Proteomic approaches to study CD4+ T cells and meningococci: New insight into life science and infection biology

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Proteomic approaches to study CD4+ T cells and meningococci:

New insight into life science and infection biology

Thesis for the degree of Philosophiae Doctor (PhD) Tahira Riaz

Department of Microbiology Institute of Clinical Medicine

University of Oslo

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© Tahira Riaz, 2019

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-448-1

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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“In every atom slumbers the might of the self.”

- Allama Iqbal

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Acknowledgments

The work presented in this Thesis was conducted at the Institute of Clinical Medicine at the University of Oslo and Oslo University Hospital-Rikshospitalet.

The work on Papers I and II was performed: at the Centre for Immune Regulation (CIR) and Department of Immunology (IMM), and on Papers III and IV: at the Department of Microbiology (MIK). I am grateful for the excellent working facilities provided at CIR/IMM and at MIK. This work has in part been facilitated through a Ph.D. scholarship from South-Eastern Norway Regional Health Authority and Research Council of Norway project 220901 for which I am most grateful.

First, my sincere gratitude goes to my main supervisor Prof. Tone Tønjum, group leader of Genome Dynamics (GD) group at MIK, and co-supervisor Dr. Ingrid Olsen. Tone, your inspiring scientific insight, curiosity and motivation is highly appreciated. Thanks for introducing me to exciting opportunities in academia and for top-notch research networking. Ingrid thanks for being an unofficial

supervisor during my time at CIR/IMM and continuing as co-supervisor together with Tone during Thesis writing and submission. Your support and guidance throughout this Thesis have been invaluable.

I would also like to acknowledge Dr. Gustavo de Souza and Prof. Ludvig M.

Sollid for the supervision during the research work conducted at CIR/IMM. My continuing thanks to all the contributing authors and to the GD family for

providing a highly enjoyable scientific environment.

Very heartfelt gratitude goes to my colleagues and friends: Maria, Astrid and Siri at CIR/IMM and to Håvard and Mari in GD group. For lunch breaks and

scientific discussions, but most of all for their endless support and friendship through moments of joy as well as in times of desperation.

Last but certainly not least, thanks to my caring parents, Nasreen and Riaz, for teaching us the value of education and for always being there. Also thanks to the rest of the family and friends who have believed in me, for their love and for cheering me on along the way. A special thanks to the two most amazing and joyful girls I know: Mariam & Amelia – you are the best!

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Abbreviations

3R DNA repair, recombination and replication processes AMR antimicrobial resistance

APC antigen presenting cell

CD Cluster of Differentiation (T cell membrane glycoproteins) CTL cytotoxic T lymphocyte

DnaX DNA polymerase III subunit gamma/tau DprA DNA processing chain A

ESI electrospray ionization

FASP filter-assisted sample preparation

GZM granzyme

HGT horizontal gene transfer

HolA DNA polymerase III, delta subunit IBD inflammatory bowel disease IFNγ interferon gamma

Ig immunoglobulin IL interleukin

IRF4 interferon regulatory factor 4 LFQ label-free quantitation

MS mass spectrometry

MS/MS tandem mass spectrometry Ng Neisseria gonorrhoeae NK natural killer

NKG2D natural killer group 2 member D Nl Neisseria lactamica

nLC nano liquid chromatograph Nm Neisseria meningitidis

PBMC peripheral blood mononuclear cell

Pil pilus biogenesis protein (i.e PilG and PilE)

PRF perforin

PTM posttranslational modification RecG ATP-dependent DNA helicase RecG SSB single strand binding protein

TCR T cell receptor TF transcription factor Tfp type IV pili

Th T helper

TopA DNA topoisomerase 1 Treg regulatory T cell UvrD DNA helicase UvrD

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Abstract

The human mucosa in both the gut and the oropharynx is constantly under exposure to commensals and opportunists and are a key site of immune responses. Homoeostasis between mucosal components play a critical role in avoiding immune mediated diseases and fighting infections. This Thesis aims to delineate the comprehensive proteome analysis of two selected mucosal entities representing the host and a potential pathogen by use of high-throughput mass spectrometry analysis.

In the first part of the Thesis (Papers I and II), the cellular composition of CD4+ T helper (Th) cells from the inflamed gut of Crohn’s disease patients were addressed. Paper I explored the phenotypic characteristics and molecular functions of Th cells by comparison of gut-derived Th cell phenotypes. Paper II addressed the relationship between the

transcriptome and proteome upon activation of gut-derived cytotoxic Th cells. In the second part of the Thesis (Papers III and IV), DNA

metabolic components from an oropharyngeal mucosal opportunist, the bacterium Neisseria meningitidis (Nm), were addressed. The aim was to understand which Nm proteins contribute to transformation of DNA (DprA, Paper III) and DNA repair/recombination (helicase RecG, Paper IV) by comparative analysis of Nm wildtype and deletion mutants.

Thus, by investigating relatively homogenous cellular assemblies, the unique properties of the host cell clone and bacterial cultures studied were characterized. In Paper I, the homogenous primary Th cells analysis led to crucial findings on a novel phenotype of cytotoxic CD4+ T cells. In Paper II, the changes observed by 'omics' analysis upon activation of cytotoxic Th cell provided a deeper description of their complexity and molecular mechanisms. In Papers III and IV, Nm proved to be a relevant genetically amenable model for investigating DNA-related proteins and a good source for protein-protein interaction studies. Collectively, these results provided new insight into life science and infection biology, focused on the gut and oral mucosal surfaces.

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List of Papers

Paper I

Quantitative Proteomics of Gut-Derived Th1 and Th1/Th17 Clones Reveal the Presence of CD28+ NKG2D- Th1 Cytotoxic CD4+ T cells

Riaz T, Sollid LM, Olsen I and de Souza GA

Molecular & Cellular Proteomics, 2016 Mar;15(3):1007-16

Paper II

Quantitative transcriptome and proteome analysis of an activated gut- derived cytotoxic Th1 cell clone

Riaz T and Olsen I

Manuscript in review in BMC Clinical Proteomics

Paper III

Comparative proteomic analysis of Neisseria meningitidis wildtype and dprA null mutant strains links DNA processing to pilus biogenesis Beyene GT, Kalayou S, Riaz T and Tønjum T

BMC Microbiology, 2017 Apr 21;17(1):96

Paper IV

Characterization of the Neisseria meningitidis Helicase RecG

Beyene GT, Balasingham SV, Frye SA, Namouchi A, Homberset H, Kalayou S, Riaz T and Tønjum T

PLoS One, 2016 Oct 13;11(10):e0164588

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Introduction

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1. Introduction

Mucosal immunology is a fascinating key part of the human immune system and essential in the protection against invading pathogens. Both immune cells and normally harmless bacteria residing on mucosal surfaces are crucial in maintaining the homeostasis necessary to fight infections, and at the same time avoid immunity-mediated diseases. This makes studying the proteome, the ultimate functional entities of immune cells and bacteria derived from the mucosa an important and exciting topic.

This bipartite Thesis arose based on two lines of proteomics experiments in mucosal biology. The proteomic signatures of host immune cells derived from the intestinal mucosa and a bacterium inhabiting the

oropharyngeal mucosa were investigated in this Thesis. The main focus here was on proteome characterization of gut-derived CD4+ Th cell clones from patients with Crohn’s disease, which is an inflammatory disorder causing chronic intestinal inflammation. Secondly, as a representative of the mucosal microbial community, the bacterium Neisseria meningitidis, an opportunistic pathogen that can asymptomatically inhabit the mucosal surface of the oropharynx, was also studied. Neisserial DNA

transformation, repair and recombination pathways were the main subject of detailed proteome analysis.

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Introduction — The immune system

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1.1 The immune system

The immune system is a fascinating and intricate system evolved to maintain homeostasis in the human body by distinguishing between self and non-self (foreign). When encountering a pathogen or other invading and harmful substances an immune response is triggered, leading to a sequence of reactions with the aim to eradicate it. The immune system is divided into the innate and the adaptive immune system which work in synergy to protect the host [1].

1.1.1 Innate immune system

The innate immune system is the first line of defense and has a quick response. Epithelial barriers, the complement system, natural killer (NK) cells and antigen presenting cells (APCs) like dendritic cells (DCs) and macrophages are essential elements of the innate immunity. Through activation by professional APCs, the innate immune responses can initiate the cascade of the adaptive immune system [2].

1.1.2 Adaptive immune system

The adaptive immune system is highly specific and improves for every successive encounter with the same pathogen. For all adaptive immune responses, lymphocytes are essential and they can be divided into two categories: B- and T-lymphocytes.

Upon encounter with an antigen, the B cells proliferate into memory B cells and antibody secreting plasma cells. B cells secrete antibodies called immunoglobulins (IgA, IgD, IgE, IgG, and IgM) [3].

T-lymphocytes on the other hand are divided into two major subgroups, CD8+ T cells and CD4+ T cells. T cells recognize antigens through major histocompatibility complexes (MHC) expressed on the surface of

professional APCs. The human leukocyte antigens (HLAs) encode MHC proteins in humans. MHC class I molecules are expressed on all nucleated

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cells in the body, and these present antigens to the CD8+ T cells. MHC class II molecules are expressed on the surface of professional APCs, which mediate antigen presentation to naive CD4+ T cells [4]. CD4+ T cells are differentiated into T helper (Th) cells that have the task to support other cells (e.g. CD8+ T cells) of the immune system during an immune response. On the other hand, CD8+ T cells recognize and destroy the infected cells directly/have cytotoxic activity [5].

1.1.2.1 CD8+ T cells

As a result of their cytotoxic action against intracellular pathogens, CD8+

T cells are often referred to as cytotoxic T lymphocytes (CTLs).

Moreover, they are important agents of tumor surveillance. CD8+ T cells have two different approaches to eradicate foreign cells: either through the release of cytotoxic granules, or through the Fas Ligand (FasL)

interaction, which is expressed on the CD8+ T cell surface [6]. In the latter mode, FasL binds to the Fas receptor (FasR) on the target cell, which then initiates the caspase cascade which subsequently leads to apoptosis of the target cell. In the first mentioned mechanism of CD8+ T cells, cytotoxic granules are released packed with perforin and granzymes. Perforin

facilitates entry of the granzymes into the target cell through formation of pores in the membrane. As serine proteases, granzymes activate the

endogenous apoptosis pathway to induce cell death. Granules packed with these proteins are also found in NK cells [7, 8].

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Figure 1. A simplified overview of CD4+ T cell differentiation. Naive CD4+ T cells can be differentiated into Th1, Th2, Th17 and Treg cells. The differentiation is initiated by antigen/TCR binding between the APC and naive CD4+ T cells. Various cytokines in the nearby environment influence the differentiation together with lineage specifying transcription factors. A set of effector cytokines are produced by the CD4+ T cell subsets to execute their role during an immune response.

1.1.2.2 CD4+ T cells

CD4+ T cells are a diverse group that differentiates into subgroups of Th cells. These are important players of the immune system and are classified according to the cytokines they release and their expression of

transcription factors. Signals from the antigen and T cell receptor (TCR) binding, together with various cytokines (such as interleukins (IL)) in the nearby milieu and transcription factors, interplay with each other to

regulate the polarization and differentiation into specific types of Th cells.

Furthermore, Th cells can as well cross regulate each other through their cytokine expression and suppress polarization of the other Th cell types [9]. In general, Th cells are grouped into Th1, Th2, Th17 and Treg cells (figure 1).

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Unique functions are attributed to the distinct Th phenotypes. Th1 cells play a role in protecting against intracellular bacterial and viral infections.

These cells are characterized by the production of IFNγ [10] and expression of the key transcription factor T-bet [11]. Th2 cells fight

against extracellular pathogens and are defined by IL4 production [10] and the transcription factor GATA-3 [12]. IL17 [13, 14] and RORγT

expression [15] characterize Th17 cells, while IL10 and FOXP3 expression define Treg cells [16, 17].

1.1.2.2.1 Transcription factors as master regulators of CD4+ T cell differentiation

The recognition of the complexity of CD4+ T cell differentiation into the subgroups of CD4+ T cells has increased vastly over the past years. For a long time, specific transcription factors (TFs) were considered as “master regulators” for CD4+ T cell differentiation [18]. The term “master

regulators” refers to TFs that are not only necessary, but also are sufficient on their own for deciding the function and phenotype of a certain type of Th cell. In addition, they were thought to be specific for the unique Th cell type.

Historically, it was believed that Th cells were committed to their lineage and their end fate was absolute. In some cases, this model proved to be valid, while in other instances cellular plasticity is observed. Recent

emerging data, showing evidence that Th cells acquire plasticity according to the function needed for the specific Th cell type, contradicts the

paradigm of “master regulators” [19-22]. Various cytokines and TFs have been proposed to impact on the reprogramming from one Th cell to

another Th cell [21, 23, 24].

Furthermore, the recognition of co-expression and interplay of TFs, which decide the role of Th cells, provides additional complexity to these cells.

Th cell types identified as “mixed” phenotypes also contribute greatly to this complexity [18]. Examples of mixed phenotypes such as Th1/Th2, Th1/Th17 have previously been described [25, 26].

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16 1.1.2.2.2 Role of Th cells in etiology

Th cells do not always function as they should in the immune system. In the cases where they have an inappropriate response, they can trigger an immune mediated disease [27-34]. Immune mediated disorders can arise from any dysregulation of the immune system [35] and are an important cause of severe and/or chronic illness. Various allergies and inflammatory conditions, as well as autoimmune diseases, are represented in the term immune mediated disorders. There are several such diseases where an irregular Th cell response plays a central role, e.g. in inflammatory bowel diseases (IBDs): ulcerative colitis (UC) and Crohn’s disease [36-38], and in autoimmune diseases: asthma [39, 40] and arthritis [41], to mention a few.

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Introduction — Mucosal immunity

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1.2 Mucosal immunity

Mucosal immunity is the body’s immune response involving mucosal surfaces. The mucosal surface has complex and diverse tasks in the body.

Mucosa associated lymphoid tissue (MALT) is the inductive site for immune responses against pathogens and other harmful molecules (e.g.

food antigens) encountered along the mucosal surfaces. Being constantly under exposure to its microbiota (commensals, symbiotic and pathogenic microorganisms), the mucosal surface plays a key role in balancing the immune homeostasis and orchestrates an intricate network of innate and adaptive responses [42, 43]. The composition of the microbiome, such as the abundant gut and oral microbiomes, also holds the key to health and disease [44-47], as well as being important as a reservoir of drug

resistance genes and transfer of antimicrobial resistance (AMR) [48, 49].

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Figure 2. Gut associated lymphoid tissue (GALT). Through specialized epithelial cells, referred to as M (microfold) cells, antigens and pathogens are taken up from the lumen and transported to organized lymphoid follicles called Peyer’s patches in the GALT. The effector site of the immune response takes place in the lamina propria. Antigens taken up from the mucosal surface activate APCs that subsequently present them to naive T and B lymphocytes. Memory and effector lymphocytes undergo terminal differentiation in the effector sites, e.g. B cells differentiate into plasma cells that secrete IgA. Reproduced from reference [50]. Copyright (2013) licensed under CC BY 4.0.

The gut, which is not only a digestive system, also counts as the largest immune organ in the body, referred to as the gut associated lymphoid tissue (GALT) (figure 2). GALT is a key part of the MALT. A thick layer of mucus covers the gastrointestinal tract, which can be maintained,

altered or degraded by a range of components, such as microbes and

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cytokines [51-54]. The gut is a rich habitat for a huge range of bacteria that can stick to the mucus layer through adhesins, and thereby preventing the bacteria to reach the epithelium and damage it [55]. Although the mucosal barrier itself is part of the innate immunity, the adaptive immunity with antibody secretion plays a significant role in protection against mucosal infections. IgA is recognized as the most prominent

antibody in the mucosal immunity and plays a major role in initial defense against pathogen colonization. Certain secretory IgA molecules can also enhance bacterial adherence to the mucosa and provide pathogen

clearance aided by intestinal peristalsis movement [56].

Another important aspect of immune response is the oral and nasal-

associated lymphoid tissue [42, 57, 58]. These regions are readily exposed to airborne challenges including pathogenic (and non-pathogenic/

commensal) bacteria. The mucus layer has an essential role in defending against potentially harmful exposure [59]. Bacterial motility (e.g.

twitching motility mediated by the retracting neisserial pilus) is an

essential virulence factor that pathogens use to acquire intimate adherence to the mucosal surface before they penetrate it and can traverse the

epithelium [60]. Commensal bacteria can act as potential pathogens as a result of dysbiosis in the microbial environment, altered bacterial gene expression or imbalance in the host health/disease state [61, 62].

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Introduction — Crohn’s disease

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1.3 Crohn’s disease

Crohn’s disease is a chronic inflammatory bowel disease that can affect any part of the gastrointestinal tract; however, the end part of the small intestine is often affected. For Crohn’s disease patients, the most common symptom is abdominal pain, while diarrhea and malabsorption are also commonly found [63, 64].

The highest incidence and prevalence found for Crohn’s disease is in the industrialized regions (Australia, Europe and North America). The third highest reported value of prevalence is found in Norway with 262 per 100 000 affected (Germany: 322 per 100 000; Canada: 318 per 100 000).

Notably, Crohn’s disease is emerging in the newly industrialized and westernized places in Africa, Asia and South America and has become a global disease at the turn of the 21^st century [65, 66].

Although the etiology of the disease is unknown and is likely to be multifactorial, excessive immune response to commensal bacteria has been reported to be a driver of the disease [64]. In particular, some reports support the potential link between Crohn’s disease andMycobacterium avium subspecies paratuberculosis (MAP) [67-70], which is the cause of Johne’s disease in sheep [71]. However, clear etiological evidence of MAP as the causative agent of inflammatory bowel disease in humans remains to be demonstrated [72-77]. Increased levels of Th responses with IFNγ and/or IL17 expression are also found in the inflamed intestine [78- 80]. Other risk factors such as smoking and low fiber/high carbohydrate diet are also linked to Crohn’s disease. Moreover, gene variants of NOD2 [81], IL23R [82, 83] and ATG16L1 [84, 85] have been shown to be

associated with susceptibility to Crohn’s disease.

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Introduction — Neisseria meningitidis

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1.4 The bacterium Neisseria meningitidis is a mucosal opportunist

Neisseria meningitidis (Nm), also called the meningococcus, is a Gram- negative bacterium that belongs to the genus Neisseria in the phylum Proteobacteria [86]. Nm cells are diplococci, as for most of the members in the genus Neisseria, however, some neisserial species are rod-shaped [87, 88].

Nm is an opportunistic pathogen that asymptomatically can inhabit the normal flora of the oropharynx and can reach the nasopharynx upon infection [89-92]. Such carriage can induce the production of protective bactericidal antibodies [93, 94]. Colonization of the closely related

commensal Neisseria lactamica (Nl) and generation of antibodies against Nl has also been shown to protect against Nm infections by generating cross-reactive protective antibodies [95-97]. Additionally, the complement system has been shown to be pivotal in protecting individuals against Nm infection [98]. Deficiency in properdin, a positive regulator of

complement activity, has been related to increased host susceptibility to fulminant meningococcal infection [99-101]. Moreover, protective immunity with increased CD4+ Th response has been detected in hosts infected with both Nm (Th1) [102-104] and the closely related obligate pathogen Neisseria gonorrhoeae (Ng) (Th17) [105-108].

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Figure 3. Neisseria meningitidis (Nm) pathogenesis. Nm is an oropharyngeal mucosal opportunist that can proliferate and cross the epithelium to invade the bloodstream where it can survive and finally reach the meninges. This can subsequently cause severe disease, e.g. sepsis and meningitis. Reproduced from reference [109] with permission from Guillaume Duménil, Institute Pasteur. Copyright (2015) by Institute Pasteur.

Invasion of Nm into the host is further promoted by a range of main virulence factors such as the pili, the polysaccharide capsule, lipo- oligosaccharide (LOS), adhesion molecules including the Type IV pili (Tfp), and IgA protease [90, 92, 110, 111]. Based on its capsule structures, Nm is divided into 13 serogroups, where six serogroups (A, B, C, W135, X and Y) are the major cause of life-threatening meningococcal disease worldwide [88]. Occasionally, in the lack of bactericidal antibodies, Nm can invade the bloodstream and meninges to cause sepsis and meningitis [88, 112] (figure 3). The highest incidence of meningococcal disease is found in the African meningitis belt and is caused by Nm serogroup A strains [113, 114]. In Europe, serogroup B is the major cause of

meningococcal disease [114]. Mucosal damage, e.g. by co-infection, low humidity, dust, smoking and overcrowded places are some of the risk factors linked to meningococcal disease.

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23 1.4.1 Neisserial genome dynamics

Neisserial genome dynamics are fueled both by intragenomic variation and by the introduction of DNA by horizontal gene transfer (HGT). Nm hosts a highly recombinogenic bacterial genome which also undergoes mutations [115]. This genome plasticity promotes Neisseria diversity, adaptation and pathogenicity, generating a massive surplus of antigenic variants that enable the bacterium to escape from host immune responses [116, 117]. Transformation and recombination processes play key roles in maintaining the phenotypic variability of Nm, as well as contributing to its genomic conservation [118]. Transformation is the mechanism by which neisserial cells take up extracellular DNA by HGT [119, 120]. In

transformation, uptake of exogenous DNA is proposed to be linked to pilus retraction [121], before the DNA taken up is recombined into the genome by homologous RecA-mediated recombination [122]. The two other mechanisms of HGT are conjugation (through direct contact) and transduction (via bacteriophages) [120].

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Figure 4. Model of type IV pilus biogenesis linked to uptake of exogenous DNA by transformation through pilus retraction. Exogenous DNA taken up by transformation is recombined into the genome by RecA-dependent homologous recombination, aided by DprA. PilG is involved in pilus-mediated translocation of DNA across the membranes.

DprA takes part in the protection and processing of incoming ssDNA. RecG is a helicase that unwinds DNA, presumed to be involved in recombination and DNA repair and could also play a role in recombination of incoming DNA into the genome. Adapted from reference [121] with permission from Tremani and Tone Tønjum. Copyright (2009) by the authors.

1.4.1.1 Pilus associated transformation and DprA

In the pathogenic Neisseria, Nm and Ng, the type 4 pilus (Tfp) promotes adherence and auto-agglutination affecting the formation of colony morphology and microcolonies. The major subunit of Tfp is the

antigenically variable PilE protein [123]. Pilus expression has also been shown to be important for efficient transformation [124]. In neisserial pilus-associated transformation, DNA containing the 10-12 base pair DNA

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uptake sequence (DUS) [125, 126] is preferentially taken up and integrated into chromosomal DNA by RecA dependent homologous recombination [122] supported by DprA [127, 128].

A whole machinery of pilus-related Pil proteins homologous to type 2 secretions is required for Tfp biogenesis and retraction across the

neisserial inner and outer membranes [129-131] (figure 4). For instance, the inner membrane protein PilG, which is required for pilus biogenesis [132], has also been shown to bind DNA in Nm transformation [133, 134].

Another key protein taking part in the Nm transformation machinery is the DNA processing chain A (DprA) [135]. DprA is a DNA binding protein (figure 4) which is ubiquitously expressed among bacteria [136]. DprA protects single-stranded DNA (ssDNA) from exonucleases by binding to it [137], and it also induces partial displacement of theSingle-Stranded

DNA-Binding protein (SSB) from ssDNA [127, 128, 138].

1.4.1.2 Genome dynamics and RecG

Neisserial DNA repair pathways can be divided into the following mechanisms: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR) and recombinational repair (RR) [139].

As well as contributing to genome dynamics, DNA repair along with recombination and replication (the so-called 3R processes) provides essential genome maintenance and stability in all living organisms. In the oral mucosal surface, Nm is constantly exposed to oxidative stress which can lead to DNA damage. A wide range of enzymes participates in DNA repair, such as the helicases which are motor proteins that use energy from ATP hydrolysis to unwind double stranded nucleic acids into single

strands [121]. Helicase activity is also required in recombination, replication and a number of other DNA as well as RNA metabolic processes [140, 141]. RecG is an ATP dependent DNA

repair/recombination helicase (figure 4) involved among other processes in the RR pathway [142] and is ubiquitously expressed in bacteria [143].

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Introduction — Proteomics

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1.5 Whole cell proteome characterization

Comprehensive studies of protein content of cells are essential to

understanding the biological functions in-depth. The proteome is defined as the total number of proteins expressed in a biological system (cell, tissue, body fluid, organism, etc.) whereas proteomics is defined as study of the proteome. Proteins are crucial biological components being the ultimate functional entities in an organism and are widely studied using mass spectrometers. Whole cell proteome characterization can provide essential insight into biology of the sample investigated, e.g. by studying the overall protein expression, protein-protein interacting partners and/or posttranslational modifications (PTMs).

1.5.1 Mass spectrometry-based proteomics

Mass spectrometry (MS)-based proteomics has evolved and advanced significantly since the discovery of ‘soft’ ionization techniques and has become an indispensable tool in life sciences. A mass spectrometer consists of an ion source, mass analyzer(s), detector and a computer to process the data. These instruments measure mass to charge ratio (m/z) of gas phase ions, which are generated in the ion source and separated in the mass analyzer according to their m/z ratios.

1.5.1.1 Ionization techniques

The two ‘soft’ ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), are commonly used in proteomics. In ESI, high voltage is applied to liquid material to generate electrically charged droplets that become smaller and smaller until they eventually evaporate to form gaseous analyte ions. The generated ions are analyzed in the mass spectrometer based on their m/z values. MALDI, on the other hand, is used for dry crystalline material that consist of the analyte mixed with a matrix. Bursts of analyte ions are produced through laser pulses. The development of these ‘soft’ ionization techniques

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(preventing fragmentation of the molecules during ionization in the

source) was awarded with the 2002 Nobel prize in chemistry to John Fenn and Koichi Tanaka [144].

Figure 5. Schematic overview of Q Exactive mass spectrometer. The analyte is ionized in the nanospray source, where it eventually desolvates and enters the mass spectrometer.

Ions are focused in the S-lens. The various Q Exactive ion optics (injection flatapole, bent flatapole, split lens, transfer multipole, C-trap and Z-lens) play their part in transmitting ions to the orbitrap, and preventing contaminants (neutrals) to enter the orbitrap mass analyzer. The precursor ion selection takes place in the quadrupole, while ion fragmentation occurs in the collision cell (HCD). In both cases, the ions are transferred to the C-trap, where they are gathered into a “small cloud” and finally subjected to the Orbitrap mass analyzer. Adapted from reference [145]. Copyright (2011) by The American Society for Biochemistry and Molecular Biology, Inc.

1.5.1.2 Mass analyzers

A vital part of the instruments is the mass analyzer(s). These exist in different variants such as Orbitrap, quadrupole, ion trap and time-of-flight (TOF) and can come together in a range of combinations (hybrid

instruments). A major breakthrough in MS-based proteomics was with the invention of Orbitrap in the late 1990s [146]. Orbitrap was commercially introduced to the proteomics field in 2005 [147]. Launch of the Q

Exactive (figure 5) which is a hybrid Quadrupole-Orbitrap came in 2011

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[145]. Some of the main features of the Orbitrap mass analyzer is high- resolution that provides increased data quality and the extremely high mass accuracy performance for high confidence in identifications for both qualitative and quantitative analysis [148].

1.5.2 Experimental approaches

Numerous proteomics approaches exist to study the complexity of the proteome. Depending on the biological question of interest, the

investigated proteome is analyzed on the protein (top-down) or peptide (bottom-up) level by MS to gain qualitative and/or quantitative data.

1.5.2.1 Top-down and bottom-up proteomics

Protein analyses by MS can be divided into top-down and bottom-up proteomic approaches. In the top-down approach intact proteins are subjected to MS analysis, while in the bottom-up approach, proteins are digested into peptides, which are then subjected to MS analysis [149, 150].

The bottom-up strategy is more popular and has been well-established in proteomics field in the last decades. Nevertheless, there is a slight trend back towards the top-down approach with increasing efforts to study proteoforms [151-155]. Proteoforms are defined as all protein forms from a single gene product, including posttranslational modifications,

alternative splice variants and changes resulting from genetic variation [156].

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Figure 6. Workflow scheme exemplified by experimental design in Paper I.

A) Biological material. Primary Th cell clones derived from gut biopsies of Crohn’s disease patients were expanded in vitro and harvested in their resting stage. B) Sample preparation for MS. Proteins were extracted and in-solution digested with trypsin. The following complex peptide mixture was fractionated on strong anion exchange (SAX) columns, followed by nLC-MS/MS analysis. C) Bioinformatics. The raw data from MS/MS analysis were uploaded to MaxQuant for protein identification and label-free quantitation (LFQ), followed by data processing in Perseus to statistically quantify the fold change differences. Supplementary figure from Paper I.

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30 1.5.2.2 Bottom-up sample preparation

In MS-based bottom-up proteome analysis, the biological material (cells, tissue, body fluid, etc.) containing the complex protein mixture is digested by a sequence-specific enzyme, mostly trypsin, to create peptides. Over the years, efforts in optimizing bottom-up sample preparation protocols have been made. Digestion of proteins is performed by in-gel digestion method [157-160] or through in-solution digestion [161, 162], e.g. by use of spin-filter columns [163] such as the filter-assisted sample preparation (FASP) method [164, 165]. The peptide mixture is then separated in a high performance nano liquid chromatograph (nLC) coupled directly to an ESI tandem mass spectrometer [166-168]. An example of bottom-up

proteomics workflow is shown in figure 6.

Identification of a peptide based on the peptide mass is called peptide mass fingerprinting (PMF). To gain comprehensive sequence information, the peptides are subjected to fragmentation by tandem MS (MS/MS). A vast number of techniques is used for fragmentation, with collision induced dissociation (CID) being the most common method. CID fragments peptides at the amide bond and generates mainly b- (charge retained at the amino-terminal) and y-ions (charge retained at the carboxy-terminal) [169, 170].

With the advancements in sample preparation, instrumentation and the subsequently huge data flow generated, increased need for automated data handling emerged. There are numerous protein sequence databases and automated computer search algorithms [171-174], such as Mascot [175], SEQUEST [176], X!Tandem [177], PeptideShaker [178] and MaxQuant software with the integrated Andromeda search engine [174, 179], that aid in managing the high-throughput proteome data.

1.5.2.3 Quantitative proteomics

Quantitative proteome analysis by MS is divided into label and label-free approaches. Metabolic labeling with stable isotopes is the earliest point of introducing a label to the sample during cell growth and division [180].

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The most popular method for metabolic labeling is the stable isotope labeling with amino acids in cell culture (SILAC) [181]. Another labeling approach is by chemical labeling the proteins or peptides after the

biosynthesis. Tandem mass tags (TMTs) [182] and isobaric tags for absolute and relative quantification (iTRAQ) [183] are two popular chemical labeling techniques. Spike-in standards of known absolute

amounts are also used to determine the absolute quantitative amounts. On the other hand, the label-free technique is the latest point for quantitation [150, 184-186].

1.5.3 Current knowledge on proteomics in CD4+ T helper cells, Crohn’s disease and Neisseria meningitidis until now

Notably, the characterization of the proteomes of both primary (in vivo differentiated) Th cells and of Nm is still in its infancy. To the best of our knowledge, there are currently no other proteomic studies published that describe the collective expression of the total protein content of primary Th cell clones. There are, however, a few MS-based Th cell proteome analysis of in vitro polarized CD4+ T cells from blood samples described in the literature [187-191] and a recent study of Th cells from splenocytes [192]. Moreover, most proteomics studies on Crohn’s disease have

focused on peripheral blood mononuclear cells (PBMC) and tissue from clinical gut biopsies [193-195].

The partial proteome of Nm serogroup A [196], whole cell proteomics of the closely related Ng [197], and identification of common proteins between Nm and Nl [198] have been presented. The PTM patterns in the form of O-linked glycosylation has been described in Ng [199] and in N.

elongata [200]. Surface exposed proteins and outer membrane vesicle (OMV) proteins are of particular interest in host-microbe interactions, immune responses and vaccine development and has therefore been the subject of proteomics studies [201-210]. Proteomics characterization of neisserial pilus compositions and PTMs [211-214] and biofilm analysis [215] have also reported. However, complete in-depth cellular analysis of

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Nm wildtype and mutant cells involved in genome dynamics and maintenance has not been performed until now.

An overview of available proteomics studies relevant for this Thesis is presented in Table 1.

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Table 1. Short description of proteomic studies on CD4+ T helper cells, Crohn’s disease and neisserial cells available before this Thesis.

Sample source Proteomics

method

Reference

CD4+ T helper cells

Comparison of activated Th1 and Th2 (70 proteins quantified)

CD4+ T cells from human blood, in vitro differentiated to Th cells

2-DE and PMF [187]

Cell surface proteins of resting Th1 and Th2 (372 proteins identified and 40

quantified) SILAC [188]

Nuclear proteome of Th2 cells (903

proteins identified) iTRAQ [189]

Comparison of microsomal fractions during Th2 cell differentiation (557

proteins identified and 304 quantified) ICAT [190]

Early Th17 cell differentiation (5,600

proteins identified and 291 quantified) LFQ [191]

Comparison of Th17 and Treg and protein changes during differentiation (4,287 proteins identified)

CD4+ T cells from murine splenocytes, in vitro differentiated to Th17 and Treg

LFQ [192]

Crohn's disease (CD)

11 proteins described to discriminate

between UC and CD Human PBMC 2-DE and

MALDI-MS/MS [193]

8 m/z peaks detected to discriminate

between UC and CD Human gut tissue PMF [194]

Neisseria species

Analysis of OMV, protein antigens and potential vaccine targets

Nl, Ng, Nm and Cuban OMV vaccine VA-MENGOC-BC

Various qualitative and quantitative methods

[201-210]

Subcellular fractionation to map the total proteome (about 1000 proteins identified)

Ng TMT [197]

Comparative proteome analysis (48

common proteins identified) Nl and Nm 2-DE MALDI-

MS/MS

[198]

Proteome analysis of serogroup A (273

proteins identified) Nm 2-DE PMF and

PSD

[196]

PTM analysis of O-linked glycosylation Ng and N. elongata Enrichment [199, 200]

Proteoform and PTM analysis of

components of type IV pili Nm Top-down

approach [211-214]

Ig-binding proteins and biofilm

formation Nm PMF [215]

2-DE: two-dimensional gel electrophoresis; ICAT: isotope-coded affinity tag; PSD:post-source decay (technique for generation of fragment ions)

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Aim of the Thesis

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2. Aim of the Thesis

The overall aim of this Thesis is to use large-scale mass spectrometry-based proteome analysis to explore qualitative and quantitative protein expression signatures in gut mucosal CD4+ T helper (Th) cells (Papers I and II) and in bacteria present in the oropharyngeal mucosa exemplified by meningococci (Papers III and IV).

2.1 Specific aims

Aim I

To investigate the in vitro effect of multiple expansions on gut-derived primary Th cell clones by proteomic analysis (Paper I)

Aim II

To compare Th1 and Th1Th17 cells at the qualitative and quantitative proteomic level to gain insight into their phenotypic characteristics

(Paper I)

Aim III

To correlate the quantitative differences in the transcriptome and proteome upon activation of a CD4+ Th cell clone (Paper II)

Aim IV

To explore the role of DprA in transformation and investigate the effects of loss of DprA on the global meningococcal proteome in the wildtype and dprA mutant strains (Paper III)

Aim V

To study the role of RecG in meningococcal DNA repair/recombination and transformation by characterizing the wildtype and recG mutant strains (Paper IV)

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Summary of Papers

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3. Summary of Papers

Paper I

In this study, the proteome of primary Th1 and Th1Th17 cells was

explored in detail. This work is the first to characterize the proteomes of gut-derived Th cell clones obtained from clinical subjects, rather than clones differentiated in vitro. Additionally, the effect of common variations in cell culture on protein expression in Th cell clones was investigated. We found that there were only minor deviations in the proteins expressed between the multiple in vitro growth of Th cell clones (less than 7%), and that these changes could not be related to the different cell culture parameters applied in the experimental setup. In addition to qualitative presence of proteins in gut-derived Th cells, with a

comprehensive dataset of more than 7400 proteins identified, quantitative information between two distinct phenotypes (Th1 and Th1Th17 cells) was provided. Through this study, major differences in expression levels of cytotoxic proteins were identified, such as granzymes and perforin, with higher expression levels in the Th1 phenotype. These results were further validated by testing a larger panel of gut-derived Th cells with different phenotypes. We discovered that a certain population of Th1 subtypes, the CD28+ and NKG2D- Th1 cells, express cytotoxic proteins. This Th1 subtype had different phenotypic markers as compared to previously described cytotoxic Th cells. With cytotoxicity being a rare trait of Th cells, these findings contribute to elucidate the broad diversity of Th cells.

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Summary of Papers

36

Paper II

This study aimed to explore the in-depth correlation between

transcriptome and proteome during activation of a Th1 cell clone. The Th1 cell clone studied here was the cytotoxic CD4+ T cell described in Paper I, which as mentioned earlier is a quite rare feature in CD4+ T cells. In

Paper II, a comprehensive dataset of the transcriptome and proteome of resting versus the activated stage of the cytotoxic Th1 cell clone is provided. In addition to qualitative presence of mRNA and protein expression, the quantitative differences upon activation of the Th1 cells are described. A total of 6296 transcripts and 1209 proteins were

significantly differentially expressed upon activation. A non-linear correlation was observed from the quantitative differences in

transcriptome and proteome during activation of Th1 cells. Comparing these large datasets revealed that the genes involved in the cytotoxic granules were upregulated at the mRNA level, while on the protein level they were downregulated. This can be explained as compensatory

upregulation of the genes upon release of the proteins with the cytotoxic granules. Another interesting finding was that the transcription factor Interferon regulatory factor 4 (IRF4) was highly upregulated during activation. This transcription factor is associated with both expression of cytotoxic proteins and cytotoxic T lymphocyte response. The major increase in IRF4 upon activation of the Th1 cell clone lead us to

hypothesize a possible future implication of IRF4 as drug target in therapy of Crohn’s disease.

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Summary of Papers

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Paper III

In Paper III, the aim was to utilize proteomic tools for studying the DNA processing chain A (DprA) protein in Nm. DprA is a ubiquitous protein with an important role in DNA transformation. To further understand its function, proteins affected by DprA deletion were identified by comparing the Nm wildtype and Nm DprA null mutant strains. Among 1057 proteins identified, 100 proteins were significantly differentially abundant with a majority being less abundant in the DprA null mutant. Within these, proteins involved in homologous recombination during transformation (RecA, UvrD and HolA), pilus biogenesis (PilG, PilT2, PilT1 and PilM), response to oxidative stress and core metabolism were found. Since the inner membrane protein PilG is also essential in transformation, we further investigated its connection with DprA. The expression levels of DprA in the Nm PilG null mutant was reduced as compared to Nm wildtype, which further strengthened a possible interactive link between DprA and PilG.

Western blot and co-immunoprecipitation (co-IP) were performed to validate the results and to characterize the association between DprA and PilG in more detail. When analyzed by co-IP and subsequent MS analysis, several of the less abundant proteins identified in the DprA null mutant, such as the pilus biogenesis proteins, were found to interact with DprA. A clear interaction of the recombination proteins RecO, RecR and TopA with DprA and PilG was found. Overall, these results demonstrated that DprA interacts with proteins which are important in Nm recombination, transformation and pilus biogenesis.

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Summary of Papers

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Paper IV

In this Paper, we investigated the role of Nm RecG in DNA

repair/recombination and transformation as well as its role during genotoxic stress. The purified recombinant RecG exhibited ATP dependent binding and unwinding ability of a wide range of DNA oligonucleotides, including a Holliday junction. The role of RecG in genotoxic stress was further studied by investigating the survival rate of Nm wildtype as compared to Nm RecG null mutants. The latter was more sensitive to genotoxic stress than the wildtype. Comparative sequence analysis of NCBI available Neisseria genomes were also included, which showed that Nm recG is the gene that harbors the most DNA uptake sequences (DUS), which enable transformation of neisserial DNA. In addition, single nucleotide polymorphisms (SNPs) in Nm recG were identified in the Neisseria genomes. Among 49 SNPs found, 37 were non- synonymous SNPs (nsSNPs), including seven nsSNPs located in

conserved active site residues. Proteomics tools were used to examine the functional role of Nm RecG in more detail. In the comparative global proteomic analysis of Nm wildtype and Nm RecG null mutant strains, 1060 and 1064 proteins were identified, respectively. Differentially abundant proteins were identified, in order to search for proteins that potentially interact with or are co-regulated with the RecG protein. 83 proteins were significantly differentially abundant, among which 29 were upregulated and 54 downregulated in the Nm RecG null mutant. The differentially expressed proteins suggested that RecGis associated to proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity.

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General Discussion

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4. General Discussion

Life science involves extensive research on all molecular levels, including proteins as the ultimate functional entities of living organisms. Proteomics research as part of life science is an exciting and growing field due to the instrumental and computational advancements. Studying the proteome can provide unique and essential knowledge of both health and pathological conditions. The mass spectrometer is a powerful tool which is widely used in proteomics. With the major advances in mass spectrometer instruments, there has been a shift in proteomics analysis from qualitative to

quantitative proteomics [184-186, 216-218].

In this Thesis, two types of biological materials related to mucosal

immunology were explored by high-throughput MS analysis.In CD4+ Th cells, phenotypic characteristics and molecular functions were studied by comparison of two gut-derived Th cell phenotypes from Crohn’s disease patients. In addition, the relationship between the transcriptome and

proteome upon activation of gut-derived cytotoxic Th cells was delineated.

In meningococci, the aim was to understand the transformation, DNA repair and recombinational molecular processes by comparative analysis of Nm DprA and RecG deletion mutants with wildtype cells. As a result, new insights into life science and infection biology, with a focus on the gut and oral mucosal surfaces, were acquired.

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General Discussion — T helper cells

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4.1 Identification of CD28+ NKG2D- Th1 CTLs

With their versatile roles, CD4+ T cells are of great importance for the immune system. In Crohn's disease, subtypes of them have been found to heavily infiltrate the inflamed tissue, especially Th1 and Th17 phenotypes, as well as Th1Th17 cells. In the study presented in Paper I, a subgroup of gut-derived Th1 cell clones from Crohn’s disease patients (CD28+

NKG2D- Th1 phenotype) was identified to be cytotoxic. It was observed that proteins with cytotoxic function, such as the granzymes and perforin, were substantially more abundant in the CD28+ NKG2D- Th1 phenotype (table 2).

Table 2. Identification of higher abundance of cytotoxic proteins in the Th1 cell clones compared to Th1Th17. Selected essential cytotoxic proteins are shown.

In Paper II, the cytotoxic CD28+ NKG2D- Th1 phenotype was further investigated both by proteomics and transcriptomics analysis. Correlating the transcriptomics and proteomics differences found upon activation of cytotoxic Th1 cell, the cytotoxic granula proteins were found to be

downregulated, while on the mRNA level an increase was observed during cell activation. This allowed us to connect the observed decrease of

granzymes and perforin with release of the cytotoxic granula with compensatory increase on the transcriptome level. TF IRF4, which is predominantly expressed by lymphocytes [219], was upregulated by activation of CD28+ NKG2D- Th1 cells. This TF has also been reported

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to have an important role in CTLs activity and memory [220], as well as being associated with immune mediated diseases [221].

MS-based proteomics clearly holds great promise for unravelling novel immune cell characteristics that can provide novel disease related

biological insights. For the development of diagnostic biomarkers, studies of PBMCs and biopsies can be most useful and proteomics studies have discriminated between ulcerative colitis and Crohn’s disease [193-195].

On the other hand, since these biological samples contain a wide range of various cell types, specific and unique information from each of the cell types may be masked due to the heterogeneity of the sample. The analysis of individual Th clones in this study allowed us to delineate the general cytotoxic features to the CD4+ Th cells, which in heterogeneous samples such as tissue or PBMC, would naturally be linked to cytotoxic CD8+ T cells or NK cells. Thereby, increased understanding on the function of distinctive features of cells of the immune system was gained.

Notably, most of the observed CD4+ CTLs described in the literature are from chronic viral infections and studies of human PBLs [222-225].

Cytotoxic CD4+ T cells have also been described in virus infection

models [226, 227]. It is assumed that in viral infections, CD4+ CTLs play a protective role. CD4+ CTLs have also been found to be induced by vaccine candidates against rotavirus with potential to lyse the infected cells [228], and some studies allude that they have an antitumor response [229, 230]. CD4+ CTLs have also been detected in chronic inflammatory conditions, where they have been suggested to have a pathogenic role [231-235]. Their potential pathogenic role in various diseases makes them of interest as drug targets. A potential drug can for instance act through inhibiting the CD4+ CTLs formation.

The proteomic analysis of gut-derived Th cell clones presented in this Thesis revealed a cytotoxic Th phenotype which has not been described in the literature before. Cytotoxic activity is generally thought to play a protective role by killing the infected cells. However, the role of cytotoxic CD4+ T cells in Crohn’s disease remains unknown. There is a possibility that CD4+ CTLs in Crohn’s disease may contribute to the pathogenesis by

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destroying normal epithelial cells. Hence, further studies with focus on the role of cytotoxic CD4+ T cells in Crohn’s disease are needed. This can help to improve more accurate therapeutic option and/or decide disease onset.

Even though an altered microbiota is linked with various diseases, its role as a cause or outcome remains controversial [236, 237]. Environmental and genetic factors, increased Th cell immune responses and altered microbiota are all factors attributed to Crohn’s disease. In order to establish if there is a certain triggering factor for Crohn’s disease or if there is an intricate vicious cycle involving CTLs and the microbiota influencing each other, remains to be delineated. Further identification of CTLs from gut biopsy of Crohn’s disease patient and their potential role (if any), will add to this complexity.

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General Discussion — Neisseria meningitidis

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4.2 The proteomic signature of meningococcal DprA and RecG null mutants

The bacterium Neisseria meningitidis (Nm), the meningococcus, is an oropharyngeal mucosal opportunist. Upon invasion into the host, aided by a number of virulence factors (e.g. the pili), Nm can cause the acute severe disease sepsis and/or meningitis [238-240]. Additionally, a major reason for counting Nm as one of the most successful human bacterial pathogens is its ability to generate vivid genome variation and subsequently induce a vast abundance of potentially virulent Nm strains [238].

Exploring the proteomic signatures of Nm allowed the identification of DprA and RecG associated proteins, which play key roles in genome diversity and maintenance. DNA processing chain A (DprA), which is involved in transformation, and DNA repair/recombination helicase RecG are both ubiquitously expressed in bacteria. Transformation and

recombination are key players in driving the evolution of virulent Nm strains through genome variation [241, 242]. The Nm DprA null mutant was absolutely non-competent for transformation, while the Nm RecG mutant only exhibited a transient reduction in transformation efficiency (Papers III and IV). In the Nm DprA and RecG deletion mutants, several proteins were found to be differentially regulated. Among these, proteins related to the transformation machinery as well as proteins belonging to the 3R processes were identified.

Complementary proteins, notably PilG, PilM and PilT involved in Tfp biogenesis and retraction were found to be affected by deletion of DprA and RecG (tables 3 and 4). The twitching motility of Tfp provided by pilus retraction [243] cause Nm to cross the mucosal layer and reach the

epithelium [244].Tfp is essential in adhesion to the host tissue and to induce growth of microcolonies on the epithelial cells [245, 246].

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Table 3. Fold change values of selected differentially abundant proteins in DprA deletion mutant (∆DprA) compared to wildtype, exemplified by pilus biogenesis and DNA recombinational-repair proteins.

For the DprA mutant, PilG was less abundant than in wildtype (table 3).

The PilG null mutant has been shown to be non-piliated, non-agglutinating and defective in competence [132, 134]. The lack of transformation was also a hallmark in the DprA deletion mutant.

Table 4. Fold change values of selected differentially abundant proteins in RecG deletion mutant (∆RecG) compared to wildtype, exemplified by pilus biogenesis and DNA repair, recombination and replication (3R) proteins.

In the RecG deletion mutant, PilE and PilX were less abundant than in wildtype Nm (table 4). RecG has been shown to be required for pilin

antigenic variation [247]. Furthermore, the phenotype of RecG null mutant

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was different from the wildtype, in showing smaller colonies and non- agglutination in addition to reduced competence for transformation. This was in concordance with previous studies which showed reduced

transformation efficiency for RecG mutants [248, 249]. Lack of auto- agglutination, transformation and adherence are key hallmarks in PilE- deficient Neisseria [250-252]. PilX has also been reported to mediate adherence of Nm to host cells and to be of importance for inter-bacterial contacts [253], with reduced piliation in PilX-deficient Neisseria [254].

The quantitative analysis showed that RecG mediated competence for transformation through recombination mechanism, in addition to its influence through altered expression of Tfp components.

Altogether, depletion of DprA exerted more severe impact on protein expression than depletion of RecG with regard to fold change differences in protein abundance. DprA depletion, however, exerted no growth effect, whereas RecG depletion had an impact on colony size. This shows that the magnitude of changes on the proteome profile does not necessarily

correspond to the changes in the phenotype.

4.3 Impact of genome dynamics on meningococcal lifestyle

The balance between genome dynamics and genome conservation generates a vast surplus of meningococcal variants, among which some neisserial cells are more fit for survival in the human oropharyngeal mucosa than others. Neisserial survival depends on the selective environmental pressures met in the mucosa, including host immune responses. The mechanisms for genome maintenance and genomic diversity are also important to understand the evolution of antimicrobial resistance (AMR). Nm is fully able to take up AMR genes by

transformation [255]. Hence, DprA may potentially be used as a drug target and hinder genome diversity and pathogenicity, e.g. through transformation and reduced expression of Tfp components without inducing a change in the Nm growth. Nm adaptation to ever changing

Proteomic approaches to study CD4+ T cells and meningococci: New insight into life science and infection biology