected female Swiss Webster mice by i.p. injection with ~5x10^7 CFU of

F11∆hlyA. Approximately 1 hour later, we administered diminazene by i.p. at 4

mg/kg or saline control and then monitored the survival of the mice over 48

hours. There was an increase in the survival of mice that were infected with

F11∆hlyA and received diminazene. However, the differences in survival

between the drug and vehicle control with F11∆hlyA infected mice were not

significant (Figure 3.7E). In contrast, mice that were infected with F11 and

received diminazene showed significantly improved survival when compared to

control vehicle-treated mice (Figure 3.7F). These results strongly support that

inhibition of HlyA-mediated Try4 activation protects the host.

Discussion

Before this work, how Try4 trafficked within cells and what it meant for the

host were unknown. The purpose of this study was to identify factors that were

important for Try4 activation, intracellular trafficking, and what were the

repercussion of activating Try4 for the host. 79

Using Try4 truncation mutants, we showed that in response to intoxication

Try4 is trafficked to the nucleolus, and that relocalization requires the mesotrysin

“core domain” (Figures 3.1 and 3.2). These results are interesting in light of a

study that showed that 28 amino acids of the N-terminal leader sequence were crucial in mouse astrocytes for targeting Try4 to the plasma membrane in response to anoxia. They further argued that the 28 amino-acid sequence was essential in mediating the eventual activation and extracellular secretion of

Try434. Our previous research suggested that Try4 became activated in response to the PFT HlyA but not other PFTs aerolysin, and alpha toxin6. However, we did

not know if Try4 activation was required for trafficking into the nucleolus or

whether other PFTs could also cause its nucleolar localization. We discovered

that Try4 trafficking into the nucleolus was independent of its catalytic activity and

its canonical activation via cleavage of the DDDDK motif. In further support of

these data, we also observed that Try4 trafficked into nucleoli upon aerolysin

intoxication, even though it was not activated.

Having defined some intrinsic factors that mediated Try4 trafficking into

nucleoli, we also discovered several extrinsic factors. Many studies have shown

that PFTs induced calcium oscillation that can activate the MAPK signaling

pathways (p38, Jun, and ERK)23-25,35-37 and potassium efflux is known to mediate

the activation NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome38. Here, we showed that the efflux of potassium but not calcium

was necessary and sufficient to induce nucleolar localization of Try4. These

results are further suggestive in the context of reports that Try4 can mediate the 80 activation of caspase-1, secretion of IL-1β and may play a role in the initial priming of the NLRP3 inflammasome39. Other studies have shown that activation of the NLRP3 inflammasome aids in resisting colonization and invasion into epithelial cells39. In light of our results and the current literature, it is possible that initially the activation of Try4, by HlyA intoxication, is beneficial to the host but as the infection progresses, the cell trafficks Try4 into the nucleolus to inhibit its function.

In our effort to find the mechanism for Try4 nucleolar localization, we identified by mass spectrometry that caspase-14 interacted with Try4 during HlyA intoxication. Caspase-14 is a cysteine protease that governs the terminal differentiation of complex epithelial cells such as the skin40-42. As keratinocytes terminally differentiate into corneocytes, they must get rid of their nuclei as well as other organelles43. Recent studies have demonstrated that mesotrypsin (a splice variant of Try4) and caspase-14 participate in parallel pathways in keratinocyte denucleation resulting in a nonapoptotic type of cell death44.

Caspase-14 has been shown to activate mesotrypsinogen, which facilitates the formation of the permeability barrier45. While caspase-14 and Try4 are expressed in skin cells, they are also both expressed in other epithelial cells suggesting that interactions between these two proteases likely have impacts in different cell types46. Similar to other caspases, caspase-14 activation involves dimerization and autocleavage in between the large and small subunits. However, to achieve optimal function, caspase-14 required a kosmotropic environment (i.e., sodium citrate and others). During keratinocyte differentiation, the water content of those 81 cells decreases, and the increase in salt concentrations promotes conformational changes in caspase-14 that mediate its full activation47. The sudden efflux of potassium and influx of sodium ions likely serves as a kosmotropic signal for caspase-14 activation. However, more experiments are needed to determine if caspase-14 is activated during HlyA intoxication.

The interactions between Try4 and caspase-14 may also explain how Try4 traffics into the nucleolus. As previously noted, Try4 has no secretion signal, nor does it have an identified NLS. However, caspase-14 does have an annotated

NLS, and in-silico analysis suggests that it can localize to the nucleus48. Limited studies have examined the exact mechanism by which caspase-14 traffics into the nucleus. However, based on our results using nuclear transport inhibitors

(Figure 3.5), we argue that transport of Try4 could be through direct binding of caspase-14 with the karyopherin B (importin β). This hypothesis is also supported by the structure and characteristics of caspase-14’s NLS, which favors an importin β nuclear import model49,50. Also, the result of our caspase-14 overexpression experiments (Figure 3.6) further suggests a role for caspase-14 in facilitating Try4 trafficking into the nucleolus (see Figure 3.8 for summary model).

The nucleolar localization of a serine protease at first appears to be a poor choice for the cell to make. Nevertheless, there are increasing reports in the literature that the proteases are recruited to the nucleoli during times of stress51,52. There is a newly described functions for the nucleolus called the nucleolar detention center” pathway (NDC)53-55, which we argue is supported by 82

our experimental observations. Despite the nucleoli’s well-established role in

ribosomal biogenesis, the subnuclear compartment has other functions56. Indeed, nucleoli are responsive to cellular stress signals such as DNA damage, nutrient stress, viral infection, and osmotic stress56,57.

Since this is the first instance reported of Try4 localizing to the nucleolus, many outstanding questions remain. For example, it is unclear if recruitment into

the nucleolus is beneficial or harmful to the cell. Since Try4 activation is not required for nucleolar targeting, it seems less likely that the cell is sequestering

Try4 to inhibit its proteolytic activity, at least with regards to intoxication by aerolysin and alpha-toxin. Instead, perhaps recruitment to the nucleolus is a programmed response to PFTs, as evidenced by our data showing at that aerolysin can also promote nucleolar localization of Try4. Nucleolar localization of Try4 could be one signal that initiates death pathways in BECs. In support of this argument two other proteases, caspase-2 and caspase-6 have been reported to localize to the nucleolus during genotoxic stress as well as extrinsic apoptotic signaling respectively51,52.

Given the inhibitor-resistant nature of Try4 and its role in many different

diseases, there is much interest in identifying effective small molecule

inhibitors58,59. Splice variants of Try4, isoforms A and B (found in the brain and

epithelial cells), are implicated in driving inflammatory processes, cancer

progression, and neurodegenerative diseases27-29,60-65. Recently Kayode et al.

used a virtual screen of two databases and identified novel inhibitors of

mesotrypsin (the spice variant of Try4). One of their hits was identified as the 83

trypanolytic agent, diminazene aceturate (Berenil)30. We tested whether inhibiting

the activation of Try4 was beneficial or harmful to the host. In our mouse model

of UTI, diminazene treatment significantly reduced signs of inflammation but

increased edema (Figure 3.7). Importantly our experiments also showed that

diminazene had no significant antimicrobial effect. Thus the reduction in

infiltrating immune cells can be attributed to the inhibition of Try4 activation. We

also demonstrated that inhibiting Try4 during a HlyA mediated model of sepsis

improved the host’s survival. Our in vivo data support the current body of

literature that Try4 is a potent driver of inflammation in various disease models.

Finally, the data presented here argue that inhibition of Try4 may be a valid

therapeutic target in the future in different models of infection.

Materials and Methods

Bacteria, Cell Culture, and Drugs

The UPEC cystitis isolate UTI89, UTI89∆HlyA 7, F11, and F11∆HlyA and were grown static at 37C° in 20 mL M9 minimal media for 48 hours before use.

K12 nonpathogenic E coli strain AAEC185 was transformed with constructs

designated (pSF4000 and pSF4000 ∆BAMHI66) to generate active and inactive

HlyA producing strains. Human bladder epithelial cells (BECs) designated 5637

(ATCC HTB-9) were obtained through the American Type Culture Collection and

maintained in RPMI 1640 (Invitrogen and Lonza) supplemented with 10% heat- inactivated fetal bovine serum (Seradigm). The following drugs were used in this study: BaptaAM, Nocodazole, and Ivermectin (Tocris), Z-VAD-FMK and 84

Valinomycin (Apex Bio), AC-YVAD-CMK (Alexis and Enzo life sciences),

Importazole, TLCK, Diminazene and Ivermectin (Cayman chemical). FSLCK and

SR-FSLCK were purchased from immunochemistry.com .

Bacteria were grown from frozen stocks at 37°C with static overnight in 20

ml modified M9 minimal medium (6 g/liter Na2HPO4, 3 g/liter KH2PO4, 1 g/liter

NH4Cl, 0.5 g/liter NaCl, 1 mM MgSO4, 0.1 mM CaCl2, 0.1% glucose, 0.0025%

nicotinic acid, 16.5 μg/ml thiamine, and 0.2% casein amino acids). Cultures were

then diluted 1:100 into the indicated medium, and the growth of quadruplicate

200-μl samples in shaking 100-well honeycomb plates at 37°C was assessed

using a Bioscreen C instrument (Growth Curves USA).

Cell Culture and Western Blotting

Human bladder epithelial cells (BECs) designated 5637 ;( ATCC HTB-9;

American Type Culture Collection,) were maintained in RPMI 1640 medium

(Invitrogen, supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2

(Seradigm, VWR). For western blot analysis, BECs cells were seeded to

confluency in 6 well tissue culture plates. ~18-24 hours after seeding, confluent

monolayers were serum starved overnight in RPMI 1640. Monolayers were then

subjected to defined experimental conditions which included bacterial challenges,

drug treatment or both. In the event of a bacterial challenge monolayers were

infected using a multiplicity of infection (MOI) of ~25-30. Plates were spun at

500g for 5 minutes to synchronize bacterial contact with host cells. After infection

or treatments, cells were washed 3 times with phosphate buffered saline (PBS) 85 containing Mg2+/Ca2+. Cells were lysed using lysis buffer containing RIPA (50 mM

Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% Sodium Deoxycholate, 0.1% SDS,

1% Triton X-100, 1X Complete protease inhibitors EDTA free (Roche), 1mM

PMSF (Sigma-Aldrich) pH 7.4. Pierce BCA Protein assay kit was utilized to determine protein concentrations of lysates and typically 30-50 µg total protein plus β-mercaptoethanol and 5 x sample buffer were resolved on a 10% or 4-20%

SDS-PAGE (Biorad TGX precast gel). Protein was then transferred onto an

Immobilon-FL PVDF membrane (Millipore Corporation) using a Mini Trans-Blot transfer cell (Bio-Rad). Membranes were blocked at room temperature for 1 hour in TBS with 5% w/v nonfat powdered milk. Immunoblotting was done by incubating PVDF membranes overnight at 4o C with indicated primary .

Antibodies used in this study were purchased from Abcam Inc. (anti-Caspase-14 ab174847, anti-Paxillin ab32084, anti- ab3280, anti-GFP ab1218 and Santa

Cruz B2 sc-9996). After overnight incubation, blots were washed 3 times in tris buffered saline (TBS) plus 0.1% Tween and probed with the secondary antibodies anti-rabbit-HRP or anti-mouse-HRP (Invitrogen and GE health sciences) using a dilution of 1:5,000-10,000 in TBS plus 0.1% Tween with 5% w/v nonfat powdered milk for 1 hour at room temperature. After secondary probing, membranes were washed 3 times in TBS plus 0.1% Tween and 2 times in TBS alone. Blots were analyzed using the BioRad ChemiDoc or by conventional film.

86

Trypsinogen4-EGFP Cloning

Trypsinogen 4 was cloned out of 5637 cDNA previously generated using

Invitrogen’s SuperScript III following the manufactures’ protocol. XhoI and Hind III restriction sites were added and the stop codon removed for expression in pEGFP-N1 (Clontech). The recombinant Tryp4-EGFP was then subcloned into pCDH-EF1-FHC (Addgene) using NheI and NotI restriction sites. All primers used in cloning Trypsinogen 4 are listed in the Table 3.1.

Immunofluorescence/ Microscopy

5637 cells were seeded onto sterile 12-mm diameter glass coverslips in

24 well plates and transfected at 70-90% confluency using Fugene6

(ThermoFisher) for 4-8 hours following the manufacturers recommended protocol at a ratio of 4:1 Fugene6 to DNA. Cells were then serum starved either for 4 hours or overnight in RPMI 1640 (Invitrogen). Twenty-four hours after transfection cells were infected with UTI89 or UTI89∆hlyA at an MOI of ~15.

Plates were spun at 500g for 5 minutes to promote interactions between the bacteria and host cells. Plates were then incubated at 37C in 5%CO2 for the duration of the experiments and 2 hours’ post infection gentamicin was added directly to the media at 100 ug/mL.

To image microtubules, cells were fixed using ~3.7% Formaldehyde/PBS for 20 minutes at 37C° followed by 3 x 5-min washes in RT PBS-D at 150 RPMs.

In samples were microtubules were not stained cells were fixed using 3.7%

Formaldehyde/PBS at RT for 20-30 minutes. Samples were permeabilized and 87

blocked for 15-30 minutes at room temperature in blocking buffer (1% powered

milk, 3% bovine serum albumin (BSA), and 0.1% saponin in PBS). Samples were

stained for 45 minutes at room temperature with mouse anti- alpha tubulin 1:250

(sigma T529) in blocking buffer. Following 3 x 5-minute washes in PBS the samples were then incubated in blocking buffer containing secondary antibodies conjugated to Alexa555 or -647 (1:500 Molecular probes) for 30 minutes. After secondary staining, samples were washed 3 x 5-minute washes in PBS-D and

nuclei were stained using Hoechst in blocking buffer at 1:1000 for 5 minutes at

room temperature. Samples were then mounted onto slides using Flurosave

reagent (Calbiochem) or in Prolong Gold (Invitrogen). Cells were imaged using

Nikon AR or AR1 at 60X or imaged on Zeiss 880 Airy Scan at 60X.

At the conclusion of the experiment the media was aspirated and cells

were incubated with SR-FLCK (Texas red covalently linked to CMK) to at the

manufacturer recommended 1:250 in serum free RPMI for 5 minutes at 37C.

Cells were then fixed, stained, and mounted as described in the previous

paragraph.

Nucleolar Puncta Quantification

Three or more independent experiments were performed where random

fields of view were taken using the Airy Scan 880 until a total of at least 50 GFP

positive cells were imaged per experiment. Using a single slice with resolutions

>1024 nuclear puncta were evaluated based on the localization of the GFP signal

with the absence of Dapi staining within the nucleus (nucleolus). Percentages of 88

nuclear puncta were calculated as number of puncta in GFP positive cells / total number of GFP positive cells. Data were graphed using PRISM8 and unpaired student’s t-tests were applied to test for significance.

Immunoprecipitation

Seventy to ninety percent confluent BEC monolayers were transfected in

T75 flasks using Fugene6 (ThermoFisher) or lipofectamine2000 (invitrogen) for

4-8 hours following the manufacturers recommended protocol at a ratio of 4:1

transfection reagent to DNA. Cells were then serum starved either for 4 hours or

overnight in RPMI 1640. Twenty-four hours after transfection cells were infected

with UTI89 or UTI89∆HlyA at an MOI of ~25. T75 flasks were rocked on

reciprocal rockers for 30 seconds each in all four directions to disperse the

bacteria over the entire flask. Flaks were then incubated at 37C in 5%CO2 for the

duration of the experiments, and 2 hours’ post infection gentamicin was added

directly to the media at 100 ug/mL. At the indicated time points cells were

washed 3x’s with ice cold PBS 2+ (PBS supplemented with Ca2+ and Mg2+) and then scraped into and lysed in ice cold TNN lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 % NP-40 supplemented with 1x complete protease inhibitors

(Roche) and 1mM phenylmethanesulfonylfluoride (Sigma Aldrich). Lysates were incubated on ice for 30 minutes and vortexed briefly 2-3 times during incubation.

Cell debris and nuclei were pelleted at 4C° 2000 RCF for 20 minutes. Protein content of the post nuclear fractions was determined by BCA assays (Pierce) and equal amounts of protein were used to IP against, typically 100-200 ug for 89

postnuclear fractions. Immunoprecipitations were carried out following the

recommended manufacturer’s protocol (Sigma-Aldrich ANTI-FLAG M2 magnetic beads) and IP proteins were eluted by competition with FLAG peptide at 100ug/ mL. Interacting partners were separated for analysis using precast TGX 4-20% gels from BioRad. Unique bands were excised after silver staining (Pierce Kit) and sent off to the University of Nevada at Reno’s Mitch Hitchcock Nevada

Proteomics center. LC/MS analysis was performed using a Thermo Scientific

Orbitrap Fusion with ETD coupled to a Thermo UltiMate 3000 nanoLC system

and a New Objectives digital PicoView source.

Mouse UTI Model

Seven- to eight-week-old female CBA/J mice (Jackson Labs; Bar Harbor,

ME) were used in accordance with IACUC-approved protocols as previously

described [29,60,61]. Mice were anesthetized using isoflurane inhalation and

inoculated via transurethral catheterization with 50 uL of a bacterial suspension

containing approximately 1X10^8 bacteria. F11 was grown statically for 24 hours

in M9 medium, pelleted by spinning at 10,000 r.c.f. for 8 min, and resuspended in

phosphate buffered saline (PBS) prior to inoculation. Bladders were recovered 1

day later and each was weighed and homogenized using the storm bead beater

in 1 mL PBS-D containing 0.025% Triton X-100. Homogenates were serially

diluted and plated on LB agar plates to determine the number of bacteria per

gram of tissue. Mouse experiments were repeated at least twice, and the total

combined data from 8 or more animals are presented. Data were graphed using 90

PRISM8 and unpaired Student’s t-test with medians.

Histology

Adult female CBA/J mice (Jackson Laboratories; Bar Harbor, ME) were inoculated via transurethral catheterization with 1X10^8 cfu of F11. F11 was grown statically for 24 hours in M9 medium, pelleted by spinning at 10,000 r.c.f.

for 8 minutes, and resuspended in phosphate buffered saline (PBS) prior to

inoculation. Twenty-four hours postinoculation bladders were aseptically

removed and fixed in 10% neutral buffered formalin. Bladders were randomly

sectioned 5-8 μ thick and stained with Haemotocylin and Eosin. Mouse

experiments were repeated twice, and the total combined data from 8 or more

animals are presented. Histology slides were blindly evaluated by Dr. Ting Liu

using the following scoring system: Intramucosal acute inflammation: neutrophils

present in the mucosa; single microscopic focus: 1; two foci: 2; more than 2 or

scattered: 3. Laminar propria: lymphocytic and neutrophilic aggregates:

microscopic focus: single small aggregate: 1; two foci or one big aggregates: 2;

more than two: 3. Mucosal edema: focal: 1; at least half circumferential: 2;

completely circumferential: 3. Data were graphed using PRISM8, and unpaired

Student’s t-test (Mann-Whitney) comparing log-rank were applied to test for

significance.

91

Mouse Sepsis Model

Female seven- to eight-week-old Swiss Webster mice from Jackson labs were infected by intraperitoneal injection with ~1x10^7 CFU of F11 or F11∆hlyA.

The inoculum was prepped by spinning down overnight bacterial cultures grown in M9 8000 RCF ten minutes. Pellets were resuspended in 8 mL of sterile PBS-D and then diluted 1:4 in PBS-D again. One hour post infection the mice were injected on the opposite side IP with either 4 mg/kg diminazene (caymen) or vehicle control saline. Mice were then monitored for survival for 48 hours.

Survival curves were plotted using PRISM8, and significance was tested using the Log-rank (Mantel-Cox) test. Mouse experiments were repeated at least twice, and the total combined data from 9 or more animals are presented.

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98

Figure 3.1. Alpha-hemolysin Intoxication of BECs Promotes the

Relocalization of Trypsinogen 4 into Nucleolar Puncta (A) Confocal

micrographs of BECs transfected with recombinant WT Try4-EGFP. Cells were

infected with UTI89 or UTI89∆hlyA for 4 hours. Cells were fixed, and nuclei were

stained with Hoechst (blue) and microtubules in red. (B) Confocal micrographs of

BECs transfected with nucleolar marker td-Tomato-Fibrillarin and stained for

Trypsinogen 4 using the surrogate marker DDDDK (green). Cells were treated as described in (A). (C) Confocal micrographs of BECs transfected with recombinant

WT Try4 -EGFP and then infected with a K-12 of E. coli engineered to express

either the active HlyA toxin (WAM582) or a catalytically inactive HlyA toxin

(WAM783). White arrowheads mark nucleolar punctae of recombinant Try4 or

endogenous Try4. Scale bar is 10µm.

99

100

Figure 3.2. The Mesocore Is Necessary and Sufficient for Targeting of Try4 into the Nucleolus (A) Graphical representation of all the constructs utilized in this study. (B) BECs cells were transfected with the indicated plasmids (green) and then infected with UTI89 or UTI89∆hlyA for 4 hours. Cells were fixed on coverslips, and nuclei were stained with Hoechst (blue). Coverslips were imaged using confocal microscopy, and a single z-slice is represented. Scale bar is

10 µm. (C) Coverslips in (B) were quantified using confocal microscopy. At least

three random fields of view and ≥50 GFP positive cells were counted in three or

more independent experiments. Unpaired Student’s t-test was used to test for

significance, and data are expressed as mean + SEM. *= >0.05 ***= >0.001

101

102

Figure 3.3. Try4 Trafficking into the Nucleolus Is Independent of Its

Activation or Catalytic Function (A) Graphical representation of the Try4

constructs. (B) BECs were transfected on coverslips with the indicated plasmids and then infected with UTI89 for 4 hours. Coverslips were quantified using confocal microscopy. At least three random fields of view and ≥50 GFP positive cells were counted in three or more independent experiments. Unpaired

Student’s t-test was used to test for significance, and data are expressed as

mean + SEM. (C) BECs were transfected on coverslips with WT Try4-EGFP.

Cells were then pretreated for 1 hour with either DMSO or TLCK (50 µM), then

infected with UTI89 for 4 hours and quantified and analyzed as described in (B).

(D) Confocal micrographs of BECs transfected with recombinant WT Try4-EGFP.

Cells were infected with UTI89 or intoxicated with 10ng/mL aerolysin or 10 ng/mL heat-killed (HK) aerolysin for 4 hours. Nuclei were stained with Hoechst (blue) and SR-FSLCK (red) which stained activated serine protease. A single z-slice is

represented. White arrowheads indicate nucleoli in zoom. Scale bar is 10 µm. (E)

Coverslips in (B) were quantified using confocal microscopy. At least 3 random fields of view and ≥50 GFP positive cells were counted in three or more independent experiments. Unpaired Student’s t-test was used to test for significance, and data are expressed as mean + SEM. (F) Activated serine

proteases were detected by FSLCK treatment in BECs infected with UTI89 or

intoxicated with aerolysin (5 ng/mL) for 4 hours. Cell lysates were then analyzed

by western blots probed by the anti-FITC antibody. *= band corresponding to

Try4. Experiments were repeated twice, and a representative image is shown. 103

(G) Paxillin and actin levels in BECs intoxicated with aerolysin at the concentrations indicated for 4 hours.

104

105

Figure 3.3 Continued

106

Figure 3.4. Potassium Efflux Is Necessary and Sufficient for Mediating Try4

Localization into the Nucleolus (A) BECs cells were transfected on coverslips

with WT Try4-EGFP. Cells were then pretreated for 1 hour with either DMSO or

BAPTA-AM (10 µM) and then infected with UTI89 for 4 hours. Coverslips were

quantified using confocal microscopy. (B) BECs were treated, infected, and

analyzed as described in (A) but with H2O or KCl (10 mM). (C) BECs were

transfected with WT Try4-EGFP and then treated with either DMSO or

Valinomycin (40 µM) for 4 hours. At least three random fields of view and ≥50

GFP positive cells were counted in three or more independent experiments.

Unpaired Student’s t-test was used to test for significance, and data are expressed as mean + SEM.

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108

Figure 3.5. Try4 Trafficking into the Nucleus Is Mediated by Importin-β

(A) BECs cells were transfected on coverslips with WT Try4-EGFP. Cells were then pretreated for 1 hour with either DMSO or Nocodazole (33 µM) and then infected with UTI89 for 4 hours. Coverslips were quantified using confocal microscopy. (B) BECs were transfected with WT Try4-EGFP and infected with

UTI89 for 4 hours in the presence of either DMSO, Ivermectin (1 µM), or

Importazole (10 µM). At least three random fields of view and ≥50 GFP positive cells were counted in three or more independent experiments. Unpaired

Student’s t-test was used to test for significance, and data are expressed as mean + SEM.

109

110

Figure 3.6. Caspase-14 Facilitates Try4 Trafficking into the Nucleolus (A)

BECs were transfected with Try4-FLAG and infected with UTI89 or UTI89∆hlyA for 3 hours. Try4 and interacting partners were co-immunoprecipitated using the

FLAG epitope and separated by SDS-page and silver stained. Band * was excised, sent for mass spectrometry analysis, and was identified as Caspase-14.

(B) BECs cells were transfected on coverslips with WT Try4-EGFP. Cells were then pretreated for 2 hours with either DMSO or Z-VAD-FMK (100 µM) or Ac-

YVAD-CMK (100 µM) and then infected with UTI89 for 4 hours. Coverslips were quantified using confocal microscopy. At least three random fields of view and

≥50 GFP positive cells were counted in three or more independent experiments.

Unpaired Student’s t-test was used to test for significance, and data are expressed as mean + SEM. (C) BECs were co-transfected with the indicated

plasmid and Try4-EGFP and then infected with the indicated strains (hlyA + or -) for 4 hours. Coverslips were counted and analyzed as in (B). Inset is a representative western blot of over-expression of caspase-14 or the empty vector control in BECs.

111

112

Figure 3.7. Inhibition of Try4 During UTI or Sepsis Protects the Host from

Inflammation and Death (A) Bladder homogenates from 24-hour infected F11

CBA/J mice were enumerated to determine the impact of diminazene on bacterial titers. Experiments were repeated at least twice, and the combined data are presented as medians. Significance was assessed by Student’s t-test. (B) UTI histology was blindly scored by a pathologist evaluating three different criteria of inflammation as indicated (for details see Materials and Methods). Multiple slides and sections were scored and totaled per mouse bladder. Data are presented as medians, and unpaired Student’s t-test (Mann-Whitney) comparing log-rank was applied to test for significance. (C) Representative images of F11 infected mouse bladder cross sections stained with H&E that were scored as described in (B). I- control infected bladder morphology at 24 hours with minimal edema. II- diminazene treatment increased edema indicated by the black arrow. III- Control treated bladder demonstrating submucosal infiltrates. IV- Control treated bladders demonstrating mucosal infiltrates. (D) Swiss Webster mice were infected by i.p with the indicated strains, and survival was monitored over 48 hours. Survival curves were plotted using PRISM8, and significance was tested using the Log-rank (Mantel-Cox) test. Mouse experiments were repeated at least twice, and the total combined data from 9 or more animals are presented. (E)

Mice were infected as with the indicated strains and 1 hour post infection received either vehicle or diminazene (4mg/kg) by i.p. Data analyses and graphing are described in (D). (F) Mice were treated as described in (E) and 113 infected with the indicated strains. Data analyses and graphing are described in

(D).

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115

Figure 3.8 Working Model of the Intracellular Trafficking of Try4 in

Response to HlyA Intoxication During infection HlyA is secreted from ExPEC.

The PFT binds, oligomerizes, and inserts into the host membrane. The immediate repurcussions of pore formation are the flow of ions down gradients, i.e., potassium rushes out. The changes in ion concentrations promote a kosmotrophic environment that causes dimerization and activation of caspase-

14. Activated caspase-14 binds to Try4 and mediates its translocation through the nuclear pore complex, via importin β, and into the nucleolar detention center.

Credit Amanda C Richards.

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117

Supplementary Figure 3.9 UTI89 Is More Sensitive to Diminazene Than F11

(A) Bioscreen growth curve of the indicated cultures over 24 hours in the presence of the indicated drugs or vehicle. As is shown in the graph UTI89 is more sensitive to diminazene than F11. The growth curve shown is representative data from two independent experiments. (B) Disk diffusion assays of the indicated UPEC isolates. D is diminazene, K is kanamycin, and C is saline control for the vehicle. The zone of clearance is smaller for F11 compared to

UTI89 indicating that UTI89 is more sensitive to diminazene than F11.

Kanamycin serves as a positive control.

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119

Table 3.1 Trypsinogen 4 Primers Used in This Study- Primer sequences of all primers used to clone Try4 and the truncation variants or activation mutants into their respective constructs are provided in the table below.

F-pEGFP- 5’GCATCTCGAGATG TGCGGACCTGACGAC N1 R-pEGFP- 5’GCATAAGCTTTCCGCTGTTGGCAGCGATGG N1 F-pCDH- 5’GCACGCTAGCATGTGCGGACCTGACGACAG EF1-FHC R-pCDH- 5’GCACGCGGCCGCCTTGTACAGCTCGTCCATGCCG EF1-FHC F-PRSS3- 5’GCACGCTAGCATGTGCGGACCTGACGA NheI F- 5’GCAGGCTAGCATGGTCCCCTTTGACGATGATGACAAG mesocore- EGFP-N1 R-PRSS3 5’GCCTCGAGCTTTCCGCTGTTGGCAGC XhoI- EGFP-N1 F-S257A 5’TGCCAGCGTGACGCTGGTGGCCCT mutagenes is R-S257A 5’CAGGGCCACCAGCGTCACGCTGGCA mutagenes is F-EEEE- 5’TGCTGTCCCCTTTGAGGAGGAGGAGGAGATTGTTGGGG mutagenes GCTACAC is R-EEEE- 5’GTGTAGCCCCCAACAATCTCCTCCTCCTCCTCAAAGGGG mutagenes ACAGCA is

CHAPTER 4

DISCUSSION

121

The Interplay Between Host Proteases and Pore-Forming Toxins

Proteases are essential in sustaining cellular life; in fact, ~2% of the genes in the human genome encode proteases and protease inhibitors1. As such, proteases regulate various cellular functions, such as reproduction, proper maturation and development, and immunity. Similarly, proteases have also been associated with the development of neurological disease and many cancers2-8.

At the heart of all these processes is the fundamental function that all proteases carry out, the proteolysis of substrates. Appropriate targeting of substrates is imparted by the specificity of the amino acid cleaved and also by secondary and tertiary protein structures.

Proteases are classified based on their catalytic mechanism, which is named for the amino acid that resides in the catalytic site or whether a metal co- factor is required. Currently, there are five recognized types of peptidases: serine, aspartic, cysteine, threonine, and metalloprotease. The serine proteases makeup over one-third of all known proteases and are further subdivided by catalytic structure into thirteen clans9. In the study of host-pathogen interactions the big protease players that are consistently mentioned are the caspases, especially for their roles in mediating apoptosis and pyroptosis10. However, now it is understood that other proteases such as the cathepsins, calpains, and serine proteases can not only aid in propagating death pathways but also in promoting immune responses11 (see Chapter 2 for references).

In Chapter 2, I discussed the unappreciated phenomenon of host proteases becoming activated in response to pore-forming toxins (PFTs). During 122

my review of the literature, it became clear to me that activation, whether

appropriate or aberrant, could play a significant role in the outcome of the infection for the host. For instance, the PFT secreted by Streptococcus

pneumoniae, pneumolysin, promoted calcium fluxes in macrophages.

Perturbations in ionic homeostasis facilitated the activation of host proteases,

calpain, and this resulted in increased tissue damage and invasion of S.

pneumoniae into deeper tissues12. In contrast, the activation of proteases can be

harnessed by the host and be protective. An exciting example of this comes from

studies with the frog species Bombina maxima. B. maxima produce a unique

pore-forming protein (PFP) called βγ-CAT13. This PFP has a combination of

protein domains that bind to host receptors for endocytosis, and a PFT domain

that forms pores in membranes. It is expressed on mucosal surfaces, the skin, blood, and gastrointestinal tract (GI), and is upregulated in response to bacterial

infection. In a frog model of peritonitis, Xiang and co-workers discovered that βγ-

CAT was secreted by the frog’s cells and was taken up by neighboring cells

through receptor-mediated endocytosis. Inside the endolysosome, βγ-CAT

destabilized the vesicular membrane allowing lysosomal contents into the cytosol

including the cysteine protease cathepsin B. They went on to demonstrate that

active cytosolic cathepsin B stimulated the NLRP3 inflammasome resulting in the

secretion of IL-1β which was crucial in the survival B. maxima13. These examples

and others discussed in Chapter 2 show that the role of host proteases during

infection deserves further attention as they have the potential to alter the course

of infections dramatically. 123

The work I present in Chapter 3 continues to ask what are the consequences of activating host protease during infection? My research focused on an unusual serine protease called Trypsinogen 4 (Try4) and how its activation by a PFT, alpha-hemolysin (HlyA), was detrimental to the host in murine models of sepsis and UTIs.

Trypsinogen 4 an Unusual Serine Protease Living a Double Life

As previously stated, serine proteases make up a significant portion of

proteases in the human genome, ~1/3. Trypsins are a type of serine protease

that are digestive enzymes, secreted by the pancreas and small intestines where

they proteolyze proteins aiding in the digestion of food. There are three trypsins

encoded genetically by PRSS1 (cationic), PRSS2 (anionic), and PRSS3

(mesotrypsinogen). Like most proteases, trypsins are translated as an inactive

zymogen (trypsinogens) and require cleavage of the prodomain to become

active. Trypsins can also self-activate through autocleavage, thereby amplifying

the catalytic cascade. In the pancreas, trypsins are activated by another serine

protease, enterokinase, which cleaves the trypsinogens at the trypsinogen

activation peptide motif (DDDDK)14,15.

In addition to the requirement of proteolytic processing, there are other

safeguards in place to ensure that the digestive enzymes do not become

activated prematurely. Endogenous trypsin inhibitors are secreted by pancreatic

cells and are potent repressors of trypsin activity. Interestingly, mesotrypsin is

resistant to most endogenous trypsin inhibitors and can degrade them16-18. This 124 feature of mesotrypsin is thought to initiate the digestive cascades in the GI tract by de-repression of PRSS1 and PRSS2.

Mesotrypsin regulation, tissue expression, and physiological functions are quite different from those of cationic and anionic trypsins. For instance, PRSS3 encodes two distinct mRNAs due to alternative splicing and the use of different start codons. The two mRNA species generate three splice forms and are designated isoforms A, B, and C. Isoforms A and B, also known as Trypsinogen

4 (Try4) or brain trypsin, use an alternative upstream exon that is outside of the

PRSS3 locus19 . The mRNA species are different from each other concerning the first exon used; however exons 2-5 are shared between the two19. Isoforms A and B differ only by their start codon usage, AUG vs. CUG, which changes the length of the N-terminal leader sequence. Isoform C is referred to as mesotrypsinogen and is encoded by exons 1-5 within the PRSS3 locus. Unlike isoforms A and B, isoform C has a typical signal sequence and is secreted into the extracellular milieu by the pancreas and small intestines18. Of note, all three isoforms are indistinguishable in amino acid sequence starting from the trypsinogen activation peptide (DDDDK) through the carboxy terminus.

Try4 is expressed in extrapancreatic tissues such as the brain, immune cells, and many different epithelial cell types20-22. Previous work in understanding the regulation and function of Try4 and mesotrypsin has primarily centered on their reported extracellular roles in various metastatic cancers, neurodegenerative diseases, pancreatitis, and their intracellular role in the skin differentiation5,16,23-25. Expression of mesotrypsin, as well as its activity, is 125 associated with increased metastasis and the progression of pancreatic, breast, and prostate cancers5,6,23. In models of pancreatitis, mesotrypsin can cleave protease-activate receptors (PAR), which increase pain, inflammation, recruitment of immune cells, and necrosis24,25. In Chapter 3, we used the anti- trypanolytic drug diminazene to inhibit Try4 in murine models of sepsis and UTIs.

We were able to significantly reduced the inflammation in the UTI model and improved overall survival in the sepsis model. In light of the reported role of mesotrypsin in mediating inflammation and pain, our results support the hypothesis that inhibiting HlyA mediated activation of Try4 is beneficial to the host.

Try4 also appears to be unique in how it can be activated. In addition to cleavage of the DDDDK motif by enterokinase, there is experimental evidence that at least two other proteases can activate or cleave Try4. These alternative methods of activation make sense in light of the broad extrapancreatic expression of Try4. In vitro and in vivo models of pancreatitis have shown that the lysosomal protease, cathepsin B, can activate all the trypsins but preferentially Try426-28. These data are intriguing in light of the links between lysosomal membrane permeabilization (LMP), where lysosomal contents leak out into the cytosol, cell death, and the activation of the NLRP3 inflammasome29-31.

These observations lead us to speculate that perhaps LMP, which occurs during intoxication with many PFTs (Chapter 2 for details), may be occurring in our infection model. If that is the case, then it is possible that cytosolic cathepsin B could activate Try4. 126

Furthermore, while cathepsin B activity within the cytosol has been shown

to activate the NLRP3 inflammasome, Try4 has also been linked to its activity32.

One could propose a model in which HlyA mediated LMP releases cathepsin B

which then activates Try4 and modulates the assembly of the NLRP3

inflammasome. Moreover, the exact mechanism of cathepsin B activation of the

NLRP3 inflammasome is not known. Perhaps a missing piece of the puzzle is

Try4.

Another protease that has links to activation of Try4 is the newly

discovered caspase-14, which is expressed only in terrestrial mammals33.

Caspase-14 is a member of the caspase aspartyl protease family that is

important for the terminal differentiation of complex epithelia, i.e., skin cells34,35.

While it clusters phylogenetically with the apoptotic caspases36, a direct role in

mediating apoptotic cell death has not been shown, and one study even showed

that it might promote survival37. Similar to the other caspases, caspase-14 is

produced as an inactive zymogen. But in addition to requiring proteolytic

cleavage in between the large and small subunits38, full activation also requires the presence of high concentrations of kosmotropic salts (i.e., Na+, carbonate,

zinc) to induce dimerization39.

In the skin caspase-14 expression is limited to the outermost layer, which

is where keratinocytes are undergoing the final steps in terminal differentiation

into corneocytes40. Keratinocyte differentiation is a programmed form of cell

death that is distinct from apoptosis. It is a complex process by which the cells

stop dividing, undergo extensive cytoskeletal changes, increase their lipid 127 content, and finally discard their intracellular organelles including the nucleus41.

The differentiated cells (corneocytes) make up the outermost layer of the skin, which is water impermeable and able to withstand mechanical stretching. Studies in keratinocytes have demonstrated that caspase-14 can activate mesotrypsin and that the two proteases mediate the processing of prosaposin and pro- filaggrin34,42,43. Filaggrin and saposin processing are critical final steps in the differentiation of keratinocytes into corneocytes.

Recent work has identified mesotrypsin and caspase-14 as central figures in facilitating the keratinocyte cell death. Many of the initial steps in keratinocyte differentiation are well characterized, but the process of denucleation is poorly understood. Yamamoto-Tanaka et al. showed that both proteases were activated during denucleation of keratinocytes and were found in the nuclear portion of extracts from keratinocytes and corneocytes. Mesotrypsin processing of pro- filaggrin released an N-terminal fragment (FLG-N) which was found to translocate into the nucleus where FLG-N initiated DNA degradation, although the mechanism of how this occurs is not known42.

In a parallel pathway, caspase-14 was shown to also promote the DNA fragmentation through a subapoptotic pathway mediated by caspase-activated

DNAses (CADs)42,44. CADs are co-transcribed with their inhibitor, inhibitor of caspase-activated DNAse (ICAD). These proteins are present in the nucleus as heterodimers. Stimulation of cells with either subapoptotic stimuli or in response to cell differentiation cues results in the caspase-mediated cleavage of ICAD, the release of CAD within the nucleus, and subsequent fragmentation of DNA. 128

Yamamoto-Tanaka and co-workers observed that caspase-14 degraded ICAD

and resulted in DNA fragmentation. Also, inhibition of mesotrypsin and caspase-

14 resulted in increased undigested nuclei in the cornified layer. In sum, the

activation of mesotrypsin and caspase-14 in keratinocytes appears to be two

separate but complementary ways in which efficient denucleation can occur42.

Taking into account the previously discussed data, we find our observation

that caspase-14 and Try4 interact during HlyA intoxication to be very plausible.

The stresses that a cell encounters during HlyA intoxication are not entirely

known, but the fluxes of ions, particularly K+, are well documented. We argue that

the efflux of K+, as a result of membrane damage, is sufficient to mimic the

kosmotropic environment that caspase-14 requires for full activation. As K+

leaves the cell, other ions rush in promoting an environment that facilitates

“ordered” protein confirmations. Active caspase-14 could cleave and activate

Try4, which results in the degradation of many host factors45, and activation of

the NLRP3 inflammasome. Since other epithelial cells express both caspase-14 and mesotrypsin20,21,46 (or Try4 splice variants), we argue that the type of cell death that is occurring in bladder epithelial cells is related to the programmed cell death of keratinocytes.

Another possible explanation for the interactions between caspase-14 and

Try4 during intoxication focuses more on our trafficking data. We observed that

PFTs cause Try4 to be recruited into the nucleus, specifically the nucleolus.

Since Try4 has no known nuclear localization signal (NLS) the mechanism by which Try4 enters the nucleus is unknown. We surmised, concerning our co- 129

immunoprecipitation results (Figure 2.6), that Try4 may utilize a protein

chaperone to enter the nucleus. There are several lines of evidence that support

this model, the first of which is that caspase-14 has an NLS suggesting that it can

enter the nucleus37. Limited studies have examined the exact mechanism by

which caspase-14 traffics into the nucleus. However, based on our studies using

nuclear transport inhibitors (Figure 2.5, Chapter 3), we argue that the transport of

caspase-14 may occur through direct binding with karyopherin B (importin β).

Furthermore, the structure and characteristics of caspase-14’s NLS favor a

nuclear import model that is mediated by importin β47,48.

Nucleolar Detention: Helpful or Harmful?

Nucleolar localization of protease in response to stress, while appearing

strange or even dangerous, is not unique to Try4. At least two other proteases,

caspase-2, and caspase-6, have been reported to localize within the nucleolus in

response to stress49,50. The caspase-2 data are particularly interesting because

of the percentage of amino acid identity that is shared with caspase-14 (~26%)37.

The similarities between the two caspases appear to be conserved even in regard to their cellular roles. For example, in response to DNA damage, caspase-

2 re-localizes into the nucleolus and associates with a supramolecular complex called the PIDDosome (PIDD-RAIDD-caspase-2 complex)49. Caspase-2

recruitment into the nucleolus resulted in the apoptosis of DNA damaged cells.

Strikingly, as evidenced by siRNA knockdown experiments, retention of caspase-

2 in the nucleolus occurred by direct interaction with a nucleolar protein 130 phosphoprotein nucleophosmin (NPM1).

Another report demonstrated that the effector caspase, caspase-6, also localized to the nucleolus in response to over-expression of the novel apoptosis signaling protein called death effector domain DNA binding protein (DEDD)50

(see 51 for review of apoptosis). Death effector domains (DED) are protein/protein interactions in molecules that participate in apoptotic signaling. For example,

DEDs are found within caspases 8 and 10, PIDD, as well as the Fas-associated death domain (FADD) adaptor protein. Upon death receptor ligation (TNFα or

FasL) FADD is recruited to the cytoplasmic tail of the receptor. The proximity of the DEDs of FADD and caspase-8 facilitate their dimerization, which activates caspase-8. Overexpression of the DEDD protein resulted in its localization with the nucleolus, the site of transcription for ribosomal DNA (rDNA). Moreover, they showed that DEDD recruited and activated caspase-6 in the nucleolus. However, the induction of apoptosis was independent of caspase activation and was instead determined to be due to DEDD inhibition of RNA polymerase I50.

These reports, along with our observations, support a newly described pathway by which cells can respond to stress that has been termed “the nucleolar detention center” (NDC)52-54. Recent studies have illuminated an exciting and uncharacteristic role for the nucleolus. Even though ribosomal biogenesis is the primary function attributed to nucleoli, the subnuclear compartment has other functions55. Indeed, nucleoli are responsive to cellular stress signals such as DNA damage, nutrient stress, viral infection, and osmotic stress55,56. For instance, exposure to either low pH or heat shock caused an 131 increase in the Pol 1 transcription of long noncoding RNAs (lncRNAs) from specific intergenic segments of rDNA53. These lncRNAs were found to mediate the static sequestration of the E3 ubiquitin ligases von Hippel-Lindau (VHL) and murine double minute protein (MDM2) which prevented the ubiquitin targeting of hypoxia-inducible factor (HIF) and p53, respectively57. Furthermore, the function and regulation of the lncRNAs were specific for each of the cellular stresses (pH vs. heat shock). The sequestration of the ubiquitin machinery in the nucleolus protected particular proteins from being targeted for destruction.

In light of what is known about the NDC pathway, we propose a model by which bladder epithelial cells can sequester Try4 in response to PFTs. Perhaps

Try4 is localized to the nucleolus by caspase-14 during intoxication to prevent its activation or further catalysis of downstream targets. In the case of HlyA intoxication, we propose that the cell is putting Try4 in a “timeout” in the NDC in hopes of allowing the stress responses to save itself from death. Or an alternative explanation is that Try4 trafficking during intoxication is more in line with that of caspase-2 and -6, where they are serving functions related to mediating cell death. The results presented in our in vivo mouse models of UTIs and sepsis (Figure 2.7, Chapter 3) would favor the “timeout” model for why Try4 is sequestered in the nucleolus. The study presented in Chapter 3 lays the groundwork for beginning to tease apart those questions molecularly.

In conclusion, the work presented here has touched upon many different aspects of cell biology in the context of PFT biology. My work discovered the domain requirements for Try4 intracellular trafficking as well as some of the 132 cellular cues that mediate translocation into the nucleus. Also, I identified two additional PFTs promote the nucleolar sequestration of Try4. However, Try4 sequestration was independent of its activation. Moreover, my research has shown that HlyA mediated activation of serine proteases, particularly Try4, results in increased inflammation in UTIs and death. I hope that my work will bring to light the importance that host proteases play during microbial infections specifically in the context of PFTs.

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