Comparative Functional Analysis of Secreted Autotransporter Toxin (Sat) in Uropathogenic E

Comparative Functional Analysis of Secreted Autotransporter Toxin (Sat) in Uropathogenic E. coli CFT073 and probiotic E.coli Nissle 1917 in mammalian cells

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

Biological Sciences

Taylor Michelle Halsey

2018

SIGNATURE PAGE

THESIS: COMPARATIVE FUNCTIONAL ANALYSIS OF SECRETED AUTOTRANSPORTER TOXIN (SAT) IN UROPATHOGENIC E. COLI CFT073 AND PROBIOTIC E. COLI NISSLE 1917 IN MAMMALIAN CELLS

AUTHOR: Taylor Michelle Halsey

DATE SUBMITTED: Fall 2018

Department of Biological Sciences

Dr. Christos Stathopoulos ______Thesis Committee Chair Biological Sciences

Dr. Nancy Buckley ______Biological Sciences

Dr. Junjun Liu ______Biological Sciences

ii Acknowledgements

I would like to sincerely thank my entire support system for walking with me through this journey. To my support system at Cal Poly Pomona, a place that I have called a home for the past eight years, I say thank you for your collective belief in my academic process and for giving me an education that will allow for so many opportunities in the future; the faculty and staff at Cal Poly Pomona have provided me with a tremendous gift and I promise to use this gift to continue to help others. To the faculty that have helped me the most, including members of my thesis committee, Drs.

Buckley, Liu, and Stathopoulos (thesis committee chair), as well as the coordinators for the NIH-RISE program Drs. Adler and Valdes, thank you all for investing your precious time in me as a student and a researcher; your guidance has shaped my path and I hope to be able to inspire a new generation of scientists just as you all have inspired me. Finally, to my family and friends, you each hold a different piece of my heart and I know I could not have gotten through this program without all of your love and support. Through all of the long days and nights in the lab and all of the times I thought I could not complete this task, it was you that made it easier and allowed me to keep my goals in sight; this great project could have never been completed without all of you.

iii Abstract

Autotransporter proteins, typically associated with virulence in pathogenic bacteria, are thought to serve a different function in probiotic bacteria. One such protein, secreted autotransporter toxin (Sat), has been shown in numerous pathogenic bacteria to cause vacuolization and cytotoxic effects on cells. Sat is also secreted in high amounts by the non-pathogenic, probiotic Escherichia coli Nissle 1917 (EcN). The use of EcN is under review as a candidate probiotic supplement in the United States and approval has been delayed in part because of genomic similarities to uropathogenic E. coli (UPEC) CFT073, which is responsible for approximately 70% of non-hospital acquired urinary tract infections nationwide. Previous generalized studies have indicated that when Sat functions collaboratively with other native proteins in EcN, its cytotoxic effects are negated, but the mechanisms behind these actions are unknown. To independently study the effects of Sat, we used a recombinant sat overexpression plasmid derived from UPEC CFT073 (pSat) transformed into the non-pathogenic E. coli strain HB101 (HB101 pSat). Concentrated cell-free supernatant was obtained from wildtype EcN and wildtype UPEC CFT073, as well as EcNDSat for assays and an SDS-PAGE and Bradford protein concentration assay were performed to confirm the expression, concentration, and secretion profiles. In vitro infection challenges using the HeLa and Vero cell lines were performed and analyzed using the MTT cytotoxicity assay. Our preliminary results showed that Sat of UPEC

CFT073 decreased viability of undifferentiated epithelial cells when subjected to challenges with concentrated cell-free supernatant. When the same challenges were given to Vero cells, a greater level of toxicity was conferred on the differentiated epithelial cell line. Cytotoxic effects in Vero cells were amplified following crude purification of cell-

iv free supernatant samples using ammonium sulfate precipitation. These results suggest that

Sat of UPEC CFT073 confers damage to host epithelial cells and may have greater effects on relevant differentiated cell lines. It is also suggested that purified Sat protein from both

EcN and UPEC CFT073 confer similar levels of cytotoxicity to Vero cells and reduce viability compared to the concentrated supernatant samples alone. Further experiments are needed to determine the mechanisms of action behind these results and target the specific factors that prevent Sat of EcN from displaying cytotoxic features in host cells.

v TABLE OF CONTENTS

SIGNATURE PAGE ...... ii

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... iv

INTRODUCTION ...... 1

1. Escherichia coli ...... 1

2. E. coli Nissle 1917 ...... 4

3. Probiotic Bacteria ...... 10

4. Uropathogenic E. coli strain CFT073 ...... 13

5. Similarities between EcN and UPEC CFT073 ...... 15

6. Bacterial Toxins ...... 16

7. Gram-negative Bacteria Secretion Mechanisms ...... 19

8. Type V (Autotransporter) Protein Secretion ...... 21

9. Adhesin Involved in Diffuse Adherence (AIDA-I) and Trimeric Autotransporter

Adhesins (TAA) ...... 28

10. Serine Protease Autotransporters of Enterobactericiaea (SPATE)...……...... …...... 29

11. Secreted Autotransporter Toxin (Sat) ...... 31

vi 12. In vitro Cell Models ...... 35

13. Previous Stathopoulos Lab Findings ...... 36

14. Thesis Scope and Focus ...... 40

MATERIALS AND METHODS ...... 41

1. Bacterial Strains and Plasmids ...... 41

a) Bacteria Media Preparation ...... 41

b) Preparations of Bacterial Overnight Cultures ...... 42

c) Preparations of Bacterial Day Cultures ...... 42

2. Bacterial Protein Work ...... 42

a) Supernatant Isolation and Concentration ...... 42

b) Isolation and Partial Purification of Secreted Autotransporter Toxin ...... 43

c) Acetone Protein Precipitation ...... 43

d) SDS-PAGE/Silver Stain Analysis ...... 44

3. Cell Lines Used in Study ...... 47

a) Cell Culture Media Preparation ...... 47

4. Oligopeptide Cleavage Assay ...... 48

5. MTT Cytotoxicity Assay ...... 48

6. Data Analysis and Statistics ...... 49

RESULTS ...... 50

1. Concentration of Sat protein from select strains using Filtered Centrifugation ...... 50

2. Effects of concentrated Sat of UPEC CFT073 on cell viability of HeLa cell line ... 54

vii 3. Concentrated Sat of UPEC CFT073 decreases cell viability in Vero cell line ...... 60

4. Supernatants of Wildtype EcN and Wildtype UPEC CFT073 and their effects Vero

cell line viability in vitro ...... 63

5. Partially purified Sat of UPEC CFT073 effects on Vero cell line from recombinant

HB101/pSat ...... 63

6. Partially purified Sat from EcN elicits similar effects as Sat from UPEC CFT073 on Vero

cells…………………………...…………………………………………………...... 65

DISCUSSION ...... 69

WORKS CITED ...... 84

viii

Introduction

1. Escherichia coli

Escherichia coli (E. coli) are a diverse family of gram-negative bacteria found in numerous environments. Gram-negative bacteria are typically characterized and differentiated from their gram-positive counterparts by the components and structure of the cell wall; Figure 1 shows the structures of both gram-positive and gram-negative bacteria cell walls. Briefly, they are composed of an inner (cytoplasmic) and outer membrane, separated by a periplasm containing a small amount of peptidoglycan.

Peptidoglycan is an essential bacterial cell envelope component that contributes to cell structure and, for gram-positive bacteria, carries teichoic acid and polysaccharide residues to the surface (Kang et al, 1998).

The main difference between gram-positive and gram-negative bacteria is the addition of an outer membrane for gram-negative species. This additional layer contains lipopolysaccharide (LPS) which contributes directly to gram-negative bacteria pathogenicity (Brown et al, 2015). LPS has three main parts: lipid A, an endotoxin that anchors LPS to the outer membrane, an oligosaccharide core, and an O-antigen (Figure 1) that is a target for host antibody recognition and displays species specificity (Raetz and

Whitfield, 2002); for E. coli alone, there are over 160 known O-antigen variants. LPS is also commonly targeted for attack by the immune system and so it can be used as a signal to the body that it is undergoing infection.

Figure 1: Schematic diagrams of gram-negative (a) and gram-positive (b) cell wall components. The figure highlights the location and thickness of the peptidoglycan layer and lipoproteins in each sub-figure (Adapted from Brown et al, 2015).

2 E. coli are found in different environments and reservoirs worldwide. Relevant species for this study include those located in humans as a part of their microbiota, the resident bacteria in a host. The total wet weight of all bacteria in the average human host is about 1.5 kg. The human microbiota is composed of hundreds of species of bacteria that have been shown to effect almost every facet of life; studies devoted to the microbiome have shown that bacterial composition, with all their byproducts, affect everything from nutrient metabolism to tumorigenesis (Breton et al, 2016; Tlaskalová-

Hogenová et al, 2004). Recent studies have shown that it is the upkeep and cooperation of the bacteria in the body that keeps many vital processes working consistently.

From a medical perspective, E. coli are most abundant in the gastrointestinal (GI) tracts of most mammals. In humans, the GI tract (with focus placed on the large intestine

(colon), small intestine, and stomach) is also the most densely concentrated area in the human microbiota; approximately 1x1011 bacteria/mL reside in the colon, with an additional 1x107 bacteria/mL combined in the stomach and small intestine. (Sender,

Fuchs, and Milo, 2016). E. coli are the most populous bacteria found in the GI tract and over 100 different sub-species live throughout the human body. It has also been shown that E. coli are among the first species to colonize infants following birth (Nowrouzian et al, 2003). Because of numerous host-microbe interactions, it has yet to be elucidated the exact impact E. coli have on overall microbiota health and integrity.

Commensal E. coli in the GI tract can be further distinguished from each other by their ability to cause disease. Non-pathogenic commensal E. coli are definitively unable to cause infection in any part of the body. These bacteria have a mutual relationship with the host and utilize host nutrients to perform metabolic functions but do not cause any

3 harm in the process. Contrarily, according to the Centers for Disease Control and

Prevention (CDC), there are six known pathotypes of pathogenic E. coli: shiga toxin- producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli

(EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC). Extra-intestinal pathogenic E. coli are members of the GI microbiota and do not cause disease in those areas; when introduced to sterile areas of the body (examples include bladder, kidney, and lungs), the bacteria act as pathogens and cause infection. The severity of those infections is dependent on the final location of colonization. Both groups of E. coli possess factors that directly assist with colonization of the GI tract; these same factors are also involved in the successful colonization of sterile environments by extra-intestinal pathogenic E. coli. The relevant E. coli species featured in this study are non-pathogenic, probiotic E. coli Nissle 1917 (EcN) and uropathogenic E. coli (UPEC) CFT073.

2. Escherichia coli Nissle 1917

E. coli Nissle 1917 (EcN) are non-pathogenic, commensal bacteria found within the GI tract of the human body. This species was discovered in 1917 by Dr. Alfred Nissle during World War I when a group of soldiers were afflicted by a diarrheal disease (most commonly thought to be dysentery caused by Shigella dysenteriae) but one soldier was unaffected (Wassenaar, 2016). Dr. Nissle was able to isolate and characterize this new strain of E. coli from the stool of the healthy soldier and identified it as a contributing factor in their protection from the disease. To confirm his results, he then fed the isolated strain to soldiers with dysentery and recorded steady improvement shortly after (Nissle,

1959). Decades later, EcN is still utilized globally in dietary supplements that treat

4 ailments of the GI tract including ulcerative colitis, inflammatory bowel disease (IBD), and adolescent diarrhea (Hancock et al, 2010), as well as general upkeep and maintenance once the diseases have been eliminated (Hering et al, 2013).

Since its discovery, EcN has been used as the active agent in several probiotic drugs across European and Asian countries. These drugs have been administered as oral supplements marketed for relief of symptoms related to gastrointestinal disorders, most notably those that incorporate chronic intestinal inflammation such as Irritable Bowel

Syndrome (IBS) for human children and adults (Mutaflor; EcN), as well as large mammals (Ponsocol); these supplements are popular in their respective countries and have been in production since 1936 (Wassenar, 2016). There are also variants of the wildtype EcN strain that are being sold as probiotic supplements; Symbioflor2 is a combination of six different commensal E. coli strains in addition to EcN while Colinfant is a mutant strain of EcN that has one of its potential toxins, toxin heat-stable lyosin knocked out (Wassenar, 2016). Both strains are marketed to relieve general symptoms of

GI tract disorders and have been in production for the past 40 years.

The full EcN genome was sequenced in 2014 (Figure 2). EcN has areas of genetic clusters, known as Genomic Islands (GEIs) and it is thought that proteins of similar size, functionality, and/or evolutionary conservation are grouped together (Grozdanov et al,

2004). The EcN genome encodes for numerous proteins that promote its viability in a host: adhesins (e.g. type 1 fimbriae, curli, Sap-like), proteases (e.g. Tsh and Sat), and iron acquisition systems (e.g. yersiniabactin, salmochelin) work together to ensure EcN survival, even in areas with less than ideal conditions and/or high bacterial competition

(Grombach et al, 2010).

Figure 2: Schematic diagram of the genome of E. coli Nissle 1917. Genomic Islands I-IV and smaller genomic islets are indicated by the triangles and code for fitness factors. The appropriate gene locations are representative of the specific chromosomal insertion site (Adapted from Grozdanov et al., 2004).

6 As a member of the GI, EcN resides primarily in the colon and assists the other residual bacteria in the reabsorption of water. It has been shown to possess fitness factors that aid in its ability to colonize its host; these factors have been summarized in Figure 3.

These fitness factors help with competitive exclusion of pathogens and immunomodulation for the host. EcN is particularly good at forming strong biofilms that consistently outcompete pathogens and other commensal bacteria residing in the gut.

(Hancock et al, 2010). Other notable characteristics of EcN include a sophisticated iron uptake system, secretion of potent microcins, and a short O-side chain contributing to a structurally unique LPS. Because LPS is one of the main identifiable traits of gram- negative bacteria by host immune defenses, it is thought that the structural differences may be one of the features that makes EcN non-pathogenic. It is also notably different from the more common probiotic supplements currently on the market (ex. Lactobacillus and Bifidiobacteria) because it is one of the few gram-negative probiotics available for purchase (Kandasamy et al, 2016).

Figure 3: Summary of common fitness factors in E. coli Nissle 1917. The probiotic EcN has most of the basic characteristics associated with E. coli species: fimbriae, iron transport systems, lipopolysaccharide (LPS) on the outer membrane, and microcin secretion. These features aid EcN in colonization and competitive exclusion of pathogens (Adapted from Behnsen et al, 2013).

8 The mechanisms of action for EcN remain mostly unknown but a few discoveries in the last few years have highlighted some of its key features. For example, it is known that EcN works most efficiently in an inflamed environment and actively reduces the production of inflammatory cytokines in the gut, likely by its ability to stimulate the production of anti-inflammatory cytokines like IL-10 (Underwood, 2014; Helwig et al,

2006). EcN also has the ability to successfully sequester iron from host proteins using high-affinity iron chelators (siderophores). These siderophores are resistant to antimicrobial peptide lipocalin 2, which is produced by the host in an attempt to protect it from further damage (Deriu et al, 2013). Other pathogenic bacteria, such as Salmonella typhimurium, also have powerful siderophores that contribute to their colonization of a host but EcN has multiple iron uptake systems that outcompete S. typhimurium and others in iron-depleted environments (Deriu et al, 2013).

An area of particular interest in EcN research is its ability to affect intestinal epithelial barrier function. Studies have shown that EcN supernatant has the ability to increase transepithelial resistance and reduce permeability by sealing paracellular passage pathways utilizing the secreted protein TpcC (Hering et al, 2013). This indicates that some elements, including secreted products, of EcN may be able to alter cellular permeability, even without whole cell contact.

Because EcN shares many of the key characteristics of a typical E. coli species, there are potential dangers to consider in its use as a primary probiotic in GI tract dysbiosis and ailments. One study, performed by Grombach et al (2010), showed that

EcN is a powerful colonizer in germ-free and immunocompromised mice which led to high mortality after rapid translocation to other areas of the body. Although tens of

9 thousands of patients worldwide have been positively affected by treatment with EcN- based probiotics since its inception as a medicinal product, three key elements are needed for Food and Drug Administration (FDA) approval in the United States: 1) proof of efficacy in controlled clinical studies, 2) proven product quality, and 3) information on mechanism of action (Lane, 2015).

3. Probiotic Bacteria

Probiotics are defined by the World Health Organization (WHO) as live organisms that, when given at appropriate amounts, provide a health benefit to the host

(Sonnenborn and Schulze, 2009). The exact benefit to the host varies between microorganisms but probiotics in mammals generally help with intestinal motility, inhibiting growth of pathogens through outcompeting for limited resources and/or production of microcins, and immunomodulation . In comparison to antibiotics

(medicinal substances used to kill/halt growth of living microorganisms) and prebiotics

(simple carbohydrates that promote probiotic and commensal bacteria growth), probiotics typically focus on prevention of pathogenic bacterial colonization and have the most potential for actively enhancing host health (Figure 4, Britton and Versalovic, 2009;

Singh et al, 2013).

More specifically, probiotics are rising in popularity partially due to their ability to provide protection from pathogens and toxins that infiltrate the GI tract, a characteristic that is very attractive as more pathogens become resistant to current antibiotic treatments; in the United States alone, approximately 3.9 million adults reported regular use of probiotic supplements in 2012, a number that is four times higher

10 than the reported use in 2007 (Clark, 2015). Typically, probiotic supplements must be

ingested regularly to maintain health benefits, so research efforts have been focused on

understanding their mechanisms of action as their appeal grows.

Figure 4: Common benefits of probiotic bacteria on gut health. The diagram above highlights the most commonly established benefits of probiotic bacteria (purple) on gut epithelial cells. Key features include interaction with the gut lumen and intestinal epithelium, anti-pathogenic functions, and immunomodulatory effects (Adapted from Britton and Versalovic, 2009).

11 It is now widely known that the composition of microbial communities in the body significantly affects the host in numerous ways. There are commensal bacteria and yeast that have been shown to have probiotic effects; different species of Lactobacillus,

Lactococcus, and Bifidiobacteria are most commonly used for human benefit (Vitetta,

Linnane, and Gobe, 2013). These bacterial families are gram-positive and typically used in food production processes (e.g. milk and dairy products, pickles) and no species in their families have been implicated in pathogenesis. These lactic acid bacteria are ideal for use as probiotics due to their absences of LPS and secreted proteases (Behnsen et al,

2013). Recently, some isolated strains of traditionally pathogenic families of bacteria have also been shown to have probiotic qualities in mammals; select strains of Bacillus,

Clostridium, and Escherichia have all been implicated for probiotic use.

Even with mounting evidence of probiotic safety, overconsumption of certain species has also been shown to display negative effects on the host. These situations are rare but adverse side effects have occurred with an excess of probiotics in immunocompromised individuals, as well as those without a well-established immune system (i.e. infants, elderly people) (Besselink et al, 2008). Generally, the four main side effects associated with some probiotic strains are 1) systemic infections and sepsis, 2) deleterious metabolic activities, 3) excessive immune stimulation, and 4) gene transfer

(Doron and Snydman, 2015). It has been determined recently that host characteristics (i.e. existing microbiota and intestinal immune integrity) play an essential role in the overall efficacy of the probiotic (Gronbach et al, 2010).

12 4. Uropathogenic Escherichia coli CFT073

Uropathogenic Escherichia coli (UPEC) CFT073 is virulent strain of uropathogenic E. coli that resides as a member of the microbiota in the GI tract of mammals and is of note due to its close genetic homology to EcN. Occasionally, UPEC

CFT073 can use its flagella to travel to sterile areas of the body; when UPEC CFT073 is specifically introduced to the urogenital tract, it causes urinary tract infections (UTI).

UPEC CFT073 is primarily responsible for 70-90% of non-hospital acquired UTIs nationwide. If left untreated, UPEC-caused UTIs can ascend into the urinary tract, colonize the bladder and kidneys, and cause potentially fatal systemic infections. (Guyer et al, 2000). This is considered particularly exceptional considering the urethral tract is considered a sterile environment of the mammalian body and is fairly well protected by non-specific defenses including antimicrobial elements within the bladder mucosa and the persistent flow of urine (Bahrani-Mougeot et al, 2002). It has also been shown that

UPEC CFT073 is capable of evading natural host defenses and can alter host physiology after significant infection time (Guyer et al, 2000). Common E. coli features such as the p fimbriae found on the outer membrane contribute directly to the ability of UPEC CFT073 to colonize extra-intestinal areas of the body; these features, now known as virulence factors for pathogenic species, are generally identical to those found in commensal, non- pathogenic strains.

The complete genome of UPEC CFT073 was sequenced and published in 2002

(Welch et al, 2002; Kao et al, 2002, Figure 5). The sequence contains ten known pathogenicity islands that encode virulence factors and a few additional housekeeping

13 proteins. These pathogenicity islands are highly conserved across different species of E.

coli and many related strains possess homologs in their own genomes.

Figure 5: Visual representation of the genome of uropathogenic E. coli (UPEC) CFT073. The entire genome of UPEC CFT073 is 5,231,428 base pairs (bp) long with at least seven distinct pathogenicity islands. Pathogenicity islands and phage regions are labeled according to their chromosomal insertion site (Adapted from Vejborg et al, 2010).

14 5. Similarities between EcN and UPEC CFT073

Both EcN and UPEC CFT073 are found naturally in the GI tract of healthy individuals and have both been shown to contribute to the fitness of the host as members of the microbiota. In addition to the obvious similarities these two strains share as members of the Escherichia coli species, some unexpected traits plague the probiotic

EcN and contribute to its potential dangers. In a study performed by Hancock, Vejborg, and Klemm (2009), it was shown that EcN grew readily in urine and outcompeted UPEC

CFT073 in planktonic growth. This is a testament to the colonization potential for EcN, a feature that is of great interest in the field of probiotics. But, additional characteristics of the E. coli species are present in EcN, including the presence of LPS on the outer membrane and secretion of microcins to ward off other species of bacteria, which ultimately acts as more of a detriment to the idea of using EcN more readily as a probiotic supplement.

The most pressing concern lies in the presence of virulence factors in the EcN genome. As mentioned above, autotransporter proteins make up the majority of virulent agents in gram-negative bacteria and play significant roles in host colonization and pathogenesis. Upon full comparative genomic hybridization studies following the sequencing of EcN genome in 2014, it was determined that EcN had several key factors that were closely related to that of UPEC CFT073 (Toloza et al, 2015). It was further elucidated that at least eleven different autotransporter proteins are present in the EcN genome and most share high genetic homology to the same proteins found in the UPEC

CFT073 genome (Table 1, Lane 2015). This is extremely uncommon for a probiotic

15 strain of bacteria and it has been suggested that certain shared characteristics that act as virulence factors can behave as fitness factors in EcN.

6. Bacterial Toxins

Bacterial toxins are readily produced by both gram-positive and gram-negative bacteria in an effort to maintain viability in and around a host organism or environment.

Common toxin producers include Corynebacterium diptheriae, Bordetella pertussis,

Vibrio cholerae, Clostridium tetani, Bacillus anthracis, and, as previously mentioned,

Escherichia coli (Schmitt, Meysick, and O’Brien, 1999). Various virulence factors exist as a way for microbes to adapt to their environments and protect themselves from harm; the virulence aspect is an unpleasant side-effect for the host (Lebrun et al, 2009). Usually bacterial toxins can be categorized by their specific effects on the host; three mechanisms of action have been noted as the most common for bacterial toxins: 1) damage to cellular membranes/matrices, 2) inhibition of host protein synthesis, and 3) activation of secondary messenger pathways (Figure 6; Schmitt, Meysick, and O’Brien, 1999). Two of the main toxins are elucidated further below:

16 Figure 6: Representations of the modes of action for bacterial toxins. There are three main classifications of bacterial toxins and their effects on host cells: damage to cell membrane (a), inhibition of vital host protein synthesis (b), and activation of secondary messenger pathways (c) (Adapted from Schmitt, Meysick, and O’Brien, 1999).

17 Immunomodulatory Toxins

Secreted products from foreign organisms in the body trigger the innate and adaptive immune systems. Some toxins, such as the pyrogenic toxin superantigens

(PTSAgs), are known primarily for their ability to act directly on antigen-presenting cells

(APCs) and T cells from the host immune system (Schmitt, Meysick, and O’Brien, 1999).

These toxins are usually enterotoxins and can gain access to immune cell machinery with specialized receptors. An example of this type of toxin is the well-studied cholera toxin

(CT) from Vibrio cholerae; this virulence factor is known to increase cytoplasmic levels of cyclic AMP in the intestinal epithelial cells which leads to excessive loss of water in the host (Queen and Satchell, 2013). More recently, this family of toxins have been harnessed for their therapeutic potential due to their ability to gain easy access into host immune cells (Donaldson and Williams, 2009).

Proteases

Proteases are a ubiquitous feature of all living organisms. They are enzymes that have specific mechanisms that cleave host proteins through hydrolysis of peptide bonds

(Cohen, 1990). The purpose of proteolytic activity is to break down complex polypeptide substrates into for bacterial metabolism but, in doing so, they contribute to pathogenesis of the host, making them the most prevalent type of virulence factor. There are 4,000 proteases currently described and commonly target host proteins involved in cell structure and blood clot formation (Kaman et al, 2014). Proteases are produced by bacteria for a variety of reasons including response to host defense mechanisms, inactivation of antimicrobial peptides (AMPs), and generalized host tissue destruction

18 (Potempa and Pike, 2009). Common proteases include elastase from P. aeruginosa that targets elastin, AMPs, and specific cytokines, Staphopain B (SspB) from S. aureas which targets kininogen and fibronectin, Lethal Factor (LF) from B. anthracis, which also targets kininogen and AMPs, and Botulinum neurotoxin (BoNT) from C. botulinum, which targets secretory vesicle protein Synaptobrevin (Kaman et al, 2014).

7. Gram-negative Bacteria Secretion Mechanisms

Secreted bacterial proteins have numerous functions once isolated from the bacteria, examples of these roles include enhancing attachment to eukaryotic host cells and uptake of scarce environmental resources (Green and Mecsas, 2016). Gram-negative bacteria have a particular challenge in distributing virulence/fitness factors to promote colonization and reproduction inside of a host due to the characteristic outer and inner phospholipid membranes. To navigate the extra challenge, bacteria have developed different mechanisms to secrete or embed their protein products into the extracellular space or just directly on the outer membrane, respectively.

There are currently nine known bacterial secretion mechanisms for gram-negative bacteria, currently classified by types I through IX (Desvaux et al, 2009; Abby et al,

2016). These nine mechanisms can generally be broken down further into two categories: one-step (span both the inner and outer membranes through one structure) and two-step mechanisms (secretion across both the membranes requires two separate structures or processes) (Costa et al, 2015). Seven of the nine mechanisms are featured prominently in

Figure 7. The most recently discovered mechanisms (types VII through IX) are currently known to have very specific products and/or bacterial hosts; types VII and VIII both

19 utilize the chaperone-usher pathway to export the type I pili and curli, respectively (Abby

et al, 2016). The type IX secretion system seems to be affiliated only in Bacteroidetes

(Abby et al, 2016).

Figure 7: Diagram of the common protein-secretion systems for Gram-negative bacteria. To date, two different types of secretion systems exist for gram-negative bacteria: one- step (types I, III, IV, and VI) and two-step (type II and V). ECM, extracellular matrix; OM, outer membrane; P, periplasm; IM, inner membrane; C, cytoplasm (Adapted from Beekman and Vanrompay, 2010).

20 The one-step secretion methods include types I, III, IV, and VI. Type I secretion utilizes a straightforward pore component on the outer membrane connected to an ATP- binding cassette (ABC) transporter on inner membrane through which products are transported directly from the bacterial cytoplasm to the extracellular space (Delepelaire,

2004; Henderson et al, 2004); the ABC transporter is a ubiquitous superfamily of directional pumps present in all single and multi-celled organisms- it is identified by its membrane-bound structures that utilize ATP-activated flipping mechanism (Costa et al,

2015). The type III secretion system has a unique injectosome that is used as a major virulence factor transport system for diverse gram-negative pathogens (Garmendia,

Frankel, and Crepin, 2005; Kendall, 2017). There are three main parts of the canonical injectosome: the basal body, needle, and translocon (Kendall, 2017); each is required for proper secretion of the protein product into the extracellular space.

8. Type V (Autotransporter) Protein Secretion

One specific mechanism for bacterial protein transport is the Type V

(Autotransporter) pathway. This pathway is known to be the most common way gram- negative pathogens secrete virulence factors to host cells (Ruiz-Perez and Nataro, 2014).

Because of the very nature of two-step secretion, this pathway is only found in gram- negative bacteria. Proteins that utilize this pathway have a conserved structure: a long signal sequence at the N terminus, a passenger domain with repeating structural units that holds the functional protein, and a beta domain at the C terminus; the beta domain encodes the beta barrel complex that aids in the translocation of the passenger domain across the outer membrane (Dautin and Bernstein, 2007; Figure 8). This particular feature is why the type V protein secretion pathway is also known as the autotransporter

21 pathway: proteins in this family partially facilitate their own release into the extracellular space which is a unique method bacterial secretion (Maroncle et al, 2006). The beta domains of proteins in the autotransporter family are fairly conserved between bacterial species which suggests the importance of its presence in all autotransporters.

Figure 8: Two-step secretion system for autotransporter proteins. The signal sequence (yellow) is cleaved upon translocation across the inner membrane. Designated chaperone proteins found within the periplasm. The functional protein (green) is seen in multiple areas of the diagram. Chaperone proteins help shuttle the protein to the outer membrane where it utilizes beta barrel pore-forming protein components (light green), in collaboration with the Bam complex, to complete transport across the outer membrane and into the extracellular space (Adapted from Leo, Grin, and Linke, 2012).

Figure 9: Three major groups of autotransporter proteins. (a) Phylogram of 230 autotransporter proteins separated based on protein domains: SPATE (red), TAA (yellow), and AIDA-I (blue). Values in parenthesis indicate the number of proteins in each group. (b) Protein organization of the three groups of autotransporter proteins with general pfam domains. Signal sequence (orange), peptidase S6 (red), pertactin (green), autotransporter/translocator domain (blue), and YadA (yellow) domains (Adapted from Wells et al., 2010).

23 The first step of the two-step mechanism is considered a sec-dependent process

(Figure 8). The signal sequence at the N terminus recognizes a member of the Sec protein complex embedded in the inner membrane of gram-negative bacteria to gain access into the periplasm. In many two-step sec-dependent mechanisms, SecB recognizes the signal sequence and delivers the full protein to SecA which is responsible for both delivering the protein to the translocase SecYEG and providing the energy required to complete this task by acting as the resident ATPase (Green and Macsas, 2016). Alternatively, the signal sequence of the autotransporter protein can also use the signal recognition particle (SRP) pathway which utilizes co-translational targeting. This is a more efficient method for proteins with hydrophobic signal sequences because SRP has a higher affinity for hydrophobic residues (Dautin, 2010). The N-terminal sequence is notably larger than a standard signal sequence; this is thought to slow down its translocation across the inner membrane in order to preserve protein integrity (Stathopoulos et al, 2008).

After initial recognition, a resident protease cleaves the signal sequence, SecA facilitates proper folding to ensure access through SecYEG, and the remaining pieces of the protein are transported into the periplasm and SecB acts as the first molecular chaperone (Henderson et al, 2004). From there, the protein is met by additional native chaperone proteins of the periplasm to facilitate transfer to the outer membrane and prevent improper folding and protein degradation.

Figure 10: Sec translocase mechanism. The pre-protein is represented in a black line with the signal sequence in gray. Targeting: (1) SecB targets N-terminal signal sequence, (2) chaperones protein to SecA, (3) protein-SecA complex binds to membrane at the SecA binding site and forms a hetero-trimeric complex with SecYEG. Translocation: (4) protein is inserted into SecYEG complex and (5) ATP is hydrolyzed. (6) ATP hydrolysis and proton motive force moves protein across the membrane, which occurs in a step-wise fashion with 20-30 amino acids at a time. Release: (7) release of protein into the periplasmic space from Sec translocase via cleavage of the signal sequence (Adapted from Mori & Ito, 2001.)

25 The final step in translocation for autotransporter proteins remains elusive. There

are currently three prevailing theories about how the beta barrel component of the protein

interacts with transmembrane proteins in the outer membrane to facilitate translocation

(Figure 9, Dautin and Bernstein, 2009). The hairpin model (Figure 10a) suggests that

autotransporter proteins are threaded through their beta barrel in the outer membrane in a

linear manner, maintained by periplasmic chaperones. Finally, combining the beta barrel

encoded for in the autotransporter protein with the Bam complex of intramembranous

proteins allows for the protein to be transported out of the periplasm (Leo, Grin, and

Linke, 2012). It is here that the protein either remains embedded on the outer membrane

or cleaved and secreted into the extracellular space.

One area of autotransporter protein research that needs more information is its

allocation of energy for transport. Because the periplasmic space is devoid of ATP, GTP,

or any kind of proton motive force, there are a few theories for how proteins are shuttled

to the outer membrane into the extracellular space (Henderson et al, 2004). Some

autotransporters utilize chaperone proteins but some proteins, such as BrkA, lack a

chaperone binding domain and are still actively secreted (Henderson et al, 2004). This

example is just one of many that need extensive additional dedicated research in order to

learn more about transport mechanisms of action and specific targets.

Proteins that utilize the type V secretion system are often further broken down

into three distinct categories: Autotransporters (type Va), two-partner secretion pathway

(type Vb), and AT-2 (type Vc) (Beeckman and Vanrompay, 2010). The two-partner secretion protein products differ from traditional autotransporters by way of their protein structure- the passenger domain and the beta domain (which will eventually form the

26 outer membrane pore for translocation) are translated in two separate proteins, TpsA and

TpsB (Beeckman and Vanrompay, 2010). Even more recently, types Vd (potential fusion of the two-partner secretion systems) and Ve have emerged as competing secretion pathways but more studies are needed to fully elucidate their mechanisms (Salacha et al,

2010; Leo, Grin, and Linke, 2012).

Figure 11: Schematic diagram of the type V secretion mechanism sub-types for gram- negative bacteria. Each of the encoded polypeptide sequences destined for translocation across the inner membrane are targeted to the Sec transmembrane protein complex. Following transport into the periplasm, proteins are shuttled with native chaperone proteins and can exit the outer membrane in one of five different mechanisms with the assistance of outer membrane transmembrane proteins, like Bam complex (Adapted from Vo et al, 2017).

27 Autotransporter proteins are best known for their roles in virulence and pathogenesis. Many proteins in this family act as immunomodulators, adhesins, and proteases that aid in bacteria’s ability to colonize a host. Autotransporter proteins can be categorized further into three distinct sub-families: Adhesin Involved in Diffuse

Adherence (AIDA-I), Trimeric Autotransporter Adhesins (TAA), and Serine Proteases

Autotransporters of the Enterobactericieae (SPATE). While all three descend from a common evolutionary point (Figure 9), each family comes with unique characteristics that are explained further below.

9. Adhesin Involved in Diffuse Adherence (AIDA-I) and Trimeric Autotransporter

Adhesins (TAA)

The AIDA-I sub-family of autotransporter proteins represent the largest group and most diverse of the three sub-families with 55 proteins from numerous types of bacteria

(Henderson et al, 2004; Figure 9). These proteins are distinctly characterized by the roles they play in adhesion and colonization of the host. AIDA-1 proteins utilize non-fimbrial adhesion, meaning they do not rely on longer (1 µm) fimbriae structures for assistance in colonization (Vo et al, 2017). Fimbriae are common in both gram-positive and gram- negative bacteria species Like many autotransporter proteins, the AIDA-1 family is understudied and research is currently being done to understand mechanisms of action for translocation to the surface of the outer membrane. Although mainly associated with adhesion, AIDA-1 proteins are also associated with bacterial toxicity, invasion, and biofilm formation (van Ulsen et al, 2014).

28 The Trimeric Autotransporter Adhesin (TAA) sub-family is characterized by homotrimeric proteins that become embedded in the outer membrane following translocation. It follows the type Vc model for secretion and three copies of a protein from a single gene are encoded for in a conserved manner (Dautin and Bernstein, 2007).

Once bound, these proteins typically assist in colonization of hosts by acting as adhesins but some also play significant roles in immune modulation by binding to specific receptors (Lyskowski, Leo, and Goldman, 2011; Kirjavainen et al, 2008). The organization of all TAA proteins can be described as head-stalk-anchor confirmation; the anchor is involved in the secretion of the head and stalk subunits while the head mediates most of the host binding properties (Lyskowski, Leo, and Goldman, 2011).

10. Serine Protease Autotransporters of Enterobactericiaea (SPATE)

SPATE proteins contain a serine protease residue nestled within a highly conserved catalytic triad (GDSDSG) that contributes directly to their proteolytic features

(Guignot et al, 2007). Mature SPATE proteins are relatively large in size (ranging from

100-110 kDa) and also possess the highly conserved beta helix region found on all autotransporter proteins (Boisen et al, 2009). This protein family utilizes the type Va secretion mechanism (also known as the classical AT secretion method) because their passenger domain and beta helix structure share a modular organization (Wells, Totsika, and Schembri, 2010). The first identified SPATE (temperature sensitive hemaglutinin-

Tsh) was discovered in avian pathogenic E. coli in 1994 by Provence and Curtiss. It was shown to have cytopathic effects on mammalian cells and is widely regarded as a powerful toxin that aids in the manifestation of avian illnesses. Since the initial discovery, several additional proteins have been identified and all contribute to

29 pathogenicity in unique ways. There are few universal traits that connect all SPATEs, but

all have been shown to genetically related and typically confer detrimental effects on the

host cells (Dutta et al, 2002).

SPATEs have been traditionally categorized into two distinct classes: Class I and

Class II SPATEs. Class I SPATEs are all cytotoxic to epithelial cells. The most well

studied of these toxins is Plasmid Encoding Toxin (Pet), originally discovered in

Enteroaggregative Escherichia coli (EAEC). Pet was found to be a main contributor to

emerging pediatric diarrheal illnesses caused by EAEC (Boisen et al, 2009; Eslava et al,

1998). Since its discovery, numerous other Class I SPATEs have been described in

pathogenic bacterial specials and all are thought to contribute to pathogenicity of the host.

Class II SPATEs can have many diverse functions; some can be immunomodulatory

while others can cleave structural proteins from the surface of the bacterial outer

membrane. One of the most well studied Class II SPATEs is Protease involved in

Intestinal Colonization (Pic), also originally discovered in EAEC. Pic has the ability to

cleave most types of mucins produced by epithelial cells in the body, as well as other

structural proteins like human spectrin and pepsin A (Dautin, 2010). Interestingly, Pic is

produced by multiple bacterial strains (Shigella flexnerii, EAEC, and UPEC) but seems to

have slightly different roles in each of them. According to current studies, Pic has no

record of acting as a cytotoxin but can mediate serum resistance and allow bacteria to utilize host mucus as a nutrient source (Dautin, 2010; Henderson et al, 1999; Harrington et al, 2009).

Until recently, SPATEs, much like other members of the autotransporter family, were only associated with pathogenic E. coli and Shigella species. This was due in part to

30 the nature of their detrimental roles to the host organism and many of the aforementioned proteins as well as others such as SigA, SepA, Tsh, and EspP (Henderson and Nataro,

2001). SPATEs are associated with host damage. Further studies have shown that

SPATES are also actively produced in Citrobacter, Edwardsiella, Salmonella, and some commensal strains of bacteria (Ruiz-Perez and Narato, 2014). There is at least one known

SPATE protein encoded for in the EcN genome, secreted autotransporter toxin (Sat), featured more below.

11. Secreted autotransporter toxin (Sat)

Sat is a member of the SPATE family of autotransporter proteins discovered in

2000 that is found in many pathogenic E. coli and at least one Shigella species (Table ##;

Guyer et al, 2000). The native (unprocessed) Sat protein is 142 kDa and possesses many of the key features typically associated with an autotransporter protein: an extended signal sequence at the N-terminal, a functional protein in the passenger domain, and an autotransporter domain at the C-terminal (Maroncle et al, 2006). Sat is considered in its functional and mature state at 107 kDa following cleavage of the signal peptide sequence and autotransporter domain (Maroncle et al, 2006). Sat traditionally acts as a class I

SPATE and has been shown to confer cytotoxic effects on mammalian cells including vacuolization, F-actin cytoskeleton disassembly, and rearrangement of tight junction- associated proteins ZO-1, ZO-2, and occludins (Toloza et al, 2015). Although the virulent functionality of Sat is consistent in pathogenic bacteria, Sat derived from probiotic EcN has not been shown to share these qualities (Toloza et al, 2015).

31 Sat was first identified in UPEC CFT073 and the sat gene is found on pathogenicity island II with other prominent SPATE virulence factors (Guyer et al,

2000). It is important to note that virulence factors are often clustered together on the same chromosome, commonly known as pathogenicity islands. Since its initial discovery, the sat gene has been found in diffusely adhering E. coli (DAEC), Sat shares similarities to other Class I SPATEs including plasmid encoding toxin (Pet) of EAEC and EspC of enteropathogenic E. coli (EPEC) which have also been shown to be cytopathic to mammalian cells. Although the mechanism of action is still being elucidated, Sat has been shown to bind to host-membrane receptors to gain access to internal host organelles.

From there, Sat triggers the autophagy cell-death pathway which includes the production of large vacuoles eventually leading to death. Vacuolization of mammalian cells are visible under microscope (Guyer et al, 2000). Sat is also associated with increased cell detachment and decrease in structural integrity throughout cell and tissue systems (Guyer et al, 2002).

It has been shown in previous studies performed by Maroncle et al that the cytopathic features of Sat are directly proportional to the activity of the serine residues present in all SPATE proteins. Mutations made in one or both serine residues in the conserved catalytic triad from Sat of UPEC CFT073 resulted in significantly reduced proteolytic activity but did not affect the ability for the functional protein to be secreted outside the bacteria and internalized by the host (Maroncle et al, 2006).

The mechanisms of action for Sat have just recently began to be elucidated. It was revealed by Lieven-Le Moal et al that the intracellular toxin contributes directly to cell detachment and rounding in vitro (2011). This feature also triggers the autophagy

32 pathway, a cell survival mechanism characterized by the creation of large vacuoles inside the cell cytoplasm for the purpose of compartmentalization of toxins for removal (Fung et al, 2008). While the host cell is attempting to save itself by eradicating the toxin via expanding of vacuoles from within in preparation for engulfing of foreign toxins and expulsion from the cell, the vacuoles can become too large and essentially suffocate other cell organelles from the inside, leading to intracellular damage and death.

Until recently, Sat was only identified in pathogenic strains of bacteria (Table 2).

Vacuolization (due to the triggering of the autophagy cell death pathway) is the most common side effect but many others have been revealed over time. Some of these additional detriments include tight junction disassembly, F-actin cytoskeletal rearrangements, and histological lesions in the kidney and bladder. The sat gene in EcN is flanked by the iuc gene cluster and iha (Grozdanov et al, 2004). It is thought to contribute to the overall fitness of EcN but the role of Sat in EcN is not well studied.

Recent literature does seem to suggest that its traditionally harmful roles may be repressed in EcN through unknown mechanisms (Toloza et al, 2015).

Table 1: Compilation of known Secreted autotransporter toxin (Sat) protein characteristics. Origin of Biochemical Functions Sat Protein Source Associated Diseases Roles of Sat in Pathogenesis References Bacteria of Sat

Cell-detachment, autophagy, Guyer et al, Chronic Urinary histological lesions in kidney and Uropathogenic E. Degradation of casein, 2002; Tract Infection ileum, fluid accumulation, coli (UPEC) Extraintestinal spectrin, fodrin, (UTI), acute vacuolating cytotoxin- kidney Lievin-Le CFT073 coagulation factor V pyelenophritis, and bladder, loosens cellular Moal et al, junctions 2011;

F-actin cytoskeleton Chronic UTIs, disassembly, rearrangements and Diffusely Adhering Diarrhea, Guignot et al, Extraintestinal Unknown lesions in tight junctions E. coli (DAEC) Gastrointestinal 2007 increasing paracellular infection permeability (polarized cells), 34

Enteroaggregative Boisen et al, Extraintestinal Diarrhea Unknown Cytotoxicity E. coli (EAEC) 2009

Extraintestinal, Shigella flexnerii aquatic Shigellosis Unknown Unknown Roy et al, 2006 environments

Cleaves spectrin, Colonization of ileum and colon, Toloza et al, hyrdolyzes modification of paracellular 2015; E. coli Nissle 1917 Normal Flora None- Probiotic methoxysuccinyl- Ala- permeability, contributes to Abdulrahim, Ala-Pro-Val p- biofilm production 2015 nitroanilide

The table above is a summary of the known characteristics of all Sat to date. The Sat protein is found in several strains of bacteria (listed above) and most have ties to pathogenicity and virulence on the host organism. Because Sat is a protease, it is regularly associated with cytotoxicity and cellular damage that ultimately leads to cell death. A notable exception is the Sat protein from E. coli Nissle 1917; this probiotic strain has active cleavage from the Sat protein but has been shown to have minimal detrimental effects of on the host.

12. In vitro cell models

In order to test the direct effects of the functionality of Sat derived from UPEC

CFT073 against those of Sat from EcN, immortalized cell lines were chosen as in vitro models for the study. Cell lines are usually derived from cancer biopsies and grow continuously under specific laboratory conditions. The origin of their use stems back to

1951 when the first sample of cells were grown successfully in lab settings. They can offer good insight into cellular systems and metabolic processes in host organs in a contained area. Of greater relevance for this study, immortalized cell lines can serve as models for experiments that may be too dangerous to perform on a mammalian organism.

For this study, cell lines representing epithelial cells in an undifferentiated state

(HeLa), as well as differentiated cells derived from African green monkey (Chlorocebus sabaeus) kidney epithelial cells (Vero) were chosen as models. Derived from human cervical cancer in 1951, the HeLa cell line was established as the first successful in vitro cells (Lucey et al, 2009). Since then, millions of researchers around the world have utilized these cells to study numerous diseases and cellular processes. The HeLa cell line comprises all the important organelles and features of a mammalian cell that can be monitored for change in viability. Previous literature has also utilized the Sat protein derived from EcN to test cytotoxicity utilizing the MTT assay; researchers were able to show that Sat from EcN confers cytotoxicity in a dose-dependent manner (Toloza et al,

2015).

The Vero cell line is more specific in that it displays typical attributes associated with the epithelial lining of the mammalian kidney. Genome analysis has recently

revealed that the lineage was originally derived from a female Chlorocebus sabaeus

(Osada et al, 2014). Vero cells are anchorage dependent and will die if they are unable to adhere to a suitable surface (Ammerman et al, 2009). The relevance to this study lies in the fact that UPEC CFT073 readily colonizes the kidney in ascending UTIs, which can lead to systemic organ failure and host death. The Sat protein in pathogenic bacteria has been shown to target epithelial cells mostly due to direct access the bacterial toxins have to the cells lining the organ.

13. Previous Stathopoulos Lab Findings

Through bioinformatical analysis, EcN was shown to have eleven different autotransporter proteins with at least 95% genetic homology to UPEC CFT073 (Table 2;

Lane, 2015). These proteins are all previously shown to be virulence factors in the pathogenic strains of bacteria in which they reside. EcN actively secretes these proteins in high amounts when compared to UPEC CFT073 (Lane, 2015). Previous studies have also shown that Sat of UPEC CFT073 contributes to biofilm formation as confirmed with tests using a mutant strain of EcN with the sat gene deleted; using these engineered strains, Sat of UPEC CFT073 was not shown to contribute significantly to bacterial motility. The basis of the current studies (which will be elaborated below) is to find the functional differences between Sat or UPEC CFT073 and Sat of EcN.

Table 2: Characteristics of prominent autotransporter proteins found in both EcN 1917 and UPEC CFT073

Amino MW Signal % Identity Protein AT group Role EcN vs. CFT073 Acids (kDa) Sequence to CFT073

4 amino acid deletion at the N- Sat 1299 140.01 49aa 99% SPATE Protease terminus, 7 different residues

PicU 1371 144.61 55aa 100% SPATE Protease Identical to CFT073

Tsh (Vat) 1376 148.04 55aa 100% SPATE Protease Identical to CFT073

1 amino acid deletion at the N- UpaB 765 79.52 26aa 100% AIDA-I Adhesin terminus

37 UpaC 995 107.05 27aa 99% AIDA-I Adhesin 1 different residue

UpaD 52 amino acid deletion at N- 1039 106.99 52aa 99% AIDA-I Adhesin (Ag43b) terminus, half signal sequence

101 amino acid deletion at N- UpaE terminus, no predicted signal 2549 260.85 N/A 96% AIDA-I Adhesin (YapH) sequence, last 262 amino acids do not match CFT073

UpaF 2 amino acid deletion at N- 1040 107.06 52aa 96% AIDA-I Adhesin (Ag43a) terminus, 37 different residues

UpaG 1778 177.7 53aa 100% TAA Adhesin Identical to CFT073

936 amino acid deletion at N- UpaH 1909 194.82 N/A 99% AIDA-I Adhesin terminus, no predicted signal sequence, 15 different residues

YfaL 1254 132.06 30aa 100% AIDA-I Adhesin Identical to CFT073

This table represents the eleven known autotransporter proteins produced by both probiotic EcN 1917 and UPEC CFT073. The features included are analyses on molecular characteristics of proteins assessed using BLAST software and compiled from published records (Adapted from Lane, 2015)

14. Thesis Scope and Focus

Sat of EcN and Sat of UPEC CFT073 share a 99% genetic homology but the two parent strains confer drastically different effects on host mammalian cell systems. A direct comparison of the effects of Sat from EcN and Sat from UPEC CFT073 has never been performed and would be valuable to assess their overall similarities and differences.

In order to add to the information present on the potential dangers of Sat from EcN, cell viability studies in multiple cell lines were utilized to determine relative cytotoxicity and functionality of the protein from the two strains. Sat has been shown to display cytotoxic effects within UPEC CFT073 that it does not show in EcN, therefore, it is essential to look at Sat independently to best understand its roles in pathogenicity and cell viability.

This study will also add to the growing amount of evidence that some secreted products of EcN do not present a danger to probiotic consumers and will hopefully lead to increased support in EcN's approval as a probiotic supplement worldwide.

Based on what is currently shown in the literature about Sat and EcN, it is our hypothesis that Sat from EcN will display a reduced magnitude of toxicity when compared to Sat from UPEC CFT073. We will test this hypothesis by performing a series of functionality-based experiments to show effects of Sat on different types of mammalian cells in vitro. An MTT Cytotoxicity Assay will be utilized to assess cell viability following nine-hour challenges with bacterial supernatant containing Sat.

Functionality of Sat proteins will also be assessed by looking at oligopeptide cleavage activity.

Materials and Methods

1. Bacterial Strains and Plasmids

Multiple strains of bacteria were used to complete this study (Table ##): E. coli HB101,

E. coli HB101 with overexpression sat plasmid derived from uropathogenic E. coli

(UPEC) CFT073 (HB101/pSat), wildtype E. coli Nissle 1917 (EcN), UPEC CFT073, and

E. coli Nissle with sat gene deletion (EcNDsat). All strains were originally obtained from

collaborators at the University of California, Irvine and are stored permanently in -80°C freezer when not in use. The mutagenesis of EcNΔSat was done by Martina Corsi via the

λ red recombinase system in which an ampicillin resistance gene was inserted into the protein sequence of interest to create a knockout mutant. In addition, UPEC CFT073 includes a kanamycin resistance gene, HB101 pSat has an ampicillin resistance gene- both belong to the Stathopoulos Laboratory collection and were constructed by previous graduate students.

a) Bacteria Media Preparation

Luria Bertani (LB) based media was prepared according to manufacturer’s (Fisher

Scientific; Hampton, NH) instructions. For liquid broth, 20 g of powdered media is added

to 1000 mL of sterile ddH2O, mixed thoroughly, and autoclaved on a liquid 30

sterilization cycle. Media for agar plates were prepared in a similar manner, with 37 g of

specific LB agar media added to 1000 mL of ddH2O. Following sterilization in the

autoclave, media was allowed to cool and approximately 15 milliliters of media is poured

into sterile petri dishes. For some plates, approximately 100 uL of stock Ampicillin was

added prior to pouring the plates.

b) Preparations of Bacterial Overnight Cultures

Bacterial strains were streaked for isolation on appropriate LB Agar plates with

appropriate antibiotics and grown at 37°C overnight in 311DS LabNet incubator.

Approximately 5 mL of sterile LB broth is transferred into a sterile test tube and one

colony of the appropriate bacteria is added to the tube aseptically. The culture tubes are

grown at 37°C, shaking at approximately 200 rpm for 16-18 hours.

c) Preparations of Bacterial Day Cultures

From overnight culture preparations, approximately 5 mL of bacterial culture is added to

500 mL autoclaved LB broth media and grown for 3-6 hours at 37°C, shaking at

approximately 150 rpm. Culture flasks are grown to an optical density (OD600nm) of ~1.0.

Samples were checked approximately every 60 minutes for adequate turbidity in a

SmartSpecä Plus Spectrophotometer (Bio-Rad Laboratories Hercules, CA).

2. Bacterial Protein Work

a) Supernatant Isolation and Concentration

Bacterial day cultures were centrifuged at 3,500 x g for 10 minutes to pellet whole cells

and obtain cell-free supernatant. Supernatant was concentrated in filtered 50 kDa-cutoff,

50 mL EMD Millipore Amicon Ultra-0.5 conical tubes with Centrifugal Filter (Fisher

Scientific, Hampton, NH) at 5000 x g for 10-20 minutes. Concentrated samples were

collected aseptically with Pasteur pipets and transferred into a new sterile tube for storage

at -20°C until further use. Relative protein concentrations were assessed using the

Bradford Assay, according to manufacturer's (Bio-Rad Laboratories, Hercules, CA)

instructions.

b) Isolation and Partial Purification of Secreted Autotransporter Toxin

The protocol was modified from the thesis of Anthony Huang (Huang, 2012). Samples

were grown utilizing overnight and day culture protocols previously mentioned.

Supernatant was removed by centrifugation at 3000 x g for 30 minutes at 4°C. A solution

of 3.8 M (NH4)2SO4 was added gradually to the supernatant solution until the entire concentration of (NH4)2SO4 was approximately 20%. The solution was stirred

continuously overnight at 4°C. The next day, the solution was centrifuged at 15,000 x g

for 30 minutes at 4°C. Resulting supernatant was saved, and the pellet was resuspended

in 20 mM Tris pH 7.4 and stored at –20°C for future use. A solution of (NH4)2SO4 was

added to supernatant from previous step until the solution was approximately 60%

(NH4)2SO4. The solution was mixed continuously overnight at 4°C. The solution was

centrifuged at 15,000 x g for 30 minutes and pellet was resuspended in 1X PBS. Dialysis

was performed utilizing Spectra/Por Dialysis Membrane, 50 kDa (Spectrum Laboratories,

Inc. Rancho Dominguez, CA) with resuspended pellet in 1X PBS on inside; outside of

the bag contained fresh 1X PBS (8.0 g of NaCl, 1.44 g of (Na)2PO4, 0.2 g of KCl, and

0.24 g of K2PO4 into 800 mL of ddH2O. The pH was adjusted to approximately 7.2 and an additional 200 mL of ddH2O to solution. The buffer was changed for two days and the

remaining solution inside the dialysis membrane bag was collected and analyzed for

protein concentration/stored for future use at –20°C.

c) Acetone Protein Precipitation

To concentrate protein products, an acetone precipitation is performed. Approximately

300 µL of previously collected supernatant samples from bacterial day cultures and 1200

µL of acetone are added to a new tube and mixed thoroughly. Samples are incubated at -

20°C for one hour. Following incubation, the samples are centrifuged at 15,000 x g for 15

minutes at 4°C. All supernatant is removed and resulting pellets are allowed to dry for approximately 10 minutes. Extra samples were stored at -20°C for future use.

d) SDS-PAGE/Silver Stain

To analyze the protein profiles of obtained concentrated supernatant products, samples

were run through a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE). The gel was produced using products from BioRad (Bio-Rad Laboratories;

Hercules, CA). A full list of ingredients for the 10% polyacrylamide gel can be found on

Table #1. Samples were prepared for resolution by taking approximately 12 µL of cell-

free supernatant previously obtained and mixing with approximately 4 µL of 4X loading dye. Samples were spun at maximum centrifuge speed for five minutes to mix and transferred to a heating block set at 95°C for five minutes. Samples were then returned to the centrifuge to spin for an additional five minutes at maximum speed. Once the gel was loaded with samples, it was run with 1X SDS buffer (28.8 g glycine, 6.04 g Tris base, 2 g

SDS, 2.0 mL ddH2O) at 20 mA for approximately 75 minutes.

Immediately following gel resolution, samples were stained using the silver stain method for small gels. In brief, the gel was treated for at least 45 minutes, shaking, in 100 mL of fixing solution (30% methanol, 10% acetic acid, 60% ddH2O). The container was emptied and the gel was washed twice in 100 mL of 10% ethanol and then washed twice

in 100 mL of ddH2O. The gel was then treated with 1 mL of 2% sodium thiosulfate in 99

mL of ddH2O for approximately one minute; the solution was immediately removed and

replaced with 2 mL of 5% silver nitrate in 98 mL of ddH2O for 20 minutes. The solutions

were removed and replaced with development solution (50 uL formaldehyde, 20 uL 2% sodium thiosulfate, 6 g sodium carbonate, 500 mL ddH2O) and allowed to shake until bands developed. Development solution was removed and reaction was stopped by adding 100 mL of 1% acetic acid for 2-5 minutes. Finally, the gel was washed three times with 100 mL of ddH2O and dehydrated using the BioRad Model 583 Gel Dryer.

Table 3: Solutions for preparing resolving and stacking gel components for tris-glycine Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE).

Solution Component (Resolving Gel) Component Volume (mL)

ddH2O 1.9

30% Acrylamide Mix 1.7

1.5 M Tris (pH 8.8) 1.3

10% SDS 0.05

10% Ammonium Persulfate 0.05

TEMED 0.002

Solution Component (Stacking Gel) Component Volume (mL)

ddH2O 0.68

30% Acrylamide Mix 0.17

1.5 M Tris (pH 8.8) 0.13

10% SDS 0.01

10% Ammonium Persulfate 0.01

TEMED 0.001

The following components were added to create one 10% polyacrylamide resolving gel with a 5% polyacrylamide stacking gel. All steps were done at room temperature and gels were allowed to solidify for approximately 30 minutes. If gels were not immediately used, they were stored for up to one week in 4°C.

3. Cell lines Used in Study

In order to test the effects of Sat in vitro, this study utilizes the HeLa and Vero cell lines.

HeLa cells are an immortal cell line derived from cervical cell tumor biopsy in 1950; they

are representative of undifferentiated epithelial cells in morphology and function (Scherer

et al, 1953). Vero cells are a lineage of cells isolated from kidney epithelial cells from an

African green monkey in 1962 (Yasumura and Kawakita, 1962). Mammalian cells for

this study were maintained in complete media in a 37°C, 5% CO2 ThermoFisher Brand

Isotemp incubator (Department of Biological Sciences, Cal Poly Pomona; Thermo Fisher,

Chino, CA). In brief, cells were removed from liquid nitrogen storage, thawed quickly, and plated in T-25cm2 tissue culture-treated flasks. When cells were approximately 80-

90% confluent, they were passaged and transported to T-75cm2 tissue culture-treated flasks. Excess cells were frozen in 1 mL aliquots using CDMEM with 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen for future use. The HeLa cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, passage

2-15) and Vero cells were obtained from the Department of Biological Sciences at Cal

Poly Pomona (estimated passage >50).

a) Cell Culture Media Preparation

In order to make complete media (CDMEM), the following ingredients were combined in

the proper ratio: 10% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin/Glutamine

(PSG), 1% Nonessential Amino Acids (NEAA), and completed with basal Dulbecco’s

Modified Eagle’s Media (DMEM). All the ingredients were purchased from Fisher

Scientific (Hampton, NH).

4. Oligopeptide Cleavage Assay

In order to compare the relative proteolytic activity of the Sat protein, a specific

oligopeptide substrate, Suc-Ala-Ala-Pro-Val, conjugated with paranitroanilide (Millipore

Sigma, Burlington, MA) was utilized. A 1mM solution was prepared by adding 100 mg

of substrate with 8.89 mL of MOPS buffer (209 mg MOPS, 117 g NaCl, 10 µL of 0.01 M

ZnSO4 in 8 mL of ddH2O). Supernatant from previous steps (100 µL) was added to a sterile flat-bottomed 96-well plate with 100 µL of prepared substrate. Sterile LB Broth was used as a negative control. The plate was covered in foil and incubated for 15 hours at 37°C. Absorbance intensities were read using SpectraMax 190 microplate reader

(Molecular Devices, Sunnyvale, CA) at 405nm. All treatments were performed in triplicate.

5. MTT Cytotoxicity Assay

Sat derived from pathogenic species is known to display cytotoxic effects against

mammalian cells; in order to compare these effects with those from Sat of EcN, cell

viability is assessed using an MTT Cytotoxicity Assay (Stockert et al, 2012). The

protocol for these studies were obtained from Invitrogen (Invitrogen; Carlsbad, CA) and

modified according to the protocol provided by Toloza et al (2015). In brief, cells were

plated in a 96-well plate at a density of approximately 2 x 105 cells per well and left to

recover overnight in DMEM. After media was removed, cells were washed three times

with 1X PBS (pH 7.4; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in 1 L in ddH2O). Cells were then given 200 µL of cell-free supernatant and 100 µL of fresh

DMEM. After a total challenge time of nine hours, treatments were removed and MTT

reagent was added to each well; the plate was left to incubate for approximately 45

minutes or until purple precipitate became visible under a microscope. Precipitate was

dissolved using 100 µL of DMSO and immediately read using SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA) at 570nm. Each treatment was performed in triplicate.

6. Data Analysis/Statistics

All data sets were analyzed using one-factor ANOVA with Tukey’s range test and p-

value significance was determined to be values <0.05. Data was interpreted and/or

visualized using the R-statistical package and 2015 Microsoft Excel.

Results

1. Concentration of Sat protein from select strains using Filtered Centrifugation

The secreted autotransporter toxin (Sat) is readily secreted by numerous strains of pathogenic bacteria and at least one known species of probiotic bacteria (Table 1). In order to test the effects of concentrated Sat protein on mammalian cell culture, Sat was concentrated utilizing filtered centrifugation tubes with 50 kDa cutoff filters. Sat is a fairly large protein product, with a functional product at about 107 kDa. Therefore, using a 50 kDa cutoff filter was justified to ensure Sat protein in the supernatant sample while simultaneously removing excess smaller secreted proteins and cell debris. Original samples were obtained from pelleted day culture collections where the bacterial cell bodies were removed and only supernatant was preserved. The samples were centrifuged at 5,000 x g and run on an SDS-PAGE for 120 minutes to account for the large molecular weight affiliated with the Sat protein.

The Mini-PROTEANÔ TGXÔ precast gels have a 4-15% acrylamide gradient that allows for uniform separation of protein products for best resolution. Figure 12 shows the results of an SDS-PAGE following silver stain analysis of all eight samples used for the study. Lanes 3 and 4 are the concentrated supernatant samples of HB101 and

HB101/pSat, respectively. These two strains were originally selected to help isolate the effects of Sat derived from UPEC CFT073; an overexpression sat plasmid was transformed into E. coli strain HB101 so that the bacteria secrete Sat from UPEC

CFT073 in abundance, which can assist with isolating the effects of the Sat protein. There is one concentrated band at approximately 107 kDa, which is indicative of processed Sat

protein secreted by HB101/pSat; there is no complimentary band in the HB101 sample,

which is consistent with what is expected for the strain.

Size 1 2 3 4 5 6 7 8 9 (kDa)

250

130

72 55

Figure 12: Silver stain analysis of concentrated supernatant samples. The 4-15% acrylamide gel loaded with 14 uL of prepared sample was stained with 5% silver nitrate following small gel staining protocol. The sample was developed in a solution of 2% sodium thiosulfate, 100 uL formaldehyde, and 12 g sodium carbonate in 1000 mL of ddH2O until prominent bands appeared. The labels correspond to the appropriate supernatant samples in each well: Lane 1- M (marker), lane 2- EMPTY, lane 3- HB101, lane 4- HB101/pSat, lane 5- wildtype EcN, lane 6- EcN[Symbol]sat, lane 7- wildtype UPEC CFT073, lane 8- LB Media + Ampicillin (10 mg/mL), and lane 9- HB101/pSat purified using increasing concentrations of 3.8 M Ammonium Sulfate. Approximately 10 uL of each sample was used with 3X loading buffer, boiled for five minutes, and loaded into the gel and run for 120 minutes prior to staining. The black box highlights the location of Sat protein in the samples.

A separate SDS-PAGE and silver stain was done to view protein profiles of the concentrated supernatant from the remaining bacterial strains of the study (Figure 12).

Prominent bands are present in lanes 3, 4, and 6 at approximately 107 kDa which is indicative of processed Sat protein secreted by each of these bacterial strains. Additional bands present in the protein profile are likely residual bacterial proteins that may not contribute to pathogenesis, such as LPS, cellular debris, and additional native secreted proteins. Because Sat is concentrated in these samples, it acts as the most relevant protein in the profile. Lane 5 does not contain a band at 107 kDa because the sample loaded inside was EcNDsat; this indicates successful deletion of the sat gene in the EcN genome which resulted in its elimination as a secreted product.

Estimates of total protein concentration was assessed using the Bio-Rad Protein

Assay, a modified version of the Bradford Assay. Figure 13 shows a standard curve produced with varying concentrations of bovine serum albumin (BSA) ranging from 0.1 mg/mL to 1.5 mg/mL. Approximately 500 µL of each previously concentrated sample was added with the sample buffer and absorbance readings were analyzed on the

Nanodrop spectrophotometer to be compared with absorbances of known concentrations of BSA solutions. The amount of total protein present was quantified by calculating the concentration with the given equation generated in accordance with the standard BSA curve.

Figure 13: Protein concentration assay to quantify total amount of protein in prepared supernatant samples. Supernatant samples were mixed with a diluted commercial dye (Bio-Rad) to assess protein concentration following centrifugation. Standards were prepared using 0.1 mg/mL, 0.5 mg/mL, and 1.0 mg/mL of bovine serum albumin (BSA). Approximately 500 µL of supernatant samples from HB101, HB101/pSat, EcN, EcNDsat, and UPEC CFT073 was added to the diluted dye, allowed to incubate at room temperature for one minute, and absorbance levels were read at 595 nm on a Nanodrop. The BSA standards were used to create the equation for the standard curve and absorbance values were converted into an approximate concentration for each sample.

Effects of concentrated Sat of UPEC CFT073 on cell viability of HeLa cell line

Once the supernatant samples of wildtype EcN, wildtype UPEC CFT073,

EcNDsat, HB101, and HB101/pSat were concentrated, they were used for additional studies that indicated the role of Sat in cell viability. The Sat protein is known to display a multitude of effects in different origin species and on different cell, tissue, and organ systems in the host. In order to test these effects generally on cell systems and structures, the HeLa cell line was used as the first in vitro model. Historically, HeLa has been featured in many studies to showcase generalized effects of different treatments on mammalian cells since they possess many of the standard features common to all cells.

The Methyl-Thiazoyl-Tetrazolium (MTT) Cell Proliferation Assay was used to determine cell viability following treatment; it is a standard colorimetric assay that measures mitochondrial dehydrogenase activity of living cells that complexes with the MTT reagent and forms a soluble violet precipitate. The experiment was setup in a 96-well plate following the diagram in Figure 16; all samples were run in triplicate. To complete this assay, HeLa cells were first grown to approximately 80% confluency (Figure 15) in a

T-75cm2, tissue culture-treated flask and transferred into a sterile 96-well plate at 2 x 105 cells/well. Cells were counted using a hemocytometer and diluted with DMEM.

1.2

0.8

405nm 0.6 OD

0.4

0.2

0 wt ECN wt UPEC HB101 pSAT ECN ΔSAT

Figure 14: Oligopeptide cleavage assay for Sat-specific protein sequences confirms Sat activity in supernatant samples. Concentrated supernatant samples from the wildtype EcN, wildtype UPEC CFT073, HB101/pSat, and EcNDsat were treated with a 1 mM solution of Sat-specific substrate Suc-Ala-Ala-Pro-Val, conjugated with paranitroanilide. Activity was measured after a 15 hour incubation period on a microplate reader at an optical density (OD) of 405nm.

a) b)

Figure 15: Images of a) HeLa cell line and b) Vero cell line in culture. Both cell lines were grown in a complete media (CDMEM): DMEM supplemented with 10% FBS, 1% NEAA, and 1% PSG. Cells were grown in identical conditions (37°C, 5% CO2) and passaged every 2-4 days. The images obtained were captured at 40x magnification.

Figure 16: Plate layout for MTT Cytotoxicity Assay with HeLa Cell line. A sterile, tissue culture- treated, 96-well plate was used for all MTT assay experiments. Plates were setup in the following manner: HeLa cells alone (no treatment—pink), HeLa cells treated with LB media supplemented with Ampicillin (10 mg/mL—yellow), HeLa cells treated with concentrated supernatant of E. coli strain HB101 (green), and HeLa cells treated with concentrated supernatant of E. coli strain HB101/pSat (blue). All treatments were performed in triplicate.

The first supernatant samples used were HB101 and HB101/pSat. Following a nine-hour challenge with the supernatant, cells were washed with 1X PBS and given approximately 30µL of the MTT reagent was removed when purple precipitate was visible under the microscope; the precipitate was dissolved using dimethyl sulfoxide

(DMSO) and read on a microplate reader at 570nm. The data suggests that the protein produced by HB101/pSat reduces cell viability significantly in HeLa cells as compared to the control, but not when compared to HB101 alone (Figure 17). Cell viability was reduced by approximately 25% when treated with either LB Media/Amp10 or HB101.

With the addition of HB101/pSat, cell viability was reduced by a total of 35% compared to the untreated control cells. The data is representative of three independent experiments.

* *

Figure 17: HeLa cell viability decreases with challenges from concentrated supernatant samples. MTT cytotoxicity assay was performed to assess cell viability following a nine-hour infection period with respective samples. Cells were plated at a density of 2 x 105 cells/well in a 96-well plate and left to recover overnight in DMEM. Following recovery, cells were treated with 150 µL of respective supernatant samples. Cell treatments were removed after nine hours and treated with MTT reagent for 1-2 hours. Resulting precipitate was dissolved using DMSO and read at 570 nm. Statistical significance was determined using one-factor ANOVA with Tukey’s HSD. (**p-value< 0.02; *p-value<0.05)

Concentrated Sat of UPEC CFT073 decreases cell viability in Vero cell line

Literature on this topic suggests that the effects of secreted toxins like Sat can change dramatically depending on the differentiation state of host cells. With this information, the Vero cell line was chosen as the second in vitro model for this study.

Vero cells, derived from the kidney of the African Green Monkey, are readily used epithelial cells that mimic the cellular environment of the lining of the mammalian kidney. This was chosen as a suitable model because of the role UPEC CFT073 plays in

UTIs and ascending infections leading to acute cystitis and pyelonephritis. A similar experiment to what was performed on the HeLa cell line was setup using Vero cells following the diagram in Figure 18 to measure cell viability following challenges with the bacterial supernatant.

Vero cells were treated with supernatant from HB101/pSat, EcN, UPEC CFT073, and EcNDsat (referred to as EcNsat- in Figure ##); untreated cells and cells treated with

LB media supplemented with 10mg/mL of Ampicillin served as controls (Figure 19). The concentrated supernatant of HB101/pSat reduced cell viability in Vero cells by approximately 50%, which was the most dramatic reduction of all the treatments (Figure

19). The supernatant with the sat gene deletion (EcNsat-) displayed minimal reduction of cell viability, almost at levels comparable with the LB media treatment.

Figure 18: Example of plate layout for MTT Cytotoxicity Assay with Vero cell line. A sterile, tissue culture- treated, 96-well plate was used for all MTT assay experiments. Plates for this experiment were setup in the following manner: Vero cells alone (no treatment—pink), Vero cells treated with LB media supplemented with Ampicillin (10 mg/mL—yellow), Vero cells treated with concentrated supernatant of E. coli strain HB101/pSat (blue), Vero cells with a HB101/pSat purified using 3.8 M Ammonium Sulfate (green), Vero cells with supernatant of wildtype E. coli Nissle 1917 (purple), Vero cells with supernatant of EcN with sat gene deletion (orange), and Vero cells with supernatant of wildtype UPEC CFT073 (maroon). Each treatment well had 100 µL of DMEM and 150 µL of respective treatment. All treatments, as well as each individual experiment, were performed in triplicate.

* *

** **

Figure 19: Vero cell viability decreases with challenges from concentrated supernatant samples. MTT cytotoxicity assay was performed to assess cell viability following a nine-hour infection period with respective samples. Cells were plated at a density of 2 x 105 cells/well in a 96-well plate and left to recover overnight in DMEM. Following recovery, cells were treated with 150 µL of respective supernatant samples. Cell treatments were removed after nine hours and treated with MTT reagent for 1-2 hours. Resulting precipitate was dissolved using DMSO and read at 570 nm. Percent cell viability (y-axis) was obtained by normalizing the data to the untreated control. Statistical significance was determined using one-factor ANOVA with Tukey’s HSD. (**p- value<0.01; *p-value<0.05)

Supernatants of Wildtype EcN and Wildtype UPEC CFT073 and their effects Vero cell line viability in vitro

A comparative analysis of Sat secreted from wildtype EcN and wildtype UPEC

CFT073 on cell viability and function is currently understudied but has implications in the use of EcN as a probiotic and in understanding disease manifestation in UPEC

CFT073. Comprehensive knowledge on the effects of Sat protein variants from different species of bacteria may help elucidate ways to alter the toxin. Figure 19 also shows the results of treatment of Vero cells with total protein supernatant samples from wildtype

EcN and wildtype UPEC CFT073; cell viability decreased by approximately 35% in both treatments. Supernatant samples were prepared in an identical manner as stated above and experimental conditions also remained the same. Figure 19 shows the results from three independent MTT assays: the concentrated supernatants from both wildtype EcN and wildtype UPEC CFT073 displayed similar levels of cytotoxicity on the Vero cell line, with a reduction at approximately 40% when compared to the control.

Partially purified Sat of UPEC CFT073 effects on Vero cell line from recombinant

HB101/pSat

The Sat protein is readily secreted in the total protein supernatant of HB101/pSat but better understanding of the Sat protein comes from studying it in isolation. Therefore, we wanted to look at the functionality of the Sat protein independently of the additional proteins present from the host bacteria. Samples were prepared in exactly the same manner as for the supernatant collection but isolated supernatant from the day cultures was treated with a 3.8 M Ammonium Sulfate solution in increments; the addition of a

highly concentrated salt solution precipitates secreted protein products out of solution.

The salting process must occur in increments over the course of two days so the proteins present in the supernatant do not denature from sudden high salt concentration.

Once the salt has been added, the sample underwent dialysis in a commercial semi-permeable membrane that has a 50 kDa protein cutoff, similarly to the 50 kDa filtered centrifuge tubes used for supernatant concentration. Samples are added to the inside of the membrane and spun overnight in 1X PBS for 48 hours. The remaining product inside the membrane is concentrated and purified following the removal of excess proteins and debris. Figure 12 shows the results of a silver stain analysis of the isolated Sat protein (Figure 12, lane 8). The newly purified Sat protein was then used to perform MTT Cytotoxicity assays using the Vero cell line. Treatments were set up in identical manners to the previous experiments and conditions were also the same.

This treatment was prepared for the concentrated supernatant containing Sat protein derived from HB101/pSat as well as wildtype UPEC CFT073. The experimental design uses concentrated supernatant from HB101/pSat as a point of comparison; Figure

20 shows that when the Sat protein is partially purified, it confers significant cytotoxicity against Vero cells. The treatment of Vero cells with the partially purified resulted in a

65% reduction in cell viability, the most dramatic response of all the treatments in this study.

Partially purified Sat from EcN elicits similar effects as Sat from UPEC CFT073 on

Vero cells

In order to compare data, supernatant from the wildtype EcN strain was prepared in an identical manner to that of HB101/pSat. Following supernatant isolation, proteins of the correct size were selected for and crudely purified using the ammonium sulfate precipitation method as described above. The Sat protein was selected for using a dialysis membrane with pores with sizes for proteins 50-kDa and larger. It is of note that wildtype

EcN is known to secrete numerous proteins in this size range, but Sat is one that is predominantly present as suggested by the protein profiles from the SDS-PAGE/Silver

Stain analysis (Figure ##). The resulting supernatant was used to treat Vero cells grown overnight following seeding in a 96-well plate at a cell density of approximately 2x105 cells/well.

*** ***

Figure 20: Partially purified Sat from control strain HB101/pSat confers significant reduction in Vero cell viability. Treatments groups included Vero cells alone, LB media (negative control), concentrated supernatant from HB101 pSat, and partially purified concentrated supernatant from HB101/pSat. All treatment groups, including the controls, were treated with 3.8 M ammonium sulfate dialysis and resulting protein products were collected, resuspended in 1X PBS, and filtered before inoculation with Vero cells in a 96- well plate. Percent cell viability (y-axis) was obtained by normalizing the data to the untreated control. Statistical significance was determined using one-factor ANOVA with Tukey’s HSD. (***p-value<0.005; **p-value<0.01)

** **

Figure 21: Partially purified Sat from EcN confers greater cytotoxicity than concentrated supernatant of Sat from EcN. Treatments groups included Vero cells alone, LB media (negative control), HB101 pSat (negative control), concentrated supernatant from EcN, and partially purified concentrated supernatant EcN. All treatment groups, including the controls, were treated with 3.8 M ammonium sulfate dialysis and resulting protein products were collected, resuspended in 1X PBS, and filtered before inoculation with Vero cells in a 96-well plate. Percent cell viability (y-axis) was obtained by normalizing the data to the untreated control. Statistical significance was determined using one-factor ANOVA with Tukey’s HSD. (**p-value<0.01)

** **

Figure 22: Partially purified Sat from UPEC CFT073 confers greater cytotoxicity than concentrated supernatant of Sat from UPEC CFT073. Treatments groups included Vero cells alone, LB media (negative control), HB101 pSat (negative control), concentrated supernatant from UPEC CFT073, and partially purified concentrated supernatant UPEC CFT073. All treatment groups, including the controls, were treated with 3.8 M ammonium sulfate dialysis and resulting protein products were collected, resuspended in 1X PBS, and filtered before inoculation with Vero cells in a 96-well plate. Percent cell viability (y-axis) was obtained by normalizing the data to the untreated control. Statistical significance was determined using one-factor ANOVA with Tukey’s HSD. (**p-value<0.01; *p- value<0.05; ns= Not Significant)

Discussion

Secreted autotransporter toxin (Sat) is expressed readily in pathogenic species of bacteria and at least one probiotic E. coli species, E. coli Nissle 1917 (EcN). While much of its mechanism of action remains elusive, key studies have been performed to begin understanding the effects of Sat on two cell lines, with specific focus on the Sat protein from pathogenic bacteria, uropathogenic E. coli (UPEC) CFT073, due to its known ability to contribute to host damage. Due to similarities in the genomes of EcN and UPEC

CFT073, the two were selected as species of interest to compare the functionality of Sat.

As shown in previous bioinformatical analyses, the sat genes of these two species share approximately 99% homology with general differences cited as a four amino acid deletion in the N-terminus (signal sequence) and a seven amino acid difference within the functional domain (Lane, 2015). Although the specific changes in amino acid changes and location differences are not yet clear, it has been hypothesized that these amino acid changes directly relate to the functionality of the processed protein. Even small differences in amino acid residues can change structural components, folding patterns, and hydrophobicity of a protein, which can potentially alter its ability to interact with the host. More studies need to be done to pinpoint all of the factors that contribute to the small differences in the sat genes of EcN and UPEC CFT073.

To gain further insight into the functional differences between the Sat protein from two distinct species, this study focused on utilizing relevant mammalian cell lines. It was hypothesized that the Sat protein confers damage independently. As the name suggests, Sat is a secreted protein, so the bacterial cell body is unnecessary to confer effects of the protein and would contribute to debris that could alter the results of the

experiments. Therefore, concentrated supernatant following day culture preparations

were used in all assays. During the secretion of Sat, mutations and damage to outer

membrane transport proteins and final cleavage mechanisms can result in a Sat protein

that stays attached to the cell wall of the bacteria. Sat is produced and secreted at high

volumes and the amount of improperly secreted products is negligible for these

experiments although it has been shown that Sat contributes to adherence of EcN to

common structural host cell proteins such as collagen and fibronectin (Lane, 2015).

Figure 12 represents the relative protein profiles of each of the supernatant samples

utilized for these studies; the black box highlights noticeably prominent bands on the

SDS-PAGE around 107 kDa, indicative of processed Sat protein.

Following successful confirmation of the presence of Sat in samples, it was

important to quantify the amount of total protein present in each present through a

commercial protein concentration assay (Figure 13). The concentration assay was utilized

to standardize the approximate amounts of total protein present in each sample; this was

vital for success of the study because improper dosage of total protein, particularly as it

relates to active toxins, can drastically alter the way the cell lines react to treatment. With

the addition of 500 µL of protein sample to the diluted protein concentration dye, it was

estimated, based on the BSA standards used, that all the concentrated supernatant

samples fell within a range of 500-600 µg/µL; individual sample concentrations were analyzed following each new supernatant isolation and concentration to ensure consistency throughout experiments.

Originally, the total amount of protein present in the samples were tested and the relative amount of Sat from these samples was estimated. Supernatant samples from

HB101/pSat, which was constructed by collaborators to overexpress the sat gene and produce a large amount of the Sat protein, consisted of a majority of Sat protein with some additional native E. coli proteins and cell debris, as shown on the silver stained

SDS-PAGE. In contrast, the samples from wildtype EcN and wildtype UPEC CFT073 visually contained less Sat protein than HB101/pSat but their overall total protein concentrations were slightly higher. This indicates that there are other large products secreted by these strains, which is consistent with what is known about EcN and UPEC

CFT073 from both published works and previous laboratory findings; these additional factors were taken into account when designing the experiments for the study by incorporating proper controls and testing the supernatant of EcN with the sat gene deleted

(EcNDsat) to directly compare the effects of Sat in wildtype EcN.

HeLa cells were chosen as the first cell line of interest for challenge with the prepared supernatant samples. Derived from cancerous cervical tissue in the 1950s, HeLa cells are a common cell line used to represent undifferentiated epithelial cells and are used often to showcase general effects on cells by a treatment or challenge. The prominent use of HeLa cells in cancer studies, vaccine trials, and AIDS research among many other medically relevant experiments have made it a staple resource for researchers for decades and is still readily used today. The effects of the concentrated supernatant samples of nonpathogenic E. coli strain HB101 and recombinant E. coli strain

HB101/pSat were tested utilizing the MTT Cytotoxicity Assay which was used throughout the study to test cell viability following challenge with prepared supernatant samples. This assay harnesses host cell machinery by converting active mitochondrial enzymes into dissolvable colorimetric crystals. Cells that have been killed by the

challenge are unable to produce the enzymes necessary to complex with the MTT reagent, which leads to a lower absorbance value when read on a microplate reader.

The cell viability for HeLa cells was reduced by approximately 25% as compared to the control for both LB Media (10 mg/mL ampicillin) and HB101, while HB101/pSat displayed 35% cell viability reduction when compared to the control (Figure ##). The data from the nine-hour challenge on the HeLa cell line suggests that HB101/pSat conferred a slight decrease in cell viability that was deemed not significant by statistical

ANOVA analysis when compared to the LB media (10 mg/mL ampicillin) and HB101.

One likely reason for the marginal differences lie within the nature of the cells used in these first experiments. As stated above, because of its cancerous origins, HeLa cells display the characteristics of undifferentiated epithelial cells; the differentiation state of a cell plays a large role in its reaction to extracellular pathogens and agents. The literature suggests that undifferentiated cells may be more susceptible to damage which is consistent with the data obtained from these experiments. The reduction of cell viability by LB media is abnormal and reflects the vulnerability of the HeLa cell line to any treatment additive, including other types of media.

The variation between trials and heightened susceptibility to damage made the

HeLa cell line less than ideal to support the overall hypothesis. Valuable information was still obtained, and cytotoxicity was conferred with the HB101/pSat challenge at a level of

35%. The decision to perform the same assay on the Vero cell line (derived from African green monkey kidney epithelial cells) came after analyzing the results from the HeLa cells. Vero cells are another common cell line that share many similarities with regard to cell type and growth conditions but differ from the HeLa cell line in a few key ways: 1)

Vero cells are differentiated and mimic the epithelial lining of a mammalian kidney, 2) the Vero cell line is derived from African green monkey tissues while HeLa is derived from human tissue samples, and 3) the original Vero cell line was established using noncancerous epithelial cells; with time, the Vero cell line proved to be tumorigenic when tested both in vivo and in vitro (Levenbook, Petricciani, and Elisberg, 1984). Both, however, are extremely susceptible to foreign pathogens and toxins and their use in toxicity studies is well documented.

In order to follow through with the overall objective of the study, an MTT cytotoxicity assay was performed with the cell-free concentrated supernatant of

HB101/pSat (used as a positive control), as well as the following additional strains:

EcNDsat, wildtype EcN, and wildtype UPEC CFT073. The data obtained from the MTT assay with the Vero cell line produced vastly different results than those obtained with the HeLa cell line. Figure ## displays the graphical average of four experiments done with the Vero cell line; supernatant from HB101/pSat produced the greatest reduction in cell viability (50%), while the supernatant from both wildtype strains interestingly reduced cell viability to similar levels, conferring approximately 42% cell viability reduction. Interestingly, the EcN strain with the sat gene deleted (EcNΔsat) had limited reduction of cell viability (>20%), comparable to the levels of cytotoxicity shown from the ampicillin-treated LB media. This suggests that Sat may play a role in EcN cytotoxicity in Vero cells, which has greater implications for its potential to harm host cells and tissues throughout the body but specifically in the kidney.

To test the relative levels of cytotoxicity specific to Sat further, the proteins were further concentrated and purified from the wildtype strains using an ammonium sulfate

precipitation method. A crude purification of the Sat protein using a 50 kDa-cutoff

dialysis membrane and passive diffusion allows for a more representative model for

direct effects of the protein of interest. Supernatant samples were treated with increasing

amounts of 3.8 M Ammonium Sulfate solution over two days and secreted proteins were

precipitated out of solution; resulting proteins have all residual cell debris, native

DNAses, and additional degradative elements removed so purity is remarkably improved

from the concentrated supernatant samples from previous trials (Figure ##).

Sat from pathogenic strains of bacteria is known for its ability to cause significant

cell detachment of epithelial cells in vitro by degradation of important structural components. This was a potential factor for the low absorbance values seen in all supernatant challenges that contained Sat protein; upon elimination of MTT reagent to reveal resulting purple precipitate, some of the cells were suspended in solution and subsequently removed from the plate. This phenomenon was directly proportional to approximate amounts of Sat present in the sample as assessed utilizing SDS-PAGE and protein concentration analyses. The supernatant samples from wildtype EcN and wildtype

UPEC CFT073 displayed similar cytotoxic effects on Vero cells in vitro which may indicate that Sat from both strains may have contributed to cell detachment; this argument is strengthened by looking at the data associated with EcNDsat (Figure 19) which does not display the same effects as the wildtype strains with Sat present and cytotoxic features are enhanced to the levels of LB media in comparison to the wildtype strains. Class I SPATES other than Sat, including Plasmid encoding toxin (Pet) and

EspC, are also shown to contribute significantly to cell detachment by loss of F-actin

fibers which leads to mass disorganization of the host cell cytoskeleton (Lieven-Le Moal et al, 2011).

Our studies have shown that the concentrated supernatant from wildtype EcN confers a significant amount of damage to mammalian kidney epithelial cells at levels comparable to supernatant from wildtype UPEC CFT073 when compared to the untreated control. The data shows an approximate 40% decrease in cell viability when treated with concentrated wildtype EcN supernatant (Figure 19). This data is consistent with previous studies done by Toloza et al that showed time-dependent cytotoxicity of concentrated Sat supernatant from EcN against HeLa cells (Toloza et al, 2015). This is the first known study that uses Vero cells as the model for cell viability following treatment with Sat supernatant from UPEC CFT073; future studies will include repeating experiments with

Sat protein derived from EcN, similar to the trials performed in the aforementioned article.

There are many factors that affect overall bacterial toxicity and the components of the supernatant, as shown in the SDS-PAGE analysis (Figure 14), show that there are additional elements within that can contribute to cellular damage. EcN produces eleven known autotransporter proteins, many of which are actively secreted into the supernatant when the bacteria are in their exponential growth phase. The experiments for wildtype strains were done using quantified total protein profiles that were concentrated through a

50-kDa cutoff filter. These samples reflect a diverse composition of both toxic and non- toxic proteins that could have contributed to some of the toxicity seen on the cells. The inclusion of the HB101/pSat samples and the EcNΔSat assisted in highlighting the

particular effects of Sat but more work should be done to truly isolate these features for more tangible results.

The implications of comparable cytotoxicity in Sat from EcN is of the utmost importance in pending EcN medicinal approval in the United States. As a probiotic, supplements containing live EcN have been commercially available for decades in many

European countries. Products such as Mutaflor, Symbioflor 2, and EcN™ for humans, and Ponsocol for animals, have contributed widespread relief of numerous gastrointestinal ailments and general gut dysbiosis with little to no adverse side effects on the host (Wassenaar, 2016). The fact that EcN readily produces eleven different autotransporter proteins associated with virulence continues to be an issue barring use of

EcN as a probiotic in the United States.

The kidney is constantly susceptible to infection due to its connection and proximity to the urethral tract, a prominent reservoir for infectious agents despite its harsh living conditions (Whiteside et al, 2015). Motile bacteria always have the opportunity to transcend from the GI tract to the sterile kidney and bladders through the urethra. Many commensal E. coli found natively in the GI tract are excellent colonizers and readily procreate so their appearance in other parts of the body are often associated with infection. Although EcN is a prominent member of the commensal gut E. coli, as a whole it characteristically does not confer cytopathic effects on the host regardless of its ultimate destination in the body. Therefore, the role of Sat, which is widely known to be toxic to mammalian cells, and other “virulence factors” in EcN remains in question.

With the results obtained from these experiments, it is suggested that Sat from

UPEC CFT073 and Sat from EcN may confer similar levels of cytotoxicity against Vero cells which are known to mimic the lining of the mammalian kidney (Figure 19). Cell viability was reduced to 40% using the purified version of the Sat protein in separate trials for both wildtype EcN and wildtype UPEC CFT073. This represents a 25% additional decrease from the concentrated supernatant samples from the same wildtype strains as established in previous trials (Figure 17). The data from these experiments suggest that the functional differences between isolated Sat protein from the probiotic and uropathogenic strains may be more similar than what has been thought in the past. It is already known that genetically, the sat gene is 99% identical between the two strains.

When compared to the sequence in UPEC CFT073, the sat gene in EcN contains a four amino acid deletion in the N-terminus, which targets the full protein to the Sec transmembrane proteins in the inner membrane of E. coli. It has been established that the deletion is deep enough within the sequence that the missing amino acids do not seem to affect the ability for the protein product to target the inner membrane (Lane, 2015). The other genetic differences lie in the seven amino acid point mutations found in the functional domain of the sat gene from EcN. This is most likely the location of the changes that may be responsible for functional differences found between bacterial species; these exact point mutations remain unknown and further experiments will need to be completed to gain further insight.

It is of note that these results were obtained using Sat in a concentrated and partially purified form using the ammonium sulfate precipitation method. The results show that although much of the residual protein products found normally towards the

bottom of the gel have been removed, the resulting protein band for Sat also had reduced intensity, indicating some removal of the protein of interest as well, as confirmed by an

SDS-PAGE and silver stain. This change in protein concentration following partial purification was considered when preparing proteins for use in the MTT cell viability assay. Concentrated protein within each sample volume were modified to ensure the treatment conditions were kept identical in order to compare results. There is so much more to be revealed regarding Sat in its fully purified, native form and how it may specifically affect host health from within a complex system. In future analyses, further purification steps, including co-immunoprecipitation or ion exchange chromatography can be utilized to precipitate and purify Sat protein from numerous strains.

Limitations to the overall experimental design presented challenges that make it impossible to have irrefutable conclusions regarding the implications of Sat protein on cytotoxicity. First, the treatments given were all standardized to give approximately 150

µg/µL of protein toxin per well for a total infection period of nine hours. This was based on a single study by Toloza et al (2015) in which HeLa cells treated in a similar manner displayed measurable cytotoxicity. In order to have a stronger claim that Sat was the main causative agent of the cytotoxicity, the experiment should have been performed in a dose-dependent manner. Positive results would have shown that increased levels of Sat would correspond with decreased cell viability.

Another limitation of the study was that the protein product treated with ammonium sulfate in order to precipitate out the protein of interest was only partially purified at the final stage. One of the hypotheses of the study indicated that the purified protein would convey greater toxicity and the expected phenotype was conferred in Vero

cells. Without a fully purified product, however, it is very challenging to deduce with certainty that the effects shown are from the Sat protein. The evidence is made stronger with the inclusion of the EcNDsat strain; the results indicate that with a sat gene deletion, cell viability is at comparable levels to the control treatments.

There are a few different directions this research could go based on the data obtained. One area of particular interest in relation to the experiments performed would be the effects of the Sat protein on different representative cell lines. This research featured tests on the HeLa and Vero cell lines, which only represent a very small percentage of areas within the body that could be exposed to the toxin at any given time.

The natural environment for both EcN and UPEC CFT073 is the mammalian gut, which harbors more than 100 trillion bacteria (Quigley, 2013; Sender, Fuchs, and Milo, 2016) and interactions between the microbiome is very likely to play a big role in how toxins are processed by the host. Therefore, the organs and systems that make up the gastrointestinal tract, including the unique microbiota that each host possesses potentially alters the way damage is conferred with consistent exposure to the Sat protein. Very few studies have tested Sat toxicity against these cells, specifically as it relates to EcN, and it would be interesting to compare cell viability results in more relevant cell lines and potentially in vivo to get the full effect on whole organisms.

There are some additional common cell lines that mimic the linings of the stomach (ges-1, AGS) and small/large intestines (HEp-2, Caco-2, HT29, HEK 293); very few studies have tested Sat toxicity against these cells, specifically as it relates to EcN, and it would be interesting to compare cell viability results in more relevant cell lines and potentially in vivo to get the full effect on whole organisms. Another important cell

population worth studying in terms of its relationship with Sat is the fibroblast community. While not the most obvious choice for cytotoxicity studies due to their secluded location in the body, fibroblast cells can provide an interesting perspective for the functional effects of Sat. Sat from pathogenic strains, including UPEC CFT073 and diffusely adherent E. coli (DAEC), have been shown to have detrimental effects on the cytoskeletal and extracellular matrix proteins that make up structural components of the cell (Table ##). To truly understand the role of Sat in damaging structural proteins, which leads to cell death, it is important to be able to look closely at the cells that produce these proteins most often. Therefore, cell viability studies utilizing a noncancerous fibroblast cell line (ex. IMR90) or primary cell culture studies could offer great insight into further mechanisms of action for Sat from any strain of bacteria in its native environment.

The structure of Sat remains elusive and is an area with much potential for additional research. It is thought that it may be similar in structure to other SPATE proteins of similar sizes or functionalities. One prominent example of a closely related protein is the plasmid-encoded toxin (Pet); this 106-kDa secreted protein is known to induce cytopathic effects on eukaryotic cells by degrading cytoskeletal proteins and increases transepithelial short-circuit current (Navarro-Garcia et al, 2001). Pet has the ability to translocate into host epithelial cells and this internalization is a requirement for cytopathic effects to happen (Navarro-Garcia et al, 1999). Previous studies have shown that Sat is also internalized by host cells and that vacuolization occurs after Sat has successfully entered cells (insert Sat citation). The full crystal structure for Pet was fully elucidated in 2008 by Scaglione et al.

A prevailing but understudied theory regarding Sat is that the protein is thought to be the combination of a highly attractive protein complex. Once secreted out of the cell membrane and targeted inside the host, although the different subunits are still unknown.

This theory is popular for explaining the differences in Sat functionality across species

(Toloza et al, 2015). Evidence of smaller Sat protein subunits can often appear as degraded protein elements present after silver staining on an SDS-PAGE. The silver stain method is highly sensitive and can pick up faint traces of proteins and amino acids.

Methods to confirm presence of Sat protein fragments or subunits can be elucidated utilizing common techniques such as pull-down assays or co-immunoprecipitation, which would both involve specific antibody binding that would be selective for the Sat protein.

Another potential method to identify components of the potential Sat protein complex is mass spectrometry to potentially break apart and visualize unique pieces.

From an immunological perspective, EcN is one of the most interesting probiotic strains available to consumers to date. Its immunomodulatory features make it a good candidate for treatment of gastrointestinal ailments and diseases because of its recruitment of pro-inflammatory and anti-inflammatory cytokines to disturbed areas of the body, depending on what is needed at the moment (Kamada et al, 2008). The secretion of autotransporter proteins by EcN, which have always been associated with virulence in pathogenic strains, result in stimulation of the host immune system. It is possible that EcN regulates modulation of the host immune system on its own when autotransporter proteins cause damage. Therefore, it would be of interest to future researchers to determine the effects of proteins like Sat on cytokine production and it would be beneficial to do comparative studies on Sat from both EcN and UPEC CFT073.

An ELISA treated with different pro- and anti-inflammatory cytokines could aid in understanding the role Sat has in the immunomodulatory characteristics of EcN, if any. It can also be speculated that additional commensal bacteria present in the GI tract produce factors that negate potential negative manifestations; more research is needed to validate these hypotheses in vitro and in vivo.

Future directions that can build upon this research include utilization of Sat as a tool to combat agents that cause disease in the host. Engineered probiotics are becoming increasingly popular as researchers look for new ways to fight infectious agents while reducing the amount of reliance on antibiotics. In a study performed by Hwang et al

(2017) engineered EcN had the ability to target P. aeruginosa in the gut and improve survival across two separate animal models. Treatment for ailments could include modifying the active serine residues in Sat to either reduce proteolytic activity before giving to patients or finding a way to enhance Sat or other cytotoxic autotransporters in the presence of foreign pathogens or malignant cells.

The data obtained from these experiments furthers the current knowledge of the functionality of Sat from EcN by extending toxicity studies to the Vero cell line. It was shown that the concentrated supernatant from the wildtype strain of EcN, which had a significant Sat protein presence as assessed by the SDS-PAGE/silver stain analysis, conferred significant cytotoxicity against the mammalian kidney epithelial cells. It was also shown that further purification of the supernatant samples using ammonium sulfate precipitation increased cytotoxicity in Sat derived from both EcN and UPEC CFT073. It seems as if the protein itself remains toxic regardless of its origin. Sat is an excellent protein to study due to the prevalence of Sat as a secreted product in EcN, its genetic

homology to sat genes found in other strains of bacteria, and its role in cell damage specifically in pathogenic strains.

The probiotic strain EcN contains other potential virulence factors shared with other E. coli strains. This presents something to be at least wary of when choosing which probiotic supplements to use for treatment of patients. EcN has a lot of potential for therapeutic benefit to combat dysbiosis and GI tract ailments. Current longitudinal data suggests that EcN as a supplemental probiotic is safe for use in immunocompetent individuals (Gronbach et al, 2010). In order to continue to show safety, as well as efficacy of this probiotic, more molecular studies involving the factors that make EcN so potent are essential.

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