Hanna Gebreegziabher Gebremariam Role of lactobacilli in pylori pathogenesis and host cell responses

Hanna Gebreegziabher Gebremariam Role of lactobacilli in Role of lactobacilli Helicobacter

Hanna Gebreegziabher Gebremariam Born in 1987 in Hawassa, Ethiopia. pathogenesis responses and host cell MSc degree from Uppsala University in 2014. Started her PhD studies at Stockholm University in 2015.

ISBN 978-91-7911-338-4

Department of Molecular Biosciences, The Wenner-Gren Institute

Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2020

Role of lactobacilli in Helicobacter pylori pathogenesis and host cell responses Hanna Gebreegziabher Gebremariam Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Friday 11 December 2020 at 09.00 in Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20.

Abstract Helicobacter pylori is well adapted to the harsh environment of the human , allowing it to persistently colonize the gastric mucosa of at least 50% of the global population for decades. Long-term colonization induces chronic that can eventually lead to development of ulcer and gastric . The interaction between host, bacterial and environmental factors are crucial for the pathogenesis of H. pylori. In contrast, species are members of the human microbiota and act as a first line of defense against . However, the underlying mechanisms behind lactobacilli-mediated inhibition still need further investigation. This thesis focuses on understanding the interplay between commensal and pathogenic with the human host. In Paper I, we investigated the effect of different Lactobacillus strains on the initial attachment of H. pylori to gastric epithelial cells and found that certain Lactobacillus strains can prevent the adhesion of the pathogen by decreasing the expression of SabA. In Paper II, the anti-inflammatory activity of Lactobacillus strains against H. pylori-induced production of proinflammatory was explored. We demonstrated the ability of L. gasseri Kx110A1 to reduce the level of TNF and IL-6 in human through suppression of ADAM17, a metalloproteinase responsible for releasing transmembrane proteins. Lactobacilli-mediated inhibition of these cytokines was not H. pylori-specific, suggesting a general anti-inflammatory property of L. gasseri Kx110A1. In Paper III, we characterized the role of sortase-dependent proteins in L. gasseri Kx110A1. We showed that the deletion of sortase A in lactobacilli resulted in the reduction of auto- aggregation and attachment to host gastric epithelial cells. Moreover, sortase A mutant lactobacilli were not effective in preventing H. pylori initial adherence. Finally, in Paper IV, we showed that lactate can affect the expression of H. pylori adherence and the production of bacterial-induced proinflammatory cytokines.

Keywords: Helicobacter pylori, Inflammation, Lactobacillus, ADAM17, Proinflammatory cytokines, Lactate.

Stockholm 2020 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-186203

ISBN 978-91-7911-338-4 ISBN 978-91-7911-339-1

Department of Molecular Biosciences, The Wenner- Gren Institute

Stockholm University, 106 91 Stockholm

ROLE OF LACTOBACILLI IN HELICOBACTER PYLORI PATHOGENESIS AND HOST CELL RESPONSES

Hanna Gebreegziabher Gebremariam

Role of lactobacilli in Helicobacter pylori pathogenesis and host cell responses

Hanna Gebreegziabher Gebremariam ©Hanna Gebreegziabher Gebremariam, Stockholm University 2020

ISBN print 978-91-7911-338-4 ISBN PDF 978-91-7911-339-1

Printed in Sweden by Universitetsservice US-AB, Stockholm 2020 To my family

“አድርገህልኛልና፡ ለዘለዓለም አመሰግንሀለሁ” መዝሙር፡52

LIST OF PUBLICATIONS

This thesis is based on the following publications and manuscripts, which are referred to in the text by roman numerals:

I. de Klerk N, Maudsdotter L, Gebreegziabher H, Saroj SD, Eriksson B, Eriksson OS, Roos S, Lindén S, Sjölinder H, Jonsson A-B. (2016) Lactobacilli reduce Helicobacter pylori attachment to host gastric epithelial cells by inhibiting adhesion expression. Infection and Immunity 84 (5):1526-1535.

II. Gebremariam HG, Qazi KR, Somiah T, Pathak SK, Sjölinder H, Sverremark-Ekström E, Jonsson A-B. (2019) Lactobacillus gasseri suppresses the production of proinflammatory cytokines in Helicobacter pylori-infected macrophages by inhibiting the expression of ADAM17. Frontiers in Immunology 10:2326.

III. Zuo F, Appaswamy A, Gebremariam HG, Jonsson A-B. (2019) Role of sortase A in Lactobacillus gasseri Kx110A1 adhesion to gastric epithelial cells and competitive exclusion of Helicobacter pylori. Frontiers in Microbiology 10:2770.

IV. Gebremariam HG#, Somiah T#, Zuo F, Jonsson A-B. (2020) The role of lactate in Helicobacter pylori and host cell interaction. Manuscript. # HGG and TS contributed equally.

Gebreegziabher H, is the same as Gebremariam HG.

i POPULÄRVETENSKAPLIG SAMMANFATTNING

Helicobacter pylori är en bakterie som koloniserar människans mage. Förekomsten av bakterien i magen leder ofta till en livslång infektion som så småningom utvecklas till kronisk inflammation och kan om den lämnas obehandlad orsaka magsår och magcancer. Bakterier som tillhör släktet Lactobacillus är en del av den normala mänskliga bakteriefloran och kan t.ex. förhindra att sjukdomsframkallande mikroorganismer får fäste i människokroppen. I denna avhandling, har vi studerat interaktionen mellan bakterier och den mänskliga värden. Vi har fokuserat på den hämmande rollen hos laktobaciller och hur de kan påverka värdresponsen mot H. pylori. I artikel I visade vi att vissa arter av laktobaciller minskar mängden vidhäftningsmolekyler hos H. pylori som då inte kan fastna lika bra till magsäckens slemhinna. I artikel II visade vi att laktobaciller kan minska produktionen av de proinflammatoriska cytokinerna TNF och IL-6 i H. pylori-infekterade humana makrofager. Detta berodde på att laktobacillerna påverkade makrofagerna att minska enzymet ADAM17 som reglerar utsöndringen av dessa cytokiner. Laktobaciller kunde även minska nivån av TNF och IL-6 efter att makrofager stimulerats med andra immunstimulerande ämnen än H. pylori, vilket visar en H. pylori-oberoende anti-inflammatorisk roll hos laktobaciller. Artikel III behandlar betydelsen av enzymet Sortase A vid interaktionen mellan laktobaciller och H. pylori. Sortase A är det enzym som kopplar Sortase-beroende proteiner till laktobacillens yta. Laktobaciller som saknade Sortase A aggregerade och vidhäftade sämre till slemhinneceller. Avsaknad av Sortase A minskade dessutom laktobacillernas förmåga att blockera bindning av H. pylori till magslemhinneceller. I artikel IV undersökte vi hur laktat påverkar H. pylori. Laktat är en molekyl som tillverkas både av människans slemhinneceller och av laktobaciller i normalfloran. Laktat påverkade produktion av vissa vidhäftningsgener hos H. pylori samt minskade det immunologiska svaret mot bakterien

ii SUMMARY

Helicobacter pylori is well adapted to the harsh environment of the human stomach, allowing it to persistently colonize the gastric mucosa of at least 50% of the global population for decades. Long-term colonization induces chronic inflammation that can eventually lead to development of peptic ulcer and gastric cancer. The interaction between host, bacterial and environmental factors are crucial for the pathogenesis of H. pylori. In contrast, Lactobacillus species are members of the human microbiota and act as a first line of defense against pathogens. However, the underlying mechanisms behind lactobacilli-mediated pathogen inhibition still need further investigation. This thesis focuses on understanding the interplay between commensal and with the human host. In Paper I, we investigated the effect of different Lactobacillus strains on the initial attachment of H. pylori to gastric epithelial cells and found that certain Lactobacillus strains can prevent the adhesion of the pathogen by decreasing the expression of SabA. In Paper II, the anti-inflammatory activity of Lactobacillus strains against H. pylori-induced production of proinflammatory cytokines was explored. We demonstrated the ability of L. gasseri Kx110A1 to reduce the level of TNF and IL-6 in human macrophages through suppression of ADAM17, a metalloproteinase responsible for releasing transmembrane proteins. Lactobacilli-mediated inhibition of these cytokines was not H. pylori-specific, suggesting a general anti-inflammatory property of L. gasseri Kx110A1. In Paper III, we characterized the role of sortase-dependent proteins in L. gasseri Kx110A1. We showed that the deletion of sortase A in lactobacilli resulted in the reduction of auto-aggregation and attachment to host gastric epithelial cells. Moreover, sortase A mutant lactobacilli were not effective in preventing H. pylori initial adherence. Finally, in Paper IV, we showed that lactate can affect the expression of H. pylori adherence genes and the production of bacterial-induced proinflammatory cytokines.

iii

iv CONTENTS

LIST OF PUBLICATIONS ...... i

POPULÄRVETENSKAPLIG SAMMANFATTNING ...... ii

SUMMARY ...... iii

CONTENTS ...... v

ABBREVATIONS ...... vii

INTRODUCTION ...... 1

Chapter 1 Helicobacter pylori ...... 2 1.1 Classification ...... 2 1.2 Epidemiology and Transmission ...... 2 1.3 Diagnosis and Treatment ...... 3 1.4 Major virulence factors ...... 5 1.4.1 Acid resistance ...... 5 1.4.2 Outer membrane proteins ...... 6 1.4.3 Cytotoxin-associated gene A ...... 7 1.4.4 Vacuolating cytotoxin A ...... 8 1.5 Environmental factors ...... 9 Chapter 2 The Host ...... 10 2.1 Innate immunity ...... 10 2.2 Macrophages ...... 11 2.2.1 polarization ...... 12 2.3 A disintegrin and metalloproteinase (ADAM) ...... 13 2.3.1 ADAM17 ...... 15 Chapter 3 H. pylori and the Host ...... 18 3.1 Interaction with host cells ...... 18 3.2 Macrophages in H. pylori infection ...... 20 3.3 Lactate in H. pylori pathogenesis ...... 21 Chapter 4 Lactobacillus ...... 23 4.1 Lactobacillus interaction with host cells ...... 24 4.2 Modulation of host responses by lactobacilli ...... 26 4.3 Protective role of lactobacilli against pathogens ...... 27 4.4 Lactobacillus and H. pylori ...... 30 PRESENT INVESTIGATION ...... 32 Aims ...... 32

v RESULTS AND DISCUSSION ...... 33 Paper I ...... 33 Paper II ...... 36 Paper III ...... 38 Paper IV ...... 40 FUTURE PERSPECTIVES ...... 42 ACKNOWLEDGEMENTS ...... 45

REFERENCES ...... 50

vi ABBREVATIONS

ADAM A disintegrin and metalloproteinase AMP BabA group -binding adhesin cag PAI cag CagA Cytotoxin-associated gene A CEACAM Carcinoembryonic antigen-related molecules DAMPs Damage-associated molecular patterns DC Dendritic cell EGFR Epidermal growth factor receptor EPS Exopolysaccharide HtrA High temperature requirement A IL Interleukin LPS LTA Lipoteichoic acid MAPK Mitogen activated PAMP Pathogen-associated molecular pattern PPI Proton pump inhibitor PRR Pattern recognition receptor S-layer Surface layer SabA Sialic-acid binding adhesin SDPs Sortase-dependent proteins T4SS Type IV system TACE converting enzyme TNF Tumor necrosis factor VacA Vacuolating cytotoxin A

vii

viii INTRODUCTION

To characterize microorganisms as a causative agent of a disease, Robert Koch formulated a few guidelines in the so-called Koch`s postulates in 1890. According to Koch`s postulate, pathogenicity was believed to be an invariant trait that was entirely determined by microorganisms, but with better technologies and scientific knowledge, we now understand that the pathogenicity of microorganisms can be influenced by other factors including the host. For example, microorganisms that can deadly diseases can be asymptomatically carried, while few commensals can cause opportunistic infections in individuals. This shows the fundamental role of the host immune response in determining disease outcome [1, 2]. In the case of Helicobacter pylori, which is the causative agent of , peptic ulcer, and gastric cancer, an long-term colonization is established in most infected individuals, where only a few percentages produce serious infections. Thus, host-pathogen interaction is a very complex and dynamic process, and understanding this interaction will greatly improve disease prevention and treatment in the future. The general aim of this thesis was to study the interaction between bacteria and the human host. Paper I and Paper II investigate the role of Lactobacillus strains on H. pylori attachment and the pathogen-induced responses, respectively. In addition, the mechanisms that lactobacilli utilize for these roles are identified. Paper III explores the effect of sortase-dependent proteins in lactobacilli adhesion and their ability to inhibit H. pylori adhesion. Lastly, Paper IV examines the influence of lactate on H. pylori virulence gene expression and host cytokine response.

1 Chapter 1 Helicobacter pylori

1.1 Classification

Due to its hostile acidic conditions, the human stomach was believed to be sterile until the discovery of Helicobacter pylori from gastric of patients in 1982. The two Australian scientists Barry Marshal and Robin Warren were awarded the Nobel prize in 2005 for their discovery. Sharing several characteristics with the caused H. pylori to be initially classified under this genus and named as Campylobacter pylori. But later, studies showed distinctions between H. pylori and members of Campylobacter, allowing the bacterium to be grouped into the new genus, Helicobacter [3, 4]. At present, the genus Helicobacter consists of over 35 validated species that are Gram- negative, motile, and non-spore-forming bacteria with the ability to colonize humans and various animals. H. pylori is the most well-studied human pathogen in this genus [5-7].

1.2 Epidemiology and Transmission

H. pylori has accompanied modern humans since before their migration out of Africa 60,000 years ago and has coevolved alongside humans [8, 9]. H. pylori infection is the most common bacterial infection worldwide, colonizing at least 50% of the global population. However, the prevalence of infection varies across geographical areas, socioeconomic status, age and ethnicity. For example, 50% of individuals in developing countries are infected with H. pylori compared to 35% in industrialized countries. Currently, a marked decline in the prevalence of H. pylori infection has been reported in various parts of the world [10-12]. It is

2 still not clear how H. pylori infection is transmitted, but oral-oral, fecal-oral, or gastro-oral routes are the main suggested transmission routes. Transmission within family members, especially from mother to child, has also been reported [13, 14]. In addition, the detection of H. pylori in drinking water, vegetables, and animal food products, raises the possibility of H. pylori transmission through contaminated water and food, but this demands further investigation [15]. Therefore, poor sanitation, hygiene, and housing conditions enhance bacterial transmission rates. H. pylori is commonly acquired during childhood and persists in the host for decades without causing any symptoms. Bacterial presence in the stomach induces chronic gastritis in nearly all infected individuals. However, continuous interactions between H. pylori and the host leads to the development of in 10%-15%, gastric cancer in 1%-3%, and mucosa-associated lymphoid tissue (MALT) lymphoma in less than 0.1% [16, 17]. In 2018, approximately 783,000 deaths were reported to be caused by , making it the third leading cause of cancer-related deaths in the world [18, 19]. H. pylori infection is the major risk factor for gastric ; because of this, the bacterium was recognized as a class I by the World Health Organization (WHO). Although a larger portion of the world`s population is infected with H. pylori, only a few percentages develop an overt disease, which could be influenced by both bacterial and host factors. Other factors, such as co- infection with helminths, , asthma, and diet, have been reported to affect the risk of H. pylori- associated disease [20, 21].

1.3 Diagnosis and Treatment

There are multiple diagnostic tests for the detection of H. pylori. These tests are commonly divided into invasive and non-invasive methods. Endoscopic examination is an example of an invasive test that is carried out to diagnose H.

3 pylori-associated infections or to obtain biopsies that can be further assessed using bacterial culture, , and molecular methods. Bacterial culture from samples provides precise results [22]. A breath test is the most widely used non-invasive test, detecting the activity of H. pylori by measuring labeled

CO2 in the breath after the hydrolysis of urea in the stomach. A stool antigen test is another useful non-invasive test for diagnosing H. pylori [23]. The increasing prevalence of resistance in H. pylori makes the eradication of the pathogen very challenging. The previously recommended treatment for eradication of H. pylori was a standard triple , including proton pump inhibitor (PPI), , and [24]. However, the eradication rate obtained with this regimen is currently less than 80% as a result of increasing drug resistance H. pylori strains. Hence, clarithromycin-resistant H. pylori was categorized by the WHO as a high priority bacterium that needed new alternative [25, 26]. At present, the first-line treatment in areas with high clarithromycin and resistance are the -containing quadruple therapy or the non-bismuth quadruple therapy consisting of a combination of PPI with two or three antibiotics (amoxicillin, clarithromycin, metronidazole, tetracycline or nitroimidazole) for 10-14 days [25, 27]. Although this treatment regimen has achieved an eradication rate of above 90%, there are multiple factors that contribute to eradication failures, such as bacterial factors (virulence factors, ability to invade the host cell and ability to form , transformation into a coccoid form), host factors (adverse effects, poor patient compliance), and environmental factors (smoking, poor sanitation) [26, 28]. The use of as an additional treatment has been receiving extensive attention recently due to improvement in H. pylori eradication and reduced drug side effects; however, this requires further research [29, 30].

4 1.4 Major virulence factors

To successfully colonize the human stomach and establish a persistent infection, H. pylori must survive in the harsh acidic environment and also circumvent the host immune system. Because the bacterium has coevolved with humans, it has developed a number of strategies to manipulate and use the host for its benefit [31]. H. pylori possesses various virulence factors that are responsible for its successful colonization and pathogenesis. However, in this chapter, only the most well-studied bacterial virulence factors will be summarized.

1.4.1 Acid resistance

The low pH in the stomach (pH 1-2) is one of the protective mechanisms limiting the growth of a wide-range of ingested microbes. H. pylori withstands gastric acidity through the production of urease enzyme, which hydrolyzes urea into and , thereby neutralizing the pH around the bacterium [32]. Urease is a crucial of H. pylori that is produced in larger quantities (approximately 10% of total protein) mainly intracellularly. However, it is also found on the bacterial cell surface because of the lysis of some [33]. In the gastric layer, there is a pH gradient where it is acidic (pH 1-2) in the lumen and almost neutral at the . External pH dictates the activity of intracellular urease and UreI (a channel for urea entry) in H. pylori, causing the adjustment of periplasmic pH only in acidic conditions, limiting lethal alkalization under neutral environments [34, 35]. Furthermore, the acid- responsive two-component system, ArsRS, has been shown to be involved in the regulation of urease [36, 37]. Urease-mediated increase in pH not only promotes H. pylori survival, but also reduces the viscoelasticity of the gastric mucus, enabling the bacterium to swim faster [38]. H. pylori motility is conferred by its 4-8 polar sheathed flagella [39]. The fundamental roles of the urease enzyme and

5 bacterial flagella have been shown in mutants lacking urease [40] or flagella [41], which are unable to successfully colonize the stomach. The helical shape of H. pylori also aids in bacterial colonization by allowing penetration of the mucus layer in a corkscrew-like motion facilitating its orientation close to the gastric epithelium [17].

1.4.2 Outer membrane proteins

The majority of H. pylori bacteria are present in the mucus layer, binding to highly glycosylated [42]. However, few of these bacteria come in close contact with the host epithelial cells and are believed to cause diseases [43, 44]. Adhesion to the gastric mucosa is a necessary step for H. pylori pathogenesis, and the bacterium utilizes several outer membrane proteins (OMPs) to achieve this. H. pylori OMPs are categorized into five families, in which the Helicobacter outer membrane proteins (Hop) family is the largest. AlpA/B, OipA, HopQ, HopZ, and the two well-characterized adhesins, BabA and SabA, belong to the Hop family. The expression of H. pylori OMPs are generally regulated via gene conversion, gene duplication, allelic variation, and phase variation [10, 45, 46]. Blood group antigen-binding adhesin (BabA) is the first identified adhesin of H. pylori that binds to Lewis b blood group antigen (Leb) and fucosylated structures on mucins and gastric epithelial cells [47]. BabA expression is regulated through several different mechanisms. For instance, the location of babA in the has been shown to affect its production in different H. pylori strains. Furthermore, changes in the number of dinucleotide cytosine-thymidine (CT) repeats in the 5`region also influence the expression of babA through phase variation [48, 49]. In different animal models (mice, gerbils, and macaques), loss of BabA expression has been demonstrated during infection, verifying its dynamic expression [50].

6 The expression of sialylated glycoconjugates in normal human gastric mucosa are sparse, but their level increases during H. pylori-induced inflammation [51, 52]. The sialic acid-binding protein (SabA) is the second well- studied adhesin of H. pylori that mediates the attachment of the bacterium to inflamed gastric mucosa by binding to sialyl-Lewis x and sialyl-Lewis a [53]. Similar to BabA, the regulation of SabA expression is very complex. The presence of a poly (T) tract in the promoter region and a (CT) repeat in the coding region of sabA can alter the length of these repeats, causing sabA to undergo phase variation through slipped-strand mispairing. In addition, the ArsRS two- component system is involved in the modulation of SabA expression in response to changes in the gastric environment, such as pH and inflammation [54-56]. Besides the above-mentioned H. pylori adhesins, there are many other proteins that are involved in the attachment of the bacterium to host cells and the mucus. For instance, the adherence-associated lipoprotein A and B (AlpA/B), lacdiNAc-binding adhesins (LabA) and HopQ mediate the interaction of the pathogen with laminin, lacdiNAc structures on gastric mucins, and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), respectively [57-59].

1.4.3 Cytotoxin-associated gene A

Depending on the possession of the CagA gene, H. pylori strains are divided into CagA positive and CagA negative subpopulations, in which the CagA positive strains are correlated with severe gastric disease outcomes. The cagA gene is located in the cag pathogenicity island (cag PAI), which also contains genes for encoding elements of the type IV secretion system (T4SS), a syringe-like structure responsible for injecting CagA into the host cell cytoplasm. The cag PAI is a 40

7 kb DNA segment that contains approximately 31 genes, including cagA [60, 61]. To translocate CagA into host cells, bacterial surface adhesins, such as HopQ or CagL, must first bind to the host cell receptors, CEACAM and integrin-b1, respectively [62-65]. Once CagA enters into host cells, it resides in the inner surface of the plasma membrane and undergoes tyrosine phosphorylation by the Src and Abl family of kinases at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in the Carboxy-terminal region [66, 67]. Based on the relative position of CagA and flanking sequence, four distinct types of EPIYA repeat motif (EPIYA- A- EPIYA-D) have been described. In most CagA proteins, three EPIYA motifs are present, but the number can vary in a strain-specific manner [68]. Phosphorylated CagA binds to Src homology 2 containing tyrosine phosphatase (SHP-2) and exerts various cellular alterations after initiating the phosphatase activity of SHP-2. The extent of SHP-2 binding is important for a CagA-induced outcome, which in turn depends on the number and sequence of CagA phosphorylation sites [69].

1.4.4 Vacuolating cytotoxin A

The well-characterized toxin of H. pylori, vacuolating cytotoxin A (VacA), causes vacuole formation in the cytoplasm of host epithelial cells, as its name implies. In addition to inducing vacuolation, it is now evident that VacA exerts multiple effects on target cells and, thus, is called a multifunctional toxin [70]. VacA is expressed as a precursor toxin consisting of a signal sequence, a passenger domain, and an auto-transporter domain. The passenger domain is then cleaved from the auto-transporter domain producing the mature 88 kDa toxin that is secreted into the extracellular space but can also remain bound on the bacterial surface [71]. The mature VacA toxin containing the p33 (N-terminal) and p55 (C- terminal) domains binds to host cell receptors, internalized via endocytosis, and

8 produces a broad range of biological functions on host cells [72]. Various host transmembrane receptors have been described for binding VacA, including the receptor protein tyrosine phosphatases (RPTP-a and -b), low-density lipoprotein receptor-related protein-1, epidermal growth factor receptor (EGFR), and heparan sulfate [70, 72]. All H. pylori strains carry the vacA gene, but strain-specific variation arises due to sequence diversity. H. pylori strains containing the allelic form of VacA s1m1 are associated with an increased risk of gastric disease [73].

1.5 Environmental factors

Not only bacterial virulence factors and host genetic variations but also environmental factors have been demonstrated to play a role in determining the risk of developing H. pylori-associated disease. In epidemiological studies and animal models, high dietary salt intake has been reported to increase the risk of gastric cancer development [74-76]. However, the exact mechanism by which a high salt diet augments bacterial pathogenesis is not fully understood. Some of the suggested possible explanations that might contribute to the development of gastric cancer are increased expressions of H. pylori virulence genes, such as VacA and CagA, in high salt conditions [77, 78]. Cigarette smoking has also been linked with an increased risk of H. pylori- induced gastric cancer development. This could be due to the higher susceptibility of the host to H. pylori infection, as various carcinogenic components from tobacco are known to damage the host gastric mucosa. In certain populations, the treatment failure rate for H. pylori infection has been correlated with smoking [79-81].

9 Chapter 2 The Host

2.1 Innate immunity

The human body is colonized and continuously exposed to a diverse array of microorganisms. However, these microorganisms are constantly surveyed by the host defense mechanism to establish a symbiotic relationship. Nevertheless, in some instances, pathogenic bacteria can escape from the host immune response and cause disease. Initially, anatomic and physiologic barriers, such as intact skin, epithelial barrier, production of mucus, gastric juices and enzymes (lysozyme in saliva and tears), play a critical role in limiting the entry of pathogens. If these barriers are breached, the innate immune system immediately generates a protective response that enhances the elimination of the invading pathogen. The innate immunity is composed of cellular components (macrophages, dendritic cells (DCs), , and NK cells) and humoral components (complement proteins, antimicrobial peptides (AMP), and C-reactive proteins). Cells of the innate immunity express receptors known as pattern recognition receptors (PRRs) that can recognize pathogen-associated molecular patterns (PAMPs). PAMPs are essential components of microorganisms that are commonly shared by large groups of microbes. Examples of PAMPs are lipopolysaccharide (LPS), , , and viral double-stranded RNA [82-84]. During microbial infection or tissue damage, our cells release components called damage- associated molecular patterns (DAMPs) that are also recognized by PRRs [85]. PRRs can be found on immune cell surfaces, endosomal compartments, cytoplasm, or as a soluble form in body fluids, making them capable of detecting intracellular and extracellular PAMPs. Toll-like receptors (TLRs) are the most well-known PRRs. For instance, the above-mentioned microbial PAMPs are

10 perceived by the following PRRs: TLR4, TLR2, TLR5, and TLR3, respectively [86]. The recognition of PAMPs by their respective PRRs triggers downstream intercellular signaling and induces various cellular responses, including secretion of cytokines and chemokines, expression of costimulatory molecules, and finally activation of the adaptive immunity [87, 88].

2.2 Macrophages

Macrophages are essential cells of the innate immunity that are present in all mammalian tissues. These cells were initially characterized by the Russian scientist Elie Metchnikoff for their phagocytic ability [89]. It is now clear that macrophages perform multiple functions in different tissues, including maintenance of tissue integrity and homeostasis, elimination of dead cells and foreign materials, regulation of inflammatory responses, and acting as a bridge between innate and adaptive immunity [90]. The macrophage population in our body develops from two lineages: tissue-resident macrophages and monocyte- derived macrophages. Tissue-resident macrophages originate from the yolk sac progenitor and have the ability to self-maintain their population. In addition, they express genes that are specific to their location and are named according to the site. For instance, alveolar macrophages reside in the lung, Kupffer cells in the liver, microglia cells in the brain, and osteoclast cells in the bone [89, 91]. As the name implies, monocyte-derived macrophages come from bone marrow-derived monocytes that are able to differentiate into macrophages and DCs. Upon infection and inflammation, circulating monocytes are recruited to the site to replenish the macrophage pool in the area [92, 93].

11 2.2.1 Macrophage polarization

Macrophages are extremely heterogeneous, and depending on the local environmental stimuli, such as cytokines and microbial products, they can polarize into classically activated or alternatively activated macrophages . Classically activated macrophages are proinflammatory, and polarization is achieved by LPS and IFN-g. Conversely, alternatively activated macrophages display anti-inflammatory functions and can be further polarized into M2a (by IL- 4 and IL-13), M2b (activation by Fc receptors and immune complexes), and M2c (by IL-10, transforming growth factor b (TGF-b) and glucocorticoids) [94, 95]. The M1/M2 polarization nomenclature was established based on the arginine by macrophages. While M1 macrophages produce nitric oxide (NO) from arginine, M2 macrophages generate ornithine and urea. Ornithine is vital for several repair processes; as a result, M2 macrophages are associated with wound healing and tissue repair. On the other hand, M1 macrophages kill microbes or infected cells through NO production. Although, classically activated macrophages are commonly termed as M1 and alternatively activated macrophages as M2, IFN-g, and IL-4, are not required for the M1 and M2 phenotype, respectively [96, 97]. M1 macrophages participate in the inflammatory process through of proinflammatory cytokines, such as TNF, IL-12, and IL-6, increasing antigen presentation through enhanced expressions of costimulatory molecules (CD86) and performing microbicidal activities [98]. On the contrary, M2 macrophages alleviate the inflammatory process, clean up apoptotic cells, and repair damaged tissue. The M1/M2 dichotomy provides a better understanding of macrophage diversity in a defined activation scenario. However, it does not reflect the complex process where different stimuli, a range of various cytokines on a larger scale, and a mixed population of macrophages exist together [99, 100]. Moreover, depending on external cues and stage of a disease, macrophages can shift their polarization state

12 from M1 to M2 or vice versa [101]. Lack of experimental standards in vitro still creates discrepancies in macrophage phenotype markers making the usage of this nomenclature challenging. Because macrophage activation affects a wide range of gene expression depending on various external factors, it is difficult to simply categorize macrophages as M1 and M2. Furthermore, detailed descriptions of methods and activators used in vitro could increase the reproducibility of results between different laboratories and may avoid confusion of the issue [102].

2.3 A disintegrin and metalloproteinase (ADAM)

For many transmembrane proteins (for example, cytokines, growth factors, various ligands, and receptors), cleavage from the cell surface is necessary for their systemic function. This is achieved by a family of known as the ADAMs (a disintegrin and metalloproteinases). The ADAMs belong to the adamalysin subfamily of proteases, which also include the snake venom metalloproteinases and the ADAMTSs (ADAM with thrombospondin domains). ADAMs and ADAMTSs share similar structural domain and protein sequence, but while most ADAMs are membrane-anchored, ADAMTSs are secreted enzymes [103, 104]. So far, 22 ADAMs have been described in humans, but only 13 are proteolytically active. The proteolytically active ADAMs are responsible for shedding ectodomains of various membrane-anchored proteins. However, the functions of the proteolytically inactive ADAMs are not well known, but they could act as peptide-binding receptors or involved in cell-cell interaction and intracellular signaling [105, 106]. More than half of ADAM enzymes are primarily present in reproductive organs, including the testis and uterus, participating in embryo development [103]. ADAMs have a multidomain structure which includes a pro-domain, a metalloproteinase (catalytic) domain, a disintegrin (-like) domain, a cysteine-rich domain, an epidermal growth factor (EGF)-like domain (the cysteine-rich domain

13 and EGF-like domain are not present in ADAM10 and 17), a transmembrane region, and a cytoplasmic tail [107] (Figure 1). ADAMs are initially synthesized in the endoplasmic reticulum (ER) with their pro-domain (to ensure proper protein folding and inhibit proteolytic activity), which is then removed in the Golgi apparatus by a pro-protein convertase conferring full proteolytic activation of ADAMs. In catalytically active ADAMs, the metalloproteinase domain is the part that processes various transmembrane substrates and it has the signature Zn2+- binding sequence. Following the metalloproteinase domain is the disintegrin-like domain, which is expressed on the cell surface and mediates the interaction of ADAMs with integrins. The hypervariable region (HVR) in the cysteine-rich domain is crucial for substrate selection and interaction. By changing their conformation, the disintegrin-like domain and the cysteine-rich domain are suggested to prevent access of substrates to the metalloproteinase domain possessing an autoregulatory role. This self-inhibitory conformation is released during substrate binding. The extracellular parts of ADAMs are connected to the intracellular components via the transmembrane domain and the cytoplasmic tail; the latter initiates downstream phosphorylation and regulates mRNA expression of various genes. However, the cytoplasmic tail is not required for proteolytic activity of some ADAMs [105, 108, 109]. Cleavage of substrates by ADAMs can occur both constitutively and after activation by a large group of stimuli. To impede unnecessary activation of these enzymes, their proteolytic function is tightly regulated through various mechanisms [110]. For instance, the pro-domain prevents the intracellular enzymatic activity of ADAMs. The Rhomboid family of proteins, iRhom1 and iRhom2, were demonstrated to be important in ADAM17 exit from the ER; without these proteins, there will not be extracellular proteolytic activity. Moreover, the tissue inhibitor of metalloproteinases (TIMPs) can also prevent the enzymatic activity of some ADAMs [109, 111, 112].

14

Pro-domain Metalloproteinase domain Zn2+

Disintegrin-like domain

Cysteine-rich domain HVR

EGF-like domain Transmembrane

Cytoplasmic domain

Figure 1. General structure of ADAM proteases (A disintegrin and metalloproteinase). EGF, epidermal growth factor. Adapted from [107]. The illustration is not drawn to scale.

2.3.1 ADAM17

ADAM17 is one of the well-characterized ADAM proteases that is present in most mammalian host cells. ADAM17 was initially named as a TNF-converting enzyme (TACE) due to its ability to shed surface-bound precursor TNF into a soluble form. To date, it is known that ADAM17 can proteolytically cleave the ectodomains of more than 80 substrates, including cytokines, growth factors, cell adhesion proteins, and cytokine receptors [113]. The most prominent examples of ADAM17 substrates are TNF and its receptors (TNF-R1 and TNF-R2), interleukin-6 receptor (IL-6R), and most ligands of EGFR. Once these components are cleaved from the cell surface, they regulate the expressions of an array of genes by binding to receptors on the same cell (autocrine effect),

15 receptors on nearby cells (paracrine effect), or entering the circulatory system (endocrine effect) [114]. The potent proinflammatory cytokine, TNF, is initially produced as a 26 kDa membrane-bound protein. Membrane-bound TNF plays a crucial role in inflammatory responses by acting as a ligand to TNF receptors on target cells and as a receptor signaling back to the producing cell in a direct cell contact-dependent manner. All nucleated cells express TNF-R1, but TNF-R2 is mainly expressed in immune cells. On the other hand, to affect distant cells, TNF needs to be proteolytically cleaved by ADAM17 and released from cell surfaces. Variations in TNF-induced responses can occur in cells depending on the type of receptors to which the cytokine binds. For instance, TNF-mediated signaling through TNF- R1 can induce , whereas, via TNF-R2-initiated signaling, TNF can produce cell survival [115, 116]. TNF is chiefly produced by monocytes and macrophages, but other cells, including neutrophils, natural killer (NK), T- and B-lymphocytes, and endothelial cells, can also release lower levels of TNF [116, 117]. IL-6 is produced by nearly all immune cells and exerts a wide-ranging effect on numerous cell types. In the human body, the level of IL-6 is normally very low (1-5 pg/ml), but its concentration increases during disease conditions [118]. IL-6 can both participate in normal homeostatic processes as well as in inflammatory and disease conditions. On target cells, IL-6 binds to IL-6R (IL-6R) on the and forms a complex. This complex then associates with the signaling receptor subunit gp130 (expressed by almost all cells in the body), leading to its dimerization and induction of downstream signaling. This signaling pathway is termed as the classical IL-6 signaling and is generally produced during initial immune responses. On the other hand, ADAM17 can cleave and release IL-6R from the cell surface as a soluble form (sIL-6R). The sIL-6R in the environment then binds to IL-6 and produces a different type of IL-6 signaling called the IL-6 trans-signaling. Through trans-signaling, almost all cells in the

16 body can respond to IL-6 through the expression of gp130. However, with classical IL-6 signaling, only cells that express IL-6R, such as hepatocytes, leukocytes, and some epithelial cells, respond to the cytokine [119, 120]. While IL-6 classical signaling is implicated to be protective, trans-signaling is mainly involved in inflammation and inflammation-associated cancer [121]. Therefore, ADAM17 is partly responsible for IL-6-mediated host damage. Both TNF and IL-6 play a crucial role in a number of inflammatory diseases, such as rheumatoid arthritis, Crohn`s disease, inflammatory bowel disease, ulcerative colitis, and various types of cancer [119, 122]. Understanding the role of these cytokines in various diseases is essential for the development of treatments. In addition to TNF and IL-6, ADAM17 is also responsible for processing and regulating an array of other transmembrane proteins. Thus, it is not surprising that the enzyme has been shown to be involved in myriad biological processes in health and disease. The pivotal function of ADAM17 in development has been demonstrated in mice lacking ADAM17, which resulted in embryonic lethality due to defects in EGFR signaling [123]. More knowledge about ADAM17 activity and regulation in vivo will improve future therapeutics for a multitude of inflammatory diseases.

17 Chapter 3 H. pylori and the Host

3.1 Interaction with host cells

The mucus layer in the stomach forms a physical barrier protecting the underlying epithelial cells from luminal contents (HCL, microorganisms, digestive enzymes). The dense, firmly adherent gel close to the epithelium is devoid of microorganisms, whereas the loosely adherent outer mucus layer contains bacteria. H. pylori, as mentioned above, changes the viscoelastic nature of the mucus by neutralizing the acidic pH via urease production and rapidly swims across the mucus layer to colonize the gastric epithelial surface [124, 125]. The bacterium then initiates its interaction for example, by attaching to epithelial surfaces and subsequently translocating bacterial products, such as CagA, into host epithelial cells through its T4SS [126]. CagA affects numerous signaling pathways and induces several responses in host cells. For example, the typical cell elongation phenotype, also known as the “hummingbird phenotype,” is caused by phosphorylated CagA and is accompanied by increased cell motility and scattering. CagA also promotes cell proliferation by inducing the Ras-ERK MAPK and Wnt-β-catenin signaling pathways. Further, CagA has also been shown to induce antiapoptotic effects, disruption of cell-cell junctions and elicit inflammatory responses (TNF, IL-1, and IL-8 production) [61, 127, 128]. As most Helicobacter bacteria reside in the mucus layer, secreted products like VacA, urease, and high-temperature requirement A (HtrA) are important in direct interaction with the host independently of H. pylori adhesion [129]. VacA can exert toxic effects in epithelial cells and various types of immune cells. For instance, after binding to sphingomyelin in lipid rafts, VacA is internalized and forms anion-selective channels in the endosome membrane causing swelling and

18 vacuolization. VacA can also be transported to the mitochondria and forms chloride channels, subsequently leading to cytochrome c release and apoptosis [130-132]. Moreover, VacA induces IL-8 and IL-6 production [133, 134] and also allows H. pylori to escape host immune responses by preventing T-cell activation and proliferation [135-138] (Figure 2). H. pylori urease is not only an essential colonization factor but also a potent immunogen that can evoke a strong inflammatory response in host cells [139, 140]. High levels of ammonium generated from hydrolysis of urea by the bacterial urease enzyme has been shown to cause tissue damage [140, 141]. Additionally, reducing the stomach acid as a result of H. pylori infection creates a favorable growth condition for other bacterial species [142].

BabA SabA Urease

Apoptosis VacA

CagA

IL-2 H. pylori Disruption of epithelial IFN-γ barrier IL-8 TNF Macrophage IL-1 PMN recruitment IL-2R

Urease T cell

H. pylori NO IL-12

Inflammation Figure 2. Summary of the interactions between H. pylori and host immune cells. H. pylori can directly translocate CagA into host cells, which leads to the production of IL-8 by the epithelial cells. IL-8 recruits polymorphonuclear cells (PMN) and macrophages to the infection site. These cells produce more cytokines (TNF, IL-1, IL-12) and cytotoxic compounds, including nitric oxide (NO), resulting in inflammation. TNF can cause apoptosis of epithelial cells which subsequently disturb the barrier function and allows the entrance of bacteria or bacterial secreted components like VacA that cause more damage to the host. The host immune response is not usually effective in clearing the pathogen, as H. pylori is able to escape host responses by various mechanisms, for example, by preventing T-cell activation and proliferation, leading to its persistence. Adapted from [138]. The illustration is not drawn to scale.

19 3.2 Macrophages in H. pylori infection

A strong innate and adaptive immune response is generated during H. pylori infection; however, in most cases, these responses are not adequate to eliminate the pathogen, leading to its persistence [143]. Upon infection, inflammatory cells such as macrophages, DCs, neutrophils, mast cells, and T- and B-cells are recruited into the site of infection. The level of infiltrating cells has been associated with the severity of mucosal inflammation and damage [144]. These effector cells, among other factors, produce proinflammatory cytokines (TNF, IL- 1β, IL-6), chemokines (CXCL8), reactive species (ROS), and NO. With the ability of H. pylori to establish a lifelong infection, the continuous release of these inflammatory mediators produces chronic inflammation and that will eventually contribute to gastric carcinogenesis [145, 146]. Chronic gastric inflammation plays a principal role in determining H. pylori-induced disease outcome [147]. Macrophages are essential for controlling H. pylori load. However, these cells also play an active role in H. pylori-induced inflammatory responses and can contribute to the severity of the disease. The transient removal of macrophages from the stomach and spleen of mice has been shown to lower H. pylori- associated pathology during the early stage of infection, demonstrating the contributory role of macrophages [148]. H. pylori induces macrophage apoptosis [149, 150], inhibits phagocytosis, and survives inside phagosomes [151, 152], which favors bacterial persistence and potentiates chronic inflammation. In addition, the ability of the pathogen to escape macrophage-mediated killing creates a sustained cycle of proinflammatory cytokines, ROS, and NO production in the stomach. These components alter gastric physiology and exacerbate H. pylori-induced inflammation that ultimately exerts deleterious effects on the host [139, 153].

20 3.3 Lactate in H. pylori pathogenesis

Lactate, the conjugate base of , was first discovered from sour milk by the Swedish chemist Carl Wilhelm. Lactate exists as two stereoisomers: L-lactate and D-lactate. Both stereoisomers are present in humans; however, our cells mainly produce L-lactate. Mammalian cells can also secrete a small amount of D- lactate, but microorganisms, especially , are the major source of D-lactate in the human body [154]. Lactate is utilized as an energy source mainly during times of metabolic stress. The utilization of lactate by various microorganisms has been associated with the induction of their pathogenicity. This has been well-studied in pathogenic Neisseria species, N. meningitidis and N. gonorrhoeae [155-157]. For instance, the ability of N. gonorrhoeae to utilize lactate promotes oxygen consumption enabling the bacterium to evade - dependent bactericidal mechanisms by competing for oxygen [158]. Lactate uptake and metabolism were also shown to be important for the survival and colonization of N. gonorrhoeae in vivo [159]. Likewise, in N. meningitidis, lactate metabolism increased bacterial tolerance to complement-mediated killing [160]. Other pathogenic microorganisms, such as Staphylococcus aureus [161] and Candida albicans [162], have also been demonstrated to utilize lactate and thereby enhance their virulence. However, the role of lactate in H. pylori pathogenicity is largely unknown. Nutrients that are released from gastric epithelial cells, including L-lactate, are shown to promote H. pylori growth [163-165]. This growth enhancement can contribute to bacterial-persistent colonization of the stomach [165]. H. pylori is able to use both L-and D-lactate and also produce lactate from metabolism [166, 167]. Other than lactate and glucose, the bacterium can also metabolize pyruvate as an energy source [168, 169]. In the gastric juice, the secreted L-lactate level can reach up to 0.3-1 mM; however, its concentration close to the gastric cells is unknown [170]. In blood

21 and normal tissue, physiological levels of lactate reaches approximately 1.5-3 mM, but it can go up to 10-40 mM at inflamed tissues [171]. Increased lactate concentration in the body has been associated with disease conditions, especially in various types of cancer. In order to maintain proliferation, cancer cells enhance glucose metabolism (glycolysis), subsequently upregulating lactate production and accumulation [172]. Accumulation of lactate is generally accompanied by lower pH that is often linked to the damaging effects on immune cells. Cellular metabolism and metabolites like lactate have been shown to affect immune cell functions, including cytokine production. Lactic acid, for example, affects DC functionality into a more tolerogenic phenotype by lowering IL-12 production and increasing IL-10 secretion [173]. The ability of lactate to reduce proinflammatory cytokines was also shown in LPS-stimulated murine macrophages and monocytes [174, 175]. Other than being an energy source for cells, lactate also acts as a signaling molecule affecting many cellular homeostatic functions. H. pylori has been shown to exhibit a chemotactic response to L-lactate [170]. In Paper IV, we have demonstrated that L-lactate, but not D-glucose, decreased the proinflammatory cytokines TNF and IL-6 in H. pylori-stimulated macrophages. Here, there might be a possibility that L-lactate acted as a signaling molecule in addition to being an energy source, but this needs further investigation.

22 Chapter 4 Lactobacillus

The genus Lactobacillus comprises of Gram-positive, nonsporulating, - negative, and mostly nonmotile bacteria. There are more than 200 species in the genus and are characterized by the production of lactic acid as the primary end product of carbohydrate fermentation. Because of these abilities, Lactobacillus species are commonly incorporated in food products for food fermentation, food preservation, and food texture [176, 177]. Lactobacilli are fastidious microorganisms, and therefore are present in nutrient-rich environments in plants, humans, vertebrate animals and invertebrate animals. In the human body, lactobacilli are part of the normal microbiota and are abundantly present in the female genital tract [178]. Lactobacilli are the principal microorganism used in probiotics. According to the Food and Agricultural Organization (FAO/WHO), probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer health benefits on the host” [177, 179]. Probiotics have been added to a variety of foods and are also commercially available as lyophilized pills, which expanded their consumption worldwide [180]. Probiotics, especially Lactobacillus strains, have been demonstrated to improve human health when they are used in the treatment of antibiotic-associated diarrhea, inflammatory bowel disease, and necrotizing enterocolitis in premature infants. For these protective roles, lactobacilli strains use different mechanisms, such as modulation of the immune response, prevention against pathogens, and improvement of the epithelial barrier function [181, 182]. It is worth noting that the efficacy of probiotics in alleviating infections varies due to numerous factors, including the host (age, diet, microbiome composition, and genetic background), types of lactobacilli strain(s), or types of disease, as a result, it is difficult to extrapolate health benefits of probiotics

23 between individuals, different types of diseases, or from one strain to another [180, 181].

4.1 Lactobacillus interaction with host cells

As members of the human microbiota or when used as probiotics, Lactobacillus species must survive, adhere, and successfully colonize the host environment. Lactobacilli cell surface-associated structures play a pivotal role in orchestrating initial interaction with the host [182]. Cell surface components, such as exopolysaccharides (EPS), sortase-dependent proteins (SDPs), lipoteichoic acid (LTA), and surface (S)-layer proteins are known to mediate adhesion of lactobacilli to host cells [183] (Figure 3). Furthermore, these surface structures can be modified in response to the surrounding environment conferring a strain- specific property to lactobacilli [184].

SDP Lipoproteins LTA

S-layer

EPS

Peptidoglycan LPXTG

Cell membrane

Figure 3. Major cell surface components of lactobacilli essential for interaction with the host. SDP, sortase-dependent protein; LTA, lipoteichoic acid; (S)-layer proteins. Adapted from [184]. The illustration is not drawn to scale.

24 The mucus layer is a protective layer that is situated between host epithelial cells and the lumen. Attachment to this layer is the first step to retain in place, mediate host-bacterial communication, and elicit specific cellular responses [185, 186]. Several cell surface proteins have been demonstrated to be involved in mediating adhesion of lactobacilli to the mucus. The mucus-binding proteins (Mubs) are the first characterized mucus binding adhesins that are also shared by other members of the lactic acid bacteria [186, 187]. The mucus adhesion-promoting protein (MapA) is another example of the surface adhesion factors that facilitate the binding of various lactobacilli strains, including L. reuteri and L. fermentum, to the mucus layer [188-190]. In Gram-positive bacteria, a class of cell surface-associated proteins, the so-called sortase-dependent proteins (SDPs) that contain the LPXTG (leucine- proline-random amino acid-threonine-glycine) motif, are covalently attached to the peptidoglycan by the class A sortase (SrtA) [191]. SrtA is a membrane- anchored housekeeping enzyme that is responsible for both cleaving the LPXTG motif on SDPs and coupling the newly formed C-terminus to the . Cleavage occurs between the threonine and glycine residue [192]. For example, L. gasseri ATCC33323 and L. acidophilus NCFM have been reported to possess six and eight SDPs, of which two and four of these SDPs were predicted as adhesion exoproteins and mub proteins, respectively. Sortase A deletion in both Lactobacillus strains caused lower adhesion to mucus and reduced persistence in vivo, showing the importance of SDPs in adhesion and colonization [193]. The S-layer is composed of self-assembled proteins or glycoproteins, which are non-covalently attached to the cell wall covering the entire bacterial surface by forming different lattice structures. Several species of Lactobacillus express S-layer proteins that are smaller in size compared to other Gram-positive bacteria. S-layer proteins are shown to play a role in key interaction with the host. In lactobacilli, they participate in their adhesion to host epithelial cells, mucins,

25 and extracellular matrix components (laminin, fibronectin and collagens) [194- 196]. Most bacteria express EPS on their cell surface to interact with their microenvironment. In lactobacilli, these carbohydrate polymers are present, either loosely bound to the cell surface or are released into the surroundings [197]. EPSs are very complex and can vary in structure composition, size, and function, causing strain-specific characteristics [198]. EPSs have been shown to be involved in the regulation of adhesion and biofilm formation in lactobacilli. For example, lower levels of EPS on L. johnsonni cell surface are reported to increase bacterial adhesion to chicken gut explants [199]. Furthermore, EPSs have been suggested to promote bacterial viability and aid persistence in the by providing protection against stresses like gastric acid and bile salts [200]. Other lactobacilli surface adhesion factors such as LTA and elongation factor-TU have been demonstrated to participate in the adhesion of lactobacilli to host cells and mucus [201, 202].

4.2 Modulation of host responses by lactobacilli

Although lactobacilli provide myriad beneficial effects for humans and are generally considered safe, their beneficial role depends on the type of lactobacilli strain and the dosage [203]. For example, the ability of L. rhamnosus GG to reduce IL-8 production in epithelial cells was reported to be lactobacilli concentration-dependent, where higher concentrations increased the cytokine release [204]. Lactobacilli, similar to other microorganisms, are recognized by our immune system and elicit innate and adaptive immune responses [205]. However, the induced response varies between Lactobacillus species and even strains [206]. In a strain-dependent manner, lactobacilli can act as either immune activators or immune suppressors [207]. This could be due to differences in their cell wall architecture and capability to secrete molecules or modify certain surface

26 components [184]. For instance, reduced incorporation of D-alanine in L. plantarum LTA evoked lower levels of pro- production [208]. The sensitivity of lactobacilli cell wall to intracellular digestion affects their ability to induce IL-12 secretion, in which lactobacilli that possess easily digestible cell wall barely cause IL-12 release. Moreover, peptidoglycan from certain Lactobacillus strains has been shown to inhibit IL-12 production [209]. In addition to the regulation of host immune responses, lactobacilli also play an essential role in enhancing epithelial barrier functions. They accomplish this through the improvement of tight junctions [210, 211], promotion of and antimicrobial peptide secretion [212-214], and inhibition of epithelial cells apoptosis [215, 216].

4.3 Protective role of lactobacilli against pathogens

Lactobacillus strains have developed multiple strategies to antagonize pathogenic bacteria and protect the human host. Some of the mechanisms used by lactobacilli to exert this barrier effect are illustrated in Figure 4 [217].

27

Enhancement of epithelial Competitive exclusion and Production of biosurfactant and barrier function Co-aggregation antimicrobial compounds

AMP secretion

Mucus layer

Maintenance of tight junctions

Lactobacillus species Helicobacter pylori Lactic acid H2O2 Bacteriocins AMP

Figure 4. Mechanisms utilized by lactobacilli to antagonize pathogens. Adapted from [217]. The illustration is not drawn to scale.

The initial colonization of many pathogens can be prevented by Lactobacillus strains by inhibiting their attachment to the surface of epithelial cells. Steric hindrance and competition for attachment sites are one of the mechanisms that lactobacilli use for blocking pathogens from binding to host cells [218, 219]. In Paper III, we have demonstrated that L. gasseri Kx110A1 was able to reduce the attachment of H. pylori to host gastric epithelial cells via competitive exclusion [220]. Most in vitro competitive exclusion assays are performed by allowing lactobacilli to adhere to host cells first, followed by the addition of pathogens. However, the probability of this occurring in vivo might be less, unless lactobacilli are the resident flora or are administered as probiotics for the treatment of perturbed microbial communities and establish themselves beforehand. Co-aggregation is another mechanism by which lactobacilli inhibit adhesion of pathogenic bacteria and promote their clearance by the mucus [221, 222]. For example, L. coryniformis or its cell culture supernatant aggregates with the pathogens Campylobacter coli, , and

28 [223]. Here, the aforementioned capabilities of lactobacilli to promote mucin production facilitates the expulsion of pathogens with the mucus [212]. Biosurfactants (amphiphilic molecules that alter surface tension and have emulsifying activities) that are produced by various lactobacilli strains are reported to have a strong anti-adhesive potential against a broad range of human pathogens. Moreover, lactobacilli-derived biosurfactants possess antimicrobial activity and are effective against bacterial [224-226]. Lactobacilli can affect the expressions of adhesion genes in pathogens and thereby inhibit their attachment. In Paper I, we have demonstrated the abilities of two Lactobacillus strains to reduce the adherence of H. pylori to host gastric epithelial cells by inhibiting the expression of its adhesin sabA [227]. Besides interfering with pathogen adherence, lactobacilli also exhibit antimicrobial activity against pathogens via the production of bactericidal compounds, including lactic acid, peroxide (H2O2), and bacteriocins [228]. Lactic acid causes membrane permeabilization in Gram-negative bacteria, which, in addition to its antimicrobial property, can promote the bactericidal effects of other compounds [229]. Lactobacilli are the dominant vaginal microbiota in most women and are believed to keep the habitat healthy by lowering the pH through production of lactic acid. Reduced pH in the vaginal environment has been suggested to reduce the susceptibility of women to Chlamydia trachomatis infection, a primary causative agent of sexually transmitted disease [230, 231]. H2O2 is another bactericidal molecule that is mainly produced by Lactobacillus species of the [232]. H2O2 and lactic acid have been shown to have a synergetic effect on killing pathogens [233, 234]. Various bacterial strains, including lactobacilli, produce a variety of small antimicrobial peptides known as bacteriocins. Bacteriocins have a limited inhibitory spectrum and are generally active against closely related species. They exert their antimicrobial activity through the induction of membrane pore formation [235, 236]. Bacteriocin production in many bacteria is regulated in a

29 cell density-dependent manner; for instance, L. acidophilus secretes its bacteriocin, lactacin B, when the bacterium is co-cultured with other bacterial strains [237]. Lactobacillus strains prevent host cell damage through inhibition of virulence factors and toxin expression in pathogens. The secretion of the two main exotoxins of Clostridium difficile (TcdA and TcdB) have been demonstrated to be repressed during the co-culture of the pathogen with lactobacilli [238]. Furthermore, the cytotoxic effect caused by these toxins was attenuated by L. delbrueckii [239]. Through production of lactic acid, lactobacilli also prevented Streptococcus pyogenes-induced cell cytotoxicity by degrading its LTA [240].

4.4 Lactobacillus and H. pylori

The antagonistic effects of Lactobacillus species towards H. pylori have been widely studied. However, the exact mechanisms involved in the lactobacilli inhibitory effect still need further investigation. Although lactobacilli are not the dominant microbiota in the stomach, they are shown to be found in the gastric biopsies of healthy as well as H. pylori-infected individuals [241, 242]. In H. pylori-infected individuals the pathogen becomes the major inhabitant of the stomach [243]. Lactobacilli can act on H. pylori either directly by inhibiting the pathogen viability, adhesion, and virulence factor expression or indirectly by affecting host cells and alleviating H. pylori-induced inflammation [244, 245]. Organic acids that are secreted by lactobacilli strains, including lactic acid, are shown to reduce the viability of H. pylori in vitro [246, 247]. In addition, the coincubation of H. pylori with lactobacilli inhibited its potent virulence factors, urease, and the majority of cag PAI genes [248-250]. Lactobacilli are further reported to ameliorate H. pylori-induced inflammation. For example, gastric inflammation

30 induced by H. pylori was attenuated by L. acidophilus through inactivation of Smad7 and NF-κB pathways [251]. In Paper II, we have shown the capability of L. gasseri Kx110A1 to decrease proinflammatory cytokine production in H. pylori-stimulated macrophages through inhibition of ADAM17. Several clinical studies have reported the beneficial effects of lactobacilli in improving H. pylori eradication rate and reducing drug side effects and suggested using probiotic Lactobacillus strains as an adjunct therapy [252, 253]. On the other hand, there are some reports claiming that supplementation of probiotics in H. pylori therapy did not really improve the eradication rate or decrease antibiotic-related side effects [254]. These conflicting observations might be due to variations in species/strains of lactobacilli studied, the type of treatment (antibiotic class), type of H. pylori strains, and the host genetic background. Supplementation of probiotic lactobacilli in H. pylori eradication therapy is thus still controversial and requires further optimization.

31 PRESENT INVESTIGATION

Aims

The overall aim of the work presented in this thesis was to gain a better understanding of the interactions between pathogenic H. pylori and commensal lactobacilli bacteria and their interactions with the host.

Specific aims:

• To investigate whether and how Lactobacillus species affect the initial adhesion of H. pylori (Paper I) and modulate H. pylori-induced inflammatory response in host cells (Paper II).

• To determine the role of sortase-dependent proteins in L. gasseri Kx110A1 adhesion and its antagonistic ability against the attachment of H. pylori to host cells (Paper III).

• To study the influence of lactate on H. pylori virulence gene expression and host cytokine production (Paper IV).

32 RESULTS AND DISCUSSION

Paper I

Adhesion to the gastric epithelium is a necessary prerequisite for H. pylori initial colonization, delivery of secreted factors to host epithelial cells and causing persistent infection. Additionally, the bacterium also gains access to various nutrients leaching from host cells that supports its growth and survival [255]. The aim of this study was to examine the effect of various Lactobacillus strains on the initial attachment of H. pylori to host gastric epithelial cells. Using urease activity as a measurement of H. pylori adhesion, we screened 28 Lactobacillus strains for their ability to reduce H. pylori attachment to host gastric epithelial cells. Of the 28 Lactobacillus strains, three strains inhibited the adhesion of the pathogen. We verified the inhibitory effect by the viable count method. Among the inhibitory lactobacilli strains, two of them were isolated from human gastric biopsies and the third one was isolated from human , demonstrating the antagonistic potential of lactobacilli to various pathogens they encounter regardless of their habitat location. By using a different H. pylori strain and another gastric epithelial cell line, we found that the lactobacilli-mediated inhibition was not dependent on a specific gastric cell line or H. pylori strain. Furthermore, the anti-adhesive property (prevention of initial colonization) of lactobacilli was confirmed in vivo, where mice inoculated with H. pylori in combination with lactobacilli had less H. pylori recovered from their stomach tissue compared to mice treated with the pathogen alone. To identify the possible inhibitory mechanism utilized by lactobacilli, we stained bacteria with fluorescent dye and examined their binding patterns on stimulated gastric epithelial cells, using microscopy. We found that lactobacilli and H. pylori bind to different sites on host cells excluding competition for space as a mechanism. In addition, the viability of H. pylori and the pH of the cell culture

33 medium were also not affected upon coincubation of H. pylori with lactobacilli, eliminating killing as a mechanism. To assess the importance of bacterial viability, we compared the inhibition capacities of dead (heat-killed or formaldehyde-fixed) and live lactobacilli. We observed a similar level of adhesion inhibition between dead and live lactobacilli, which suggests the involvement of a cell surface molecule. Interestingly, when the conditioned medium (CM) from lactobacilli was used together with H. pylori, the adhesion of the pathogen to host gastric epithelial cells was prevented to a similar extent as whole lactobacilli. This result suggests that the anti-adhesive molecule is released from lactobacilli into the environment. To determine the involvement of host cells in this inhibitory process, we fixed host epithelial cells with formaldehyde and quantified adhered H. pylori bacteria in the presence or absence of lactobacilli. Surprisingly, H. pylori adhered to fixed cells equally as well as untreated cells, and lactobacilli consistently blocked its attachment, indicating that the epithelial cells do not affect the pathogen adhesion. We further confirmed this result by stimulating epithelial cells with H. pylori that had already been incubated in lactobacilli CM. H. pylori pre- incubated in lactobacilli CM showed reduced adhesion to host cells, supporting the direct inhibitory effect of lactobacilli. Because lactobacilli directly inhibited H. pylori adhesion to host epithelial cells, we quantified the expressions of various H. pylori adhesins by qPCR after incubating the pathogen in lactobacilli CM. Most of the adhesin genes were differentially regulated by lactobacilli, however the inhibition of sabA expression matched the adhesion reduction result. We further verified this inhibition using a sabA deletion mutant, in which the inhibitory lactobacilli did not alter its binding capacity to host cells. We also measured protein levels of SabA by Western blot and obtained similar results.

34 In conclusion, these data demonstrate a novel mechanism by which lactobacilli directly prevent sabA expression and thereby reduce the attachment of the bacterium to host gastric epithelial cells.

35 Paper II

In Paper I, we have demonstrated the ability of L. gasseri Kx110A1 to inhibit H. pylori attachment to gastric epithelial cells through inhibition of the pathogen adhesin, SabA. Here (in Paper II), we examined the anti-inflammatory activity of four different Lactobacillus strains, including L. gasseri Kx110A. Using ELISA, we quantified the levels of H. pylori-induced proinflammatory cytokines, TNF, and IL-6 in human macrophages. Interestingly, out of the four lactobacilli strains, only L. gasseri Kx110A1 consistently decreased the production of these cytokines in macrophages. H. pylori-induced expression of TNF was not affected at the transcriptional level, while IL-6 was downregulated in the presence of L. gasseri Kx110A1. Both TNF and IL-6 reduction by L. gasseri Kx110A1 was confirmed by using monocyte-derived macrophages isolated from buffy coats. We investigated whether heat-killed lactobacilli can suppress the production of TNF and IL-6. We found that only live lactobacilli were able to reduce the secretion of these cytokines in H. pylori-stimulated macrophages, indicating that a heat-sensitive effector molecule is involved in the immunomodulatory activity of L. gasseri Kx110A1. Next, we determined the role of direct contact by using a 0.4 µm filter, which allows the diffusion of released molecules, but physically separates lactobacilli from H. pylori and host cells. This abolished the reduction of proinflammatory cytokines, suggesting that a secreted molecule is not involved and contact between lactobacilli and H. pylori or lactobacilli and host cells is necessary. Stimulation of host cells with lactobacilli for 2 hours prior to adding H. pylori prevented the release of H. pylori-induced TNF and IL-6. In addition, the inhibition of these cytokines was lactobacilli dose- dependent. Since contact with either H. pylori or host cells was needed for the anti- inflammatory effect of lactobacilli, we measured the levels of TNF and IL-6 after stimulating macrophages with LTA or LPS instead of H. pylori. Surprisingly, both

36 cytokine levels were decreased in LPS- and LTA-stimulated macrophages, showing the H. pylori independent anti-inflammatory role of L. gasseri Kx110A1. Prevention of cytokine production by lactobacilli can be mediated by affecting the viability of H. pylori or macrophages. However, the viability of H. pylori and macrophages was not affected upon coincubation with L. gasseri Kx110A1, ruling out killing as a mechanism of action. Macrophages can be polarized into M1 or M2 phenotype when they are incubated with different cytokines in vitro. By using flow cytometry, we determined different polarization markers expressed in macrophages upon stimulation of cells with either H. pylori or in combination with lactobacilli. The polarization markers tested were differentially regulated by the presence of each species, suggesting that the M1-M2 switch does not play a role. ADAM17 is the responsible enzyme that converts membrane-bound pro- TNF to a soluble form that is released into the environment [114]. Therefore, we analyzed the expression of ADAM17 using qPCR and ELISA. The expression of ADAM17, both at the transcriptional and protein level, was reduced only in cells incubated with H. pylori together with the inhibitory lactobacilli but not the non- inhibitory. We also observed a similar reduction pattern in monocyte-derived macrophages. To verify whether the observed suppression of ADAM17 by L. gasseri Kx110A1 is the cause of the cytokine inhibition, we blocked ADAM17 with a specific chemical inhibitor. We found that the blocking of ADAM17 decreased the level of TNF, confirming the essential role of the enzyme. L. gasseri Kx110A1 inhibited H. pylori-induced TNF to a similar level as the chemical inhibitor, and there was an additive effect when they were added together. In summary, we show the ability of lactobacilli strains to block the production of TNF and IL-6 by directly affecting host cells through inhibition of ADAM17 expression. Our results provide a novel immunomodulatory activity of lactobacilli.

37 Paper III

Lactobacilli interact with host cells through their cell surface components and thereby influence various cellular processes. In Paper I and Paper II, we explored the anti-adhesive function and the immunomodulatory activity of L. gasseri Kx110A1 against H. pylori, respectively. In this study, we investigated the involvement of SDPs by constructing a mutant that is deficient in sortase A (DsrtA). Sortase A is the enzyme that anchors SDPs to the bacterial cell wall. First, we examined whether the sortase A deficient L. gasseri Kx110A1 strain has a growth defect by comparing its growth rate to the wild-type and complemented lactobacilli strain (DsrtA/srtA). The mutant showed a slower growth rate and less auto-aggregative phenotype when grown overnight in MRS broth. We further determined the auto-aggregation of the mutant by sedimentation assay in PBS. Similar auto-aggregation was observed at pH 7.4; however, at pH 4.2, the DsrtA had less auto-aggregation compared to L. gasseri Kx110A1 wild- type and complemented strain, showing a pH-dependent aggregation and suggesting the involvement of sortase A in L. gasseri Kx110A1 aggregation. In comparison to pH 7.4, both the wild-type and the complemented strain exhibited improved aggregation at pH 4.2. SDPs mediate the adhesion of lactobacilli to host cells. Therefore, we assessed the adhesion abilities of wild-type L. gasseri Kx110A1 and its derivatives to host gastric epithelial cells. The adhesion of the srtA deletion mutant to gastric epithelial cells was reduced as compared to the wild-type and complemented strain. This suggests that SDPs are important in mediating adhesion of L. gasseri Kx110A1 to gastric epithelial cells. To investigate the ability of lactobacilli to co-aggregate with H. pylori, we performed a sedimentation assay at pH 7.4 and 4.2. As expected, at pH 7.4 there was no difference in co-aggregation, whereas, at pH 4.2, the co-aggregation of the srtA mutant with H. pylori was reduced compared with the wild-type and

38 complemented strain. Using microscopy, we verified the co-aggregation ability of L. gasseri Kx110A1 with H. pylori and we observed that the pathogen was trapped in aggregates of the wild-type and the complemented strain, but not the srtA mutant. Since co-aggregation is one of the mechanisms that lactobacilli uses to prevent pathogen adhesion to host cells, we quantified adhered bacteria using the viable count method after incubating the host cells with H. pylori in the presence or absence of lactobacilli. The attachment of H. pylori was inhibited by the wild- type and the complemented lactobacilli strain, when lactobacilli were either added simultaneously with H. pylori or preincubated with cells for 2 hours before adding H. pylori. However, there was no reduction in H. pylori adhesion when the pathogen was allowed to adhere first before adding lactobacilli. The srtA mutant only prevented the pathogen adherence to host gastric epithelial cells when they were added at the same time. Taken together, our results demonstrate the essential role of sortase A in lactobacilli-lactobacilli and lactobacilli-H. pylori interaction. Furthermore, it contributes to lactobacilli-mediated inhibition of H. pylori attachment to host cells.

39 Paper IV

Utilization of lactate in various pathogenic bacteria has been shown to enhance their pathogenicity [155]. However, the role of lactate in H. pylori pathogenicity is not well-known. Since adherence is a critical initial step for H. pylori virulence, we first analyzed the expression levels of various adhesins in the bacterium by incubating the bacterium in the cell culture medium with or without lactate. Most bacterial adhesins were differentially regulated in the presence of lactate, but both L- and D-lactate reduced the gene expressions of sabA and labA. Since SabA mediates the adhesion of H. pylori to sialylated glycoconjugates on host cells, we examined the adherence of the bacterium to host gastric epithelial cells by the viable count method after incubating H. pylori with cells in the presence or absence of L-lactate. The adhesion of the bacteria was not affected by L-lactate, indicating that other bacterial adhesins are involved in the attachment of H. pylori. The expressions of both sabA and labA are regulated by different mechanisms, and the pH-responsive bacterial two- component system, ArsRS, is one of them. Therefore, we measured the pH of the cell culture medium after 2 hours of incubation, but did not observe a difference when lactate was present. Furthermore, the expression of sabA and labA gene were compared between H. pylori wild-type and mutant deficient with the histidine kinase arsS (DarsS). Interestingly, the expression of sabA was only decreased in the wild-type H. pylori but not in the arsS deletion mutant in the presence of lactate, suggesting that the ArsRS two-component system is involved in the regulation of SabA. We hypothesize that lactate might be the signaling molecule that is detected by this system, but further experiments are required to support this hypothesis. In contrast, labA was downregulated in both wild type and DarsS mutant, indicating that the expression of the adhesin might not be regulated through the ArsRS two-component system and lactate might affect labA through a different mechanism.

40 We investigated the influence of lactate on macrophages ability of proinflammatory cytokine production as metabolism affects immune cell function. L-lactate reduced the levels of TNF and IL-6 in macrophages stimulated with H. pylori. Interestingly, D-glucose increased cytokine production in these cells in response to H. pylori. In summary, both L- and D-lactate inhibited the gene expression of sabA and labA in H. pylori. This inhibition, however, did not affect the attachment of H. pylori to host gastric epithelial cells. The ArsRS bacterial two-component system is involved in the regulation of sabA expression, but pH was not the signal. It is tempting to speculate that lactate might be the signal molecule detected by the ArsRS thereby affecting sabA expression. In addition, L-lactate decreased the production of TNF and IL-6 in H. pylori-stimulated macrophages.

41 FUTURE PERSPECTIVES

In this work, we have gained much knowledge by answering several questions, but new questions were also raised that still need further investigation.

In Paper I, we have demonstrated the ability of certain Lactobacillus strains to prevent the attachment of H. pylori to host gastric epithelial cells. This lactobacilli-mediated inhibition of adhesion was through reduction of the expression of H. pylori adhesin-encoding gene sabA. sabA expression in H. pylori is regulated through different mechanisms, including phase variation and the ArsRS two-component system [54]. We examined the involvement of the ArsRS system in lactobacilli-mediated SabA reduction by comparing the expression of the adhesin in the wild-type and arsS deficient mutant. The expression of sabA was equally downregulated in both wild- type H. pylori and the arsS mutant upon coincubation with only inhibitory lactobacilli but not the non-inhibitory. This suggests that the reduction of sabA by lactobacilli strains was not by affecting the ArsRS system (unpublished data). Because live lactobacilli prevented the binding of H. pylori to a similar level as dead lactobacilli, we hypothesized that the effector molecule is likely a cell surface component that is shed into the environment. However, peptidoglycan and S-layer isolated from inhibitory lactobacilli could not prevent the binding of H. pylori to host epithelial cells (unpublished data). Results also show that the anti-adhesive component of lactobacilli is larger than 50 kDa. The effect of lipoteichoic acid, an important cell surface component of lactobacilli, is currently being investigated. By treating lactobacilli CM with different enzymes, such as proteinase K, DNase, RNase, and sodium periodate, we are trying to further characterize the lactobacilli effector molecule.

42 In Paper II, we showed the anti-inflammatory activity of L. gasseri Kx1101A1 in H. pylori-stimulated macrophages via suppression of ADAM17. It would be interesting to study how ADAM17 is regulated and the involvement of various signaling pathways in the regulation of TNF and IL-6. Because the inhibition of ADAM17 by lactobacilli seems a general effect (not H. pylori dependent) and ADAM17 can process multiple substrates, it is important to study whether other known ADAM17 substrates will also be affected by lactobacilli. In addition, in vivo experiments using animal models for assessing inflammation, such as by measuring proinflammatory cytokines or infiltrating cells, would increase the relevance of our findings. Moreover, identifying and characterizing the lactobacilli effector molecule in the future will increase the potential of developing additional new treatment options for inflammatory disorders. In Paper IV, we explored the role of lactate in H. pylori virulence gene expression. We observed a decrease in sabA and labA mRNA level in the presence of lactate. It is relevant to confirm the reduction of SabA at the protein level, for example using Western Blot. The expression of other virulence factors necessary for the pathogenicity of H. pylori, including CagA, urease (ureA), flagella (flaA), and TNF-inducing protein (Tip-α) would also be interesting to test in the presence of lactate. The attachment of H. pylori to gastric mucin could be explored by using mucin isolates or tissue samples since LabA mediates binding of the bacterium to MUC5AC, and the expression is reduced in the presence of lactate. In addition, how lactate assists H. pylori to evade host immune evasion, such as phagocytosis, is very interesting to study because lactate has been shown to enhance immune escape by pathogenic bacteria.

This thesis provides novel findings on the role of commensal lactobacilli in the modulation of host cells responses and the prevention of pathogens. We specifically have shown the anti-adhesive and anti-inflammatory effect of L. gasseri Kx1101A1 against H. pylori. The identification and characterization of

43 the effector molecule from this strain will increase its usage as probiotics or alternative treatments in H. pylori infection.

44 ACKNOWLEDGEMENTS

This work would not have been accomplished without the help, support and encouragement of my amazing family, friends and people I have met along the way.

I would first like to thank my supervisor, Ann-Beth Jonsson, for giving me the opportunity to do a PhD in your group. I am grateful for your scientific guidance, feedback, and discussions. You always have trust and confidence in me, which encouraged me to work hard and become a better researcher. Thank you for being understanding and helpful, especially during my pregnancy.

To my co-supervisors, Sushil Pathak, Hong Sjölinder and Eva Sverremark Ekström, I am so grateful for your encouragement, immense scientific advice, and for always being available for questions and discussions.

In my five-years PhD journey I have had the opportunity to work with some amazing people. Nele, thank you for teaching me different lab skills when I was your Masters student and for making me interested in H. pylori, for which I have discovered a passion to explore and better understand. You are the most organized person I know and I have learned so much from you. Sunil, you are such a happy, kind, helpful, and knowledgeable person who gives your time to help others. Thank you for teaching me so many valuable lessons both in science and life. Sara, although it was for a very short time, I was under your supervision when I joined the group. Your hard work is really inspiring and I am glad that we continue to be friends. Thank you for all the great discussions about science, movies, sports

45 and food. Gaby, Yezzzz! I admired your enthusiasm for running and thank you for trying so hard to make me love outdoor running which I never thought I would like. You were always interested about Ethiopia, Enjera, and Amharic, and it was fun teaching you Amharic and sharing recipes for cooking “shiro wot.” I am so proud that you made your “van life” dream a reality. I am so thankful that I know you. Raquel, thank you for sharing the difficult “H. pylori” journey, dealing with nitrogen gas, and working late hours and weekends. You have such a kind and caring heart and I am grateful for your friendship. Pilar, for sharing your knowledge about ELISA and THP-1 cells. Thank you for the delicious cakes, good company and great talk on our way home, movie nights, after work and potlucks. Jakob and Xiao, I really appreciate all our interesting discussions. Fanglei, for being helpful and great scientific discussion. Tanvi, I was not really lucky with students, but you have changed this with your hard work and dedication, especially in our ADAM17 paper. I am happy to see what you have accomplished. Kenny, for good company during lunch, your advice on shopping baby products, baby nest was such a great tip! Jonas, although we shared the office for just a few months, I am thankful for your company and friendly conversation.

I am grateful to all former and current members of the F5 corridor, Yeneneh, I missed speaking Amharic with you. You are such a kind and polite person who always sees the positive in everything! Manu, thank you for great conversation and good company especially in my last year of my PhD. Mukti, for helping me with FACS. Ginte, Alex, and Sandra, teaching with you was fun and I am grateful for the great conversations during lunch time. Einar, thank you for informing me about ADAM17, which is my paper II now. Mattias, for your amazing cakes and great conversation.

46 I would also like to thank the technical and administration staff of MBW, especially Bea for always being very helpful and happy-spirited and for the pleasant conversations about life. Gelana, it was really nice to have someone to talk Amharic with, thank you for being kind and helpful.

በመጀመሪያ አለማዊ ሕይወታችን ብቻ ሳይሆን ሰማያዊውም ሕይወታችን የተስተካከለ እና ያማረ እንዲሆን ቀን ከለሊት በፀሎታቸው ለሚተጉልን አባታችን አባ-ገብረጊዮርጊስ እድሜ እና ጤና ይስጥልን። ለኪዳነምህረት አባላት በሙሉ ላሳለፍናቸዉ መልካም ጊዜያቶች ምስጋናዬ ከልብ የመነጨ ነው። ሙሴ፡ ሁልጊዜም ለምትመክረኝ ምክሮችህ ፣ላካፈልከኝ የህይወት ተሞክሮዎች፣ቁምነገሮች እና ቀልዶች አመሰግናለሁ። ሔሊ፡ ስላሳለፍነዉ ጥሩ ጊዜያቶች፣ኤሉን እናት ከመሆኔ በፊት አምነሽ ስለሰጠሸኝ፣ ሁሌም በትምህርቴ እንድንበረታ አይዞሽ ስላልሽኝ ከልብ አመሰግናለሁ። ያሬድ፡ አንዳንዴ መንታ የሆንን ያህል እስከሚመስለኝ ድረስ የምናስበው ነገሮች መመሳሰል ሁሌም ይገርመኛል። ላወራናቸዉ ቁም ነገሮችና ቀልዶች፣ ላሳለፍነው መልካም ጊዜያቶች ለሁሉም ነገሮች አመሰግናለሁ። አጁ፡ እንደምናስበው ብቻችንን በሰፊዉ ጌዜ ባናገኝም ሁልጊዜም አንቺ ካለሽ ሳቅ፣ ጨዋታ፣ደስታ አለ። ላለፉት ጥሩ ጊዜያችን፣ ለቋጠርሽልኝ ምሳቃዎች፣ ናኑን በፈለግነው ጊዜ ወስደሽ ስለተንከባከብሻት ከልብ አመሰግናለሁ። እዮባ፡ ለነገሮች ሁልጊዜም በጎ አስተሳሰብ ያለዉ፣ የሰውን ደስታ እና መልካም ነገረ ማየት የሚወድ ቅን እና ጠንካራ ወንድም፣ አንተን በማግኘቴ በጣም እድለኛ ነኝ። ገኒ፣ ደግነትሽ፣ያለሽን ሁሉ ማካፈል መዉደድሽ እኔንም ጨምሮ ለብዙ ሰዎች አረአያ ምትሆኚ ነሽ። እነዛ በመሀል ጣል የምታደርጊያቸዉ ቀልዶች ሳስባቸው አሁንም ያስቁኛል። ሳሚ፣ ህይወትን እየሳቁ፣ እየቀለዱ ከባድ ነገሮችን እንኳን ሳይጨነቁ ማሳለፍ እንደሚቻል ስላስተማርከኝ አመሰግናለሁ። ሒዊ፣ ይሔን ሁሉ አመት አልወደውም እያልኩ ያመለጠኝን ክትፎ በአንቺ ምክንያት መብላት ስለጀመርኩኝ አመሰግንሻለሁ። በቢኒ ምክንያት ተዋዉቀን መልካም ወዳጅ ለሆናችሁኝ እና ሁልጊዜም አይዞሽ በርቺ ላላችሁኝ፡ ሀሰን፣ ሀዪ፣ ሩት፣ ዳዊት፣ አሮን፣ ፓፒ፣ ኤርሚ፣ ቴዲ፣ ሳሚ፣ ተስፍሽ፣ መኩ፣ ዮኒ፣ ናስር፣ ሂዊ፣ አደም፣ እዮብ፣ ኤልያስ፣ ጆኒ፣ ማርቲ፣ እና ምሕረት ከልብ ላመሰግናችሁ እወዳለሁ።

For my awesome friends back home, Teche, Beki, Mes, Ase, Mati and Geme thank you for the fun times and amazing memories we shared.

To my family in Ethiopia. ከምንም በላይ ጠንካራ እና አፍቃሪ ቤተሰብ ስለሰጠኝ ፈጣሪዬን እጅግ አድርጌ አመሰግነዋለው። አባዬ፣ በሃይማኖቴ እንድበረታ እና ፈሪሃ እግዚአብሔር እንዲያድርብኝ ስላስተማርከኝ እና ስለመከርከኝ ሁልጊዜም አመሰግናለሁ። እቴቴ፣ ከልጅነቴ ጀምሮ በትምህርቴ ጠንካራ እንድሆን ስለረዳሽኝ፣ ጠንክሮ መስራት ለፍሬ እንደሚያበቃ በተግባር ስላስተማርሽኝ፣ ሁልጊዜም ስለምትረጂኝ እና እንደ ጔደኛ ቀርበሽ ስለምትመክሪኝ እድሜዬን ሙሉ አመሰግንሻለሁ። ዳኒዬ፣ የሁልጊዜም ምኞትሕ እኛ ቁምነገር ላይ ደርሰን ማየት ነበር፡ በህይወት ኖረህልኝ ይሔን ብታይ ምንኛ በታደልኩ ዳኒዬ እግዚአብሔር በገነት ያኑርህ ሁሌም እወድሃለው። To my beautiful siblings Tsega, Tsion and Biniyam, thank you for always calling and checking up on me, for encouraging and supporting me both in life and in my studies. እወዳችዋለው! To my other family: አቡ፣ ቶሜ እና እሙ ሁልጊዜም አይዞሽ በርቺ ስላላችሁኝ አመሰግናለሁ፣ እወዳችዋለው!

47

And most of all, I want to thank my amazing husband Biniyam, for your love, support, patience and encouragement in every step of the way. Thank you for always being on my side in times of happiness, failure and sadness. I am the luckiest person on earth to share life with you and to be blessed with our little addition to the family, princess Mikaela. ፍቅር አረጋችዋለው!

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