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Influence of host and bacterial factors during meningitidis colonization Sara Sigurlásdóttir Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Tuesday 18 December 2018 at 10.00 in Vivi Täckholm-salen (Q-salen), NPQ-huset, Svante Arrhenius vä g 20.

Abstract The human-restricted Neisseria meningitidis is a major cause of bacterial and worldwide. Colonization of the mucosal layer in the upper respiratory tract is essential to establish an state and invasive disease. N. meningitidis encounters diverse environmental challenges during colonization and has evolved multiple strategies and virulence factors to survive and adapt within the host. Upon initial adhesion to the host epithelial cells, N. meningitidis forms -mediated aggregates called microcolonies, which are characterized by interbacterial and host- interactions. Microcolonies promote long-term asymptomatic colonization within the host. However, the dispersal of single from microcolonies can help N. meningitidis to develop close contact with host cells and facilitate the invasion of mucosal surfaces or to a new host. This thesis focuses on understanding how the interplay between the host, environment, and virulence factors influences N. meningitidis colonization. Paper I shows that the host-derived metabolite lactate induces rapid dispersal of N. meningitidis microcolonies. Further molecular characterization in Paper II revealed that lactate-induced dispersal is mediated by pilus retraction, occurs in a density-dependent manner, and is responsive to temperature. Paper III shows that the deletion of D-lactate dehydrogenase LdhA in N. meningitidis promotes aggregation and biofilm formation through an increase in the autolysis-mediated release of extracellular DNA. Finally, Paper IV examines the role of polynucleotide phosphorylase (PNPase) in the virulence of N. meningitidis. The deletion of PNPase resulted in a pilus-dependent increase in the aggregation and adhesion to epithelial cells. A PNPase mutant was growth deficient and highly attenuated in an in vivo mouse model. Transcriptional analysis revealed that PNPase plays a role as a major regulator in N. meningitidis.

Keywords: Neisseria meningitidis, Colonization, Host-bacteria interactions, Lactate, Biofilms, Polynucleotide phosphorylase.

Stockholm 2018 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-161566

ISBN 978-91-7797-474-1 ISBN 978-91-7797-475-8

Department of Molecular Biosciences, The Wenner- Gren Institute

Stockholm University, 106 91 Stockholm

INFLUENCE OF HOST AND BACTERIAL FACTORS DURING NEISSERIA MENINGITIDIS COLONIZATION

Sara Sigurlásdóttir

Influence of host and bacterial factors during Neisseria meningitidis colonization

Sara Sigurlásdóttir ©Sara Sigurlásdóttir, Stockholm University 2018

ISBN print 978-91-7797-474-1 ISBN PDF 978-91-7797-475-8

Cover Illustration by Sara Sigurlásdóttir

Printed in Sweden by Universitetsservice US-AB, Stockholm 2018 Distributor: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University Fyrir pabba

SUMMARY

The human-restricted pathogen Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Colonization of the mucosal layer in the upper respiratory tract is essential to establish an asymptomatic carrier state and invasive disease. N. meningitidis encounters diverse environmental challenges during colonization and has evolved multiple strategies and virulence factors to survive and adapt within the host. Upon initial adhesion to the host epithelial cells, N. meningitidis forms pilus-mediated aggregates called microcolonies, which are characterized by interbacterial and host-cell interactions. Microcolonies promote long-term asymptomatic colonization within the host. However, the dispersal of single bacteria from microcolonies can help N. meningitidis to develop close contact with host cells and facilitate the invasion of mucosal surfaces or transmission to a new host. This thesis focuses on understanding how the interplay between the host, environment, and virulence factors influences N. meningitidis colonization. Paper I shows that the host-derived metabolite lactate induces rapid dispersal of N. meningitidis microcolonies. Further molecular characterization in Paper II revealed that lactate-induced dispersal is mediated by pilus retraction, occurs in a density-dependent manner, and is responsive to temperature. Paper III shows that the deletion of D-lactate dehydrogenase LdhA in N. meningitidis promotes aggregation and biofilm formation through an increase in the autolysis- mediated release of extracellular DNA. Finally, Paper IV examines the role of polynucleotide phosphorylase (PNPase) in the virulence of N. meningitidis. The deletion of PNPase resulted in a pilus-dependent increase in the aggregation and adhesion to epithelial cells. A PNPase mutant was growth deficient and highly attenuated in an in vivo mouse model. Transcriptional analysis revealed that PNPase plays a role as a major regulator in N. meningitidis.

i POPULÄRVETENSKAPLIG SAMMANFATTNING

Neisseria meningitidis är en bakterie som ger sjukdom endast i människor. Bakterien finns utan att ge symptom i svalget och bakre delen av näshålorna hos ungefär var tionde person. Bakterien sprids mellan individer genom upprepad närkontakt och genom luftburen droppsmitta. Även om N. meningitidis sällan orsakar sjukdom är den fortfarande en av de främsta orsakerna till hjärnhinneinflammation och blodförgiftning världen över. N. meningitidis måste överleva och undkomma de utmaningar den möter i människokroppen. Bakterien måste vidhäfta till slemhinnan för att inte sköljas bort. Bakterien binder in till slemhinnor med hårliknande utskott som kallas pili eller fimbrier. Under denna process binder bakterier till varandra och bildar ett stort antal så kallade mikrokolonier bestående av upp till hundratals bakterier vardera. På så sätt skyddas bakterierna från immunförsvaret. För att kunna ta sig vidare in i människokroppen måste bakterierna släppa taget och sprida ut sig som enskilda bakterier. Det är inte mycket känt om vad som orsakar att bakterierna att sprider ut sig och i nästa steg ger livshotande sjukdom. Denna avhandling fokuserar på att förstå samspelet mellan slemhinneceller och sjukdomsframkallande egenskaper hos bakterierna vid N. meningitidis infektion. I artikel I visade vi att laktat, en molekyl tillverkad av värdceller, förorsakar att N. meningitidis släpper taget från mikrokolonier. I artikel II visade vi vidare att bakteriespridningen styrs av omgivande temperatur och bakteriekoncentration samt att fungerande pili-utskott är nödvändigt. I artikel III visar vi att bristen på det metabola enzymet LdhA i N. meningitidis ökar bildandet av de bakteriella samhällen som kallas biofilmer. Brist på LdhA gjorde att bakterierna gick sönder och släppte ut extracellulärt DNA (eDNA). Utsläppet av eDNA var orsaken till ökad förmåga att bilda biofilm. I artikel IV studerade vi polynukleotidfosforylas (PNPase), som bryter ner RNA. Avsaknad av PNPase ökade vidhäftning och klumpbildning av bakterierna. Utan PNPase växte bakterierna sämre och visade färre sjukdomsegenskaper i modellsystem.

ii CONTENTS

Summary ...... i Populärvetenskaplig sammanfattning ...... ii List of publications ...... v Abbrevations ...... vi Introduction ...... 1 Chapter 1: Neisseria meningitidis, the meningococcus ...... 2 1.1 Classification ...... 2 1.2 Transmission and carriage ...... 3 1.3 Disease ...... 3 1.4 Treatment and prevention ...... 5 1.5 Epidemiology ...... 7 Chapter 2: Factors involved in N. meningitidis colonization and virulence ...... 9 2.1 capsule ...... 10 2.2 ...... 11 2.3 Type IV pili ...... 12 2.4 Opacity proteins ...... 16 2.5 Two-component regulatory systems ...... 17 Chapter 3: N. meningitidis colonization of the nasopharyngeal epithelium ...... 19 3.1 The nasopharyngeal mucosa ...... 19 3.2 Initial adherence and microcolony formation ...... 20 3.3 Microcolony dispersal and intimate adhesion ...... 21 3.4 Biofilm formation ...... 23 Chapter 4: Lactate and bacterial virulence ...... 27 4.1 Lactate utilization in pathogenic Neisseria ...... 27 4.2 Effect of lactate on bacterial pathogenesis ...... 28 Chapter 5: Polynucleotide phosphorylase ...... 31 5.1 The degradosome ...... 31 5.2 Polynucleotide phosphorylase structure and activity ...... 32 5.3 Role of polynucleotide phosphorylase in ...... 33 Present Investigation ...... 35 Aims ...... 35 Results and discussion ...... 36

iii Paper I ...... 36 Paper II ...... 38 Paper III ...... 40 Paper IV ...... 42 Future perspectives ...... 45 Acknowledgements ...... 50 References ...... 53

iv LIST OF PUBLICATIONS

This thesis is based on the following work, which is referred to in the text by roman numerals:

I. Sara Sigurlásdóttir, Jakob Engman, Olaspers Sara Eriksson, Sunil D. Saroj, Nadezda Zguna, Pilar Lloris-Garcerá, Leopold L. Ilag, Ann-Beth Jonsson (2017). Host cell derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal. PLoS 13(4): e1006251.

II. Sara Sigurlásdóttir and Ann-Beth Jonsson. Lactate-induced dispersal of Neisseria meningitidis microcolonies is mediated by changes in cell density and pilus retraction and is influenced by temperature change. Manuscript.

III. Sara Sigurlásdóttir, Gabriela M. Wassing, Fanglei Zuo, Melanie Arts and Ann-Beth Jonsson. Deletion of D-lactate dehydrogenase LdhA in Neisseria meningitidis promotes biofilm formation through increased rates of autolysis and extracellular DNA release. Manuscript.

IV. Jakob Engman, Aurel Negrea, Sara Sigurlásdóttir, Miriam Geörg, Jens Eriksson, Olaspers Sara Eriksson, Asaomi Kuwae, Hong Sjölinder and Ann-Beth Jonsson (2016). Neisseria meningitidis polynucleotide phosphorylase affects aggregation, adhesion, and virulence. Infection and Immunity 84(5): 1501–1513.

v ABBREVATIONS

AI-2 Autoinducer-2 AniA Nitrite reductase App Adhesion and penetrating protein CSF CM Conditioned medium CrgA Contact regulated gene A eDNA Extracellular DNA EPS Extracellular polymeric substances fHbp binding protein FNR Fumarate and nitrate reductase regulator GGI Gonococcal genetic island LctP Lactate permease LDH Lactate dehydrogenase LdhA Cytoplasmic D-lactate dehydrogenase LdhD Respiratory D-lactate dehydrogenase LldA Respiratory L-lactate dehydrogenase LNnT Lacto-N-neotetraose LPS Lipopolysaccharide MspA Meningococcal serine protease A NadA Neisseria adhesin A NhbA Neisserial heparin-binding NhhA Neisseria Hia/Hsf homolog A NO Nitric oxide NorB Nitric oxide reductase Opa Opacity proteins PNPase Polynucleotide phosphorylase PptB phosphotransferase B QS Quorum sensing RNase Ribonuclease ST Sequence type TCS Two-component system Tfp Type IV pili

vi INTRODUCTION

The creation of Koch´s postulates in the late 1800s laid the foundation for the diagnosis and management of infectious diseases. The postulates were guidelines that associated microorganisms with the outcomes of a disease [1]. The field of host-pathogen interactions later expanded, and limitations of the guidelines were identified. Pathogenicity became accepted as a variant trait and is determined by the virulence of a microorganism and the host’s immune status. Pathogens do not cause a disease in every host, and commensal bacteria can cause opportunistic infections [2]. The pathogenic bacterium Neisseria meningitidis does not fulfill Koch´s postulates since it is a commensal and primarily colonizes the human upper respiratory tract. In rare cases, it can cause invasive diseases, sepsis and/or meningitis [2, 3]. N. meningitidis can establish long-term colonization within the host and tightly adhere to nasopharyngeal mucosa by forming multibacterial aggregates or biofilms [4]. However, dissociation from aggregates is essential to invade the mucosa and cause a disease [5, 6]. The aim of this thesis was to identify factors that contribute to N. meningitidis colonization. Paper I and Paper II examine the role of the host´s metabolite lactate and the involvement of meningococcal virulence factors in microcolony dispersal. Paper III investigates the influence of the lactate dehydrogenase LdhA on biofilm formation. Finally, Paper IV characterizes the polynucleotide phosphorylase (PNPase) in N. meningitidis and examines its role in the aggregation and host-cell interaction.

1 Chapter 1: Neisseria meningitidis, the meningococcus

The genus Neisseria is named after the German bacteriologist Albert Neisser, who discovered the first member, , in 1879. The Austrian bacteriologist Anton Weichselbaum discovered intracellularis meningitidis in 1887, which was later reclassified within the genus Neisseria and is now known as Neisseria meningitidis [7]. Today, the genus is composed of at least 25 species that are Gram-negative, aerobic, and oxidase positive, and usually appear as diplococcic [8, 9]. N. gonorrhoeae (gonococcus) and N. meningitidis (meningococcus) are currently the only pathogenic species identified [6]. However, commensal strains have been implicated in several opportunistic infections [7]. All species exhibit adherence to the mucosal epithelium of a specific niche within the host. Species from the genus are found in various animals, including humans, but N. meningitidis and N. gonorrhoeae are human-specific pathogens [6, 7]. Even though there is extremely high genetic similarity between the two pathogens, their colonization sites and disease outcomes are distinct [10]. N. gonorrhoeae resides in the urogenital tract and is the causative agent of , the most common sexually transmitted disease. N. meningitidis, is one of the main causes of bacterial meningitidis and sepsis and is mostly present in the upper respiratory tract in an asymptomatic carrier state together with commensal bacteria [6].

1.1 Classification

Meningococci are divided into serogroups based on the structure of the polysaccharide capsule. The serogroups are classified into serosubtypes and serotypes based on differences in porins (PorA or PorB) present in the outer membrane and into immunotypes by the lipopolysaccharide (LPS) structure. There are 13 serogroups of meningococcus, with six groups that are mainly responsible for invasive disease. Serogroups A, B, C, W-135, X and Y are responsible for 90% of disease cases worldwide although there are

2 considerable epidemiological differences between countries and continents [11-14]. In addition, meningococcal isolates can be divided by a genetic approach using multilocus sequence typing (MLST) into clonal groups or complexes (CCs). The approach is based on allelic variation (sequence type, ST) in seven housekeeping genes and can be a valuable source to improve the understanding of epidemiology and clonal expansion. Amongst the 46 CCs identified so far, 41 have been linked to invasive disease [12, 15].

1.2 Transmission and carriage

N. meningitidis asymptomatically colonizes approximately 10% of the human population [16]. The upper respiratory tract is their primary reservoir, although meningococci have also been observed in the buccal mucosa, anus, urethra, urogenital mucosa, and dental plaque [17]. Transmission of meningococci occurs between asymptomatic carriers in close contact with droplets of saliva. After colonization, the bacteria can persist for months in the nasopharynx. However, disease usually develops within 1-14 days, and colonization rarely results in invasive disease [11]. Carriage is highest in adolescents and adults. Despite a low rate of carriage, newborns and children from 1-4 years are at the highest risk of developing invasive disease. The immature humoral and cellular in newborns is the main reason for their high risk of disease development. Adolescents also have a high risk of developing the disease due to a higher carrier rate [14, 18]. Additionally, factors such as gender, health, smoking, and socio-economic status can have an effect on carriage and disease status [11, 18].

1.3 Disease

The meningococcal carrier rate is high, but the transition to invasive disease is rare [12]. To cause a disease, the bacteria must penetrate the mucosal layer after initial attachment, resist the immune attack from the host, replicate, and survive in the bloodstream. Meningococcal virulence factors, host environmental factors, and immune defense efficacy can all contribute to disease development. The first symptoms of can be

3 unspecific flu-like symptoms, such as fever, chills, muscle ache, lower back pain and thigh pain [12, 19]. Therefore, it can be difficult to diagnose the disease and treat it at early stage. If not treated, the disease can progress very quickly and develop into sepsis and/or meningitis within few hours after the initial symptoms [17]. A quote from 1919, “No other infection so quickly slays,” still remains true today, as the fatality rate continues to be high despite the increasing knowledge of the pathogenesis. The disease can manifest as bacteremia, meningitis, or both. Although less common, meningococci can also cause inflammation in the pericardium, skin, joints, eyes, lungs, salivary glands, and female genital tract [12]. Unusual symptoms such as diarrhea and vomiting, caused by serogroup W, have also been reported recently [20]. Meningitis is the most frequent form of disease and occurs in at least 50% of cases [18]. Once the bacteria pass the blood-brain barrier and enter the subarachnoid space, they are able to divide uncontrollably due to a lack of both humoral and cellular immunity. Bacterial endotoxins induce the activation of proinflammatory cytokines, which affect the permeability of the blood-brain barrier. Cytokines recruit neutrophils and other immune cells through the blood-brain barrier to attack the microbe. However, this increases the inflammation in the central nervous system and further contributes to the severe clinical symptoms of the infection. The inflammation causes tissue edema and can lead to intracranial and cerebral perfusion pressure [12]. The mortality rate of patients with meningococcal meningitis is 5-20%, with 10-30% of survivors suffering from neurological deficiencies [21, 22]. Meningococcemia, or sepsis, develops in approximately 20%-40% cases upon meningococcal infection [18, 23]. Symptoms are characterized by a rapid onset of fever, rash, low blood pressure, shock, disseminated intravascular coagulation (DIC), multiple organ failure, and in the worst cases, avascular necrosis [11, 23]. Fulminant sepsis can develop within a few hours. Once bacteria reach the bloodstream, they proliferate rapidly and release high amounts of endotoxin in the form of blebs, followed by complement activation, the production of proinflammatory cytokines, and other factors that can induce shock. The severity of the disease is highly dependent on the cytokine concentration and the activity of the . Excessive

4 inflammatory response in the bloodstream can lead to circulatory collapse and an abnormal increase in the activation of coagulation pathways [12]. Circulatory collapse is the main cause of deaths in developed countries [11]. Compared to those with meningitis alone, patients that develop sepsis have a higher mortality rate, which varies from 20 to 80%. In addition, survivors can suffer from severe physical and mental disabilities [12].

1.4 Treatment and prevention

The disease progression from N. meningitidis infections occurs rapidly, and therefore, early diagnosis and treatment are important. Early diagnosis can be a challenge since symptoms can initially be unspecific or not present at all [12]. The diagnosis can easily be overlooked when altered mental status and hemorrhagic skin lesions are not present. When diagnosed, the patient should be given antibiotics immediately to stop meningococcal proliferation. Subsequently, the level of endotoxin in the plasma will drop along with cytokine and chemokine concentrations in the blood and cerebrospinal fluid [11]. , , and are the antibiotics recommended by the World Health Organization [24]. In addition, ceftriaxone treatment is recommended as the first choice for in Africa [24]. However, antibiotic resistance is a growing concern, especially for the closely related N. gonorrhoeae, which is listed as one of the three microorganisms that pose the most critical threats to human health due to their extensive resistance [25]. Although no meningococcal resistance against third-generation has been reported yet, and the frequency of chloramphenicol resistance is very low, resistance has been reported to penicillin, ciprofloxacin, , and fluoroquinolones [11, 14, 26, 27]. Chemoprophylaxis is also important to eliminate carriers and can thus be useful for the prevention of outbreaks [11]. Vaccination is the most effective to prevent of meningococcal disease, as individuals producing bactericidal or opsonizing antibodies cannot develop the disease [16]. The first attempts to develop whole-cell meningococcal vaccines began in the early 1900s. However, the first licensed vaccines were not accessible until the 1970s, which are based on the polysaccharide capsule. They are now available in bivalent (groups A and C), trivalent (A,

5 C, and W-135) or tetravalent (A, C, Y, and W-135) forms. Polysaccharide vaccines have been shown to have very limited value as they are poorly immunogenic in young children, they are T-cell independent, they can cause a hyper-response after repetition of dosage, and they do not affect nasopharyngeal carriage [16, 28]. Currently, the polysaccharide vaccines are not used in immunization programs, but can be useful for high-risk groups or to control outbreaks. More successful protein-conjugated vaccines with polysaccharide attached to tetanus or diphtheria toxoid first became available in the late 1990s and have mostly replaced polysaccharide vaccines [28]. The first MenC immunization program was implemented in 1999 in the United Kingdom and resulted in a remarkable decrease in both carriage and disease, in addition to providing herd protection [29]. Available conjugate vaccines are monovalent (A or C), tetravalent (A, C, Y, and W-135), and combined with vaccines [16]. The efficiency of the MenC vaccine has facilitated further development, and MenAfriVac was eventually developed and implemented as a low-cost conjugated vaccine in an immunization program in Africa in 2010. The program was successful and led to a 99.8% reduction in risk of developing meningitis [30]. Two years after introduction of the vaccine, a reduction in serogroup A carriage was discovered, indicating the establishment of herd immunity [31]. Vaccine development against serogroup B has been a challenge because the polysaccharide imitates glycoprotein adhesin in the human brain. Vaccination against the capsule could induce autoimmunity or disturb physiological function in the brain [32]. With reverse vaccinology, five conserved surface-exposed have been identified and combined in a new vaccine, rMenB: Neisseria adhesin A (NadA), factor H-binding protein (fHbp), neisserial heparin-binding antigen (NhbA), GNA2091 and GNA1030 [29]. A new vaccine, 4CMenB (Bexsero), was later developed, which contains the same components as rMenB in addition to outer membrane vesicles to increase strain coverage. 4CMenB has been reported to provide good protection and is approved for immunization in at least 39 countries [33]. In addition to 4CMenB, the bivalent vaccine rLP2086 (Trumenba) based on two lipidated variants of fHbp has also been developed and approved in the United States [34]. The availability of two serogroup B vaccines and the tetravalent vaccine against serogroups A, C, Y, and W

6 might be a major breakthrough towards the elimination of meningococcal disease worldwide [33].

1.5 Epidemiology

N. meningitidis is one of the most common cause of meningitis, with 500,000-1,200,000 cases of invasive meningococcal disease reported yearly worldwide, resulting in 50,000-135,000 deaths. The distribution of cases varies among different geographical areas and seasons. Sub-Saharan Africa is often called the “meningitis belt” and has the highest number of cases (up to 10-1,000 cases per 100,000 inhabitants during epidemics). As of 2009, serogroup A had been dominant in Africa and responsible for 10-20 cases per 100,000 inhabitants in dry seasons [18]. Since the introduction of the MenAfriVac campaign in 2010, however, the of serogroup A infections in the meningitis belt have decreased dramatically. A 57% reduction in overall cases has been estimated, along with a 60% reduction during winter when disease risk is highest. Instead, serogroups C, W, and X emerged in 2010. Outbreaks caused by serogroups W and C have occurred in 2012 and 2015 [35]. Unpredictable epidemics occasionally occur in South Africa, yet no epidemics have been reported in Northern Africa so far. Serogroup A is also responsible for most epidemics in Asia, although data from the continent are limited [17, 36]. In the developed world, cases of meningococcal disease are relatively uncommon (≤ 5 per 100,000 inhabitants) with serogroups B, C, and Y, mainly occurring sporadically [36, 37]. In 2011, 0.77 cases per 100,000 inhabitants were reported in 29 European countries, which were caused by serogroups B (73.6%), C (14.4%), and Y (8.2%) [18]. With introduction of the MenC vaccine program, the disease rate caused by serogroup C has declined. However, the number of cases caused by other serogroups has rapidly increased [36]. For example, in Sweden, the incidences of meningococcal disease have been quite stable, and serogroup B and C have been responsible for a majority of cases. A clear increase in the number of cases in serogroup Y has been observed in 2009-2013, and serogroup W emerged in 2015. In addition, meningococcal disease caused by serogroup W has increased in England, Wales, Finland, the Netherlands, and Australia

7 [16, 38-41]. It is indisputable that the devastating diseases caused by N. meningitidis can be contained by the use of effective immunizations.

8 Chapter 2: Factors involved in N. meningitidis colonization and virulence

To cause an invasive disease and survive, N. meningitidis must be able to adapt to different environmental challenges in the upper respiratory tract, bloodstream, and the cerebrospinal fluid (CSF). As a result of constant selective pressure in the environment, a number of strategies have developed to escape the host immune system. Meningococci are naturally competent and can therefore easily acquire new traits. The major virulence factors include the polysaccharide capsule, lipopolysaccharide, type IV pili, and opacity proteins [42] (Figure 1). It is a challenge to identify and define meningococcal virulence factors since the majority of the designated genes are also present in the genome of the commensal Neisseria, with the exception of the capsule and the opacity protein Opc. Therefore, it is suggested that sequence variation and gene regulation are linked to invasive disease rather than the presence of certain virulence genes [43, 44]. N. meningitidis has developed mechanisms to alter the antigenicity of its surface components. can arise through recombination, which alters particular antigens or levels of biosynthesis genes that can modify the antigens. The gene expression of surface components can also be controlled by phase variation, in an on/off mode through slipped strand mispairing or reversible insertion of transposable elements [42]. This chapter describes the structure and function of the major virulence factors of N. meningitidis that contribute to colonization and virulence. Furthermore, the present knowledge of the two-component systems encoded by meningococci is summarized. N. meningitidis depends on numerous virulence factors for successful colonization. However, a detailed description of these factors is beyond the scope of this thesis. They include fHbp, porins (Figure 1), and novel adhesins like NadA, adhesion and penetration protein (App), Neisseria hia/hsf homologue (NhhA) and meningococcal serine proteinase A (MspA) [9, 45].

9 IM

PP

OM Opa Opc Porin

LPS Cap Type IV pili

Figure 1. Major virulence factors on N. meningitidis surface. IM, inner membrane; PP, periplasm; OM, outer membrane; Cap, capsule; LPS, lipopolysaccharide; Opa and Opc, Opacity proteins. Adapted from [46, 47]. This illustration is not drawn to scale.

2.1 Polysaccharide capsule

The polysaccharide capsule is one of major meningococcal virulence factors. Invasive disease caused by strains lacking the capsule is very uncommon, and only a few cases have been reported in healthy individuals [48-50]. The polysaccharide capsule is not expressed by N. gonorrhoeae. Meningococci are thought to have acquired the capsule from Pasteurella through [51]. As mentioned previously, only six of the 13 total known meningococcal serogroups are responsible for invasive disease [11]. Four of the disease-associated serogroups produce capsules made of sialic acid (N- acetylneuraminic acid). Polysialic acid is the main component of the capsule expressed by serogroups B and C. Sialic acid is connected to D- in serogroup Y and to D-galactose in serogroup W. The capsule consists of N- acetyl-D-mannosamine-1-phosphate in serogroup A and N-acetyl- glucosamine-1-phosphate in serogroup X [17]. The resemblance of the capsular structure between serogroups allows capsule switching through horizontal gene transfer, which is a strategy to

10 evade antibody-mediated immunity [52]. The main advantages of capsule- expressing strains are the different strategies to escape the immune system. Human cells express sialic acids on their surfaces, and by using the mechanism of molecular mimicry, meningococci can use this to escape complement activation [32]. The capsule protects meningococci from opsonization, , and bacteriolysis, which mainly occurs through inhibition of the complement system [53-56]. The capsule can also contribute to antimicrobial peptide resistance and enhance intracellular survival [54, 57, 58]. The capsule can also influence adhesion and biofilm formation. Modifications in the capsular structure and the ability to hide membrane-bound adhesins can contribute to altered adhesion and facilitate the invasion of host cells [59-64]. Because the meningococcal capsule plays diverse roles, its expression must be tightly regulated. Serogroup switching mediated by transformation and phase variation either enables meningococci to change the structure of the capsule or affects its expression [52, 64-67]. Furthermore, the capsule expression can be regulated transcriptionally. Upon host cell contact, the response regulator MisR and the transcriptional regulator CrgA (Contact regulated gene A) have been shown to downregulate capsule biosynthesis [62, 68, 69]. Capsule expression can also be regulated by thermosensors located upstream of the capsule biosynthesis gene cssA [70].

2.2 Lipopolysaccharide

The main determinants of meningococcal disease severity and mortality are the circulating levels of endotoxin, which is also called lipopolysaccharide (LPS) [71]. Meningococcal LPS is similar to enteric LPS and composed of lipid A connected to core oligosaccharides. However, the lack of O-antigen repeats makes meningococcal LPS unique [17, 72]. The lipid A endotoxin is the main contributor of meningococcal toxicity by upregulation of inflammatory mediators through Toll-like receptor 4. Changes in the lipid A structure have been shown to affect inflammatory responses [73, 74]. Lipid A is connected to a diheptose through KDO (3-deoxy-D-manno-2- octulosonic acid). The number and composition of sugars extending from heptoses can vary, mostly due to phase variation of lgt genes encoding glycosyltransferease. The variability can result in antigenic diversity and

11 affect meningococcal virulence [72]. Based on the variation in LPS, meningococci are divided into 12 immunotypes, although a few of them (L3, L7, L9) predominate among disease isolates [75]. Similar to the capsule, sialic acid can be added to the epitope of the meningococcal LPS. Disease-associated meningococcal strains contain an LNnT (lacto-N-neotetraose) acceptor for sialic acid that is linked to heptose I on the LPS. Sialic acid can be either synthesized or acquired from the host and transferred to LPS by α-2,3-sialyltransferase (Lst) [72]. Both sialyated and nonsialyated LNnT have been shown to play a role in molecular mimicry [76]. The role of sialyated LPS in resistance against complement in N. gonorrhoeae has been clearly described [77-79]. Meningococcal studies on LPS sialyation have given conflicting results. It has been shown that the level of sialyation masking the LNnT epitope contributes to resistance to complement-mediated killing [80-82]. In contrast, other reports have shown that mutants defective in LPS sialyation do not increase in serum sensitivity [83, 84]. However, meningococcal LPS has been shown to contribute to resistance against antimicrobial peptides and neutrophil extracellular traps (NETs) [57, 85, 86]. In addition to modification by sugars and sialic acids, lipid A can be modified with phosphoethanolamine (PEA), which promotes the adhesion of meningococci to both epithelial and endothelial cells [87]. Impaired adherence and invasion of host epithelial cells in LPS-deficient strains have also been reported [88, 89].

2.3 Type IV pili

Type IV pili (Tfp) are the most widely used factor for bacterial attachment and the only type of pili expressed by both Gram-positive and Gram- negative bacteria [90]. Tfp are the most essential for initial adherence in encapsulated meningococci. Furthermore, Tfp play an important role in motility, aggregation, biofilm formation, natural transformation, and other functions that promote survival within the host [91, 92]. Of the 23 proteins that have been associated with Tfp, 15 are required for successful biogenesis, and seven are involved in fine-tuning its function [93, 94] (14 of the Tfp-associated proteins are shown in Figure 2). Tfp are flexible 6-nm-thick filaments that can extend several micrometers from the outer membrane. The predominant subunit PilE is first synthesized

12 as a prepilin precursor protein with a N-terminal signal peptide that is translocated upon recognition across the inner membrane by the Sec machinery [90]. Tfp are divided into two subgroups according to the length of both the leader peptide and the mature protein into short Type IVa and long Type IVb. N. meningitidis and N. gonorrhoeae contain Type IVa pili, which are further grouped into class I and class II [95, 96]. After translocation of prepilin across the inner membrane by Sec machinery, the prepilin is cleaved by prepilin peptidase PilD. Once cleaved, pilus units are assembled and transported through the outer membrane. These processes are dependent the cytoplasmic ATPase PilF and the PilQ secretin [90, 97, 98]. PilM, PilN, PilO and PilP are also required for successful Tfp assembly, but their functional roles have not been completely determined [94]. Moreover, PilW is important for Tfp stability and functionality, but not for assembly [99]. PilT is another ATPase, that is present in both the cytosol and the cytoplasmic membrane [100]. PilT is not involved in Tfp biogenesis but is essential for twitching motility through its ability to retract the Tfp [101]. PilT-deficient mutants are hyper-piliated, hyper-aggregative, non-motile, and have lost their natural competence [101, 102]. Furthermore, PilT mutants have lost the ability to detach from aggregates in later stages of infection, which enables close contact with host cells [102]. PilT2 and PilU are two other ATPases that are expressed by Neisseria and are highly conserved among species expressing Tfp. Although not fully characterized yet, reports on mutants deficient in PilT2 and PilU suggest they have the capacity to fine-tune adherence and motility [93, 103-105]. The Tfp-associated protein PilC is located both at the tip of the filament and on the outer membrane and can function as an adhesin [106-110]. Two homologous variants of PilC are expressed: PilC1 and PilC2 [111]. The PilC genes are expressed independently. Three promoters control PilC1 expression, and one controls PilC2 expression [112]. Expression of PilC is subject to phase variation, and at least one variant is essential for successful Tfp biogenesis as mutants deficient in both of them are non-piliated. PilC1 is required for Tfp-mediation adhesion, but not PilC2 [107, 110, 111]. The role of PilC1 in meningococcal adhesion is likely the basis for two additional PilC1-specific promoters that are responsible for host-cell induced transcriptional upregulation [113]. However, both variants of gonococcal

13 PilC play a role as adhesins, and meningococcal PilC2-mediated adhesion to the epithelial cell line ME180 has been described [107, 114].

Cap

PilC PilW PilQ OM

ComP PilV PilX PilE PP M, N, O, P

PilD IM

PilF PilT

ATP ATP ADP+Pi ADP+Pi Elongation Retraction

Figure 2. Illustration of the type IV pilus (Tfp) machinery in pathogenic Neisseria. IM, inner membrane; PP, periplasm; OM, outer membrane; Cap, capsule. The major subunit PilE and minor ComP, PilV, and PilX are processed by PilD. The ATPase PilF transports the processed units through the secretin PilQ in the OM with help from PilM, PilN, PilO and PilP. PilC functions as an adhesin at the tip of the Tfp and in the outer membrane. PilW is important for Tfp stability. The ATPase PilT is responsible for Tfp retraction. Adapted from [115]. This illustration is not drawn to scale.

As mentioned earlier, Tfp are a versatile virulence factor involved in a number of processes, and therefore, its expression and properties must be modulated. Tfp are one of the most variable virulence factors in meningococci due to their phase and antigenic variation. To undergo antigenic variation, several promoterless pilS sequences must be present in the genome. The pilS and pilE sequences share homology and can be exchanged by RecA-mediated recombination, resulting in variable versions of PilE C-termini. Nonpiliated strains can also result from this event [116-

14 118]. Class I Tfp, including those in N. gonorrhoeae, can undergo antigenic variation at high frequency, but class II Tfp lack this ability [95, 119-121]. Post-translational modifications on PilE can also influence Tfp functions. Serine on PilE can be modified by phosphocholine (PC), phosphoethanolamine (PE), O-linked glycosylation and phosphoglycerol (PG) [122]. Class I Tfp only carry only one glycosylation site at serine 63, and the lack of O-linked glycosylation results in increased piliation and enhanced adherence to epithelial cells [123]. It was recently shown that class II Tfp contain up to 5 glycosylation sites, which might make up for the lack of antigenic variation. Deficiencies in glycosyltransfereases can alter proper pilus assembly, aggregation, and meningococcal adherence to host cells [124]. A lack of modification on serine 68 by PC or PE affects the bundle formation of Tfp but there are no detectable effects on the function [125]. Lastly, the addition of PG to serine 93 changes the charge of the Tfp fiber and blocks bundle formation, which results in the detachment of bacteria from aggregates [5]. Low-abundance pilins or minor pilins can be incorporated into the Tfp filament and modulate its properties. Minor pilins share sequence identity with PilE and are cleaved by PilD [93] (Figure 2). Tfp play a key role in bacterial aggregation caused by anti-parallel interactions between pili. Meningococcal interaction with host cells is enhanced by an increase in bacterial aggregation [102, 126, 127]. The minor pilin PilX can mediate aggregation by connecting Tfp together through its surface-exposed D- region, which resembles a hook. Bacteria aggregate in PilX/PilT-deficient strains, indicating a role of PilX in counteracting PilT retraction of the Tfp [126, 128]. Another minor pilin, PilV, is involved in the induction of cytoskeleton rearrangement in endothelial cells. Cortical plaques are formed after the recruitment of cellular components beneath bacterial aggregates and provide protection from shear stress [129, 130]. Additionally, PilV plays a role in epithelial and endothelial cell invasion [131]. However, a recent study indicated that PilX and PilV are not incorporated in the Tfp filament but are present in the periplasm and participate in pilus biogenesis, thereby affecting the number of surface-exposed Tfp. Subsequently, the Tfp-mediated function can be modulated by a change in the number of surface-exposed filaments [132].

15 One mechanism that contributes to the fast adaptation of Neisseria is the ability to take up external DNA and incorporate it into their genomes by homologous recombination. This function is dependent on PilE, PilT, and the minor pilin ComP [101, 133, 134]. Neisseria species contain multiple copies of short DNA Uptake Sequence (DUS) in their genomes, which play a protective role against foreign DNA and are recognized and favored during transformation. ComP plays a role as a surface-exposed DNA receptor on Tfp and controls transformation both dependently and independently of the DUS [135-137].

2.4 Opacity proteins

Capsule and Tfp are downregulated upon initial adhesion to host cells. Consequently, surface-exposed outer-membrane proteins are uncovered and can play a role in host-cell interaction [69]. The opacity proteins Opa and Opc are phase-variable outer-membrane proteins with an important role in the host-cell interaction of unencapsulated meningococci. However, they do not play important roles in initial adhesion [138-140]. The meningococcal genome can encode up to three to four Opa genes and the gonococcal genome can encode up to 12. Opa proteins are composed of membrane-spanning domains that form a β barrel containing four surface loops, and two of them are hypervariable and give rise to the variation between Opa proteins. The hypervariable domains determine which receptor on host cells is utilized on host cells. Opa proteins are divided according to receptor specificity into two major classes: heparansulphate-specific OpaHS and carcinoembryonic (CEACAM/CD66)-specific OpaCEA [141]. OpaHS can also interact and bind to the extracellular matrix [142-144]. Opa proteins can mediate both intimate adhesion and invasion into epithelial, endothelial, and phagocytic cells by turning on the cellular signaling networks needed for bacterial internalization [141, 145]. Meningococcal-specific Opc is encoded by one gene, and is antigenically distinct from Opa proteins. Opc also plays a role in intimate adhesion and invasion by the interaction of heparansulfate, fribronectin, and vitronectin with endothelial and epithelial cells. Thus, Opc can be considered a functional homolog of OpaHS [138, 146-148].

16 2.5 Two-component regulatory systems

Bacteria use a two-component system (TCS) to respond to changes in the environment. Generally, the TCS comprises a membrane-bound sensor histidine kinase and a response regulator. In response to environmental stimuli, the sensor autophosphorylates and transfers a phosphoryl group to the regulator. The phosphorylation results in a conformational change and the regulator can control expression of target genes by its DNA-binding activity. Besides, post-translational modifications can also modulate the sensor and regulator [149]. N. meningitidis is known to encode only four TCS [150, 151]. The NarP/NarQ system in pathogenic Neisseria regulates denitrification and thereby supports growth in microaerobic conditions. The NarP/NarQ system is activated by an increase in the environmental nitrite concentration. Under microaerobic conditions, fumarate and nitrate reductase regulator (FNR) is activated, and together with NarP, it can induce the transcription of aniA, which encodes nitrite reductase. AniA can thus convert nitrite into nitric oxide (NO). In the presence of NO, activated nitric oxide reductase (NorB) catalyzes the conversion to nitrous oxide [152-156]. The role of NarP, AniA, and NorB in adaptation to low oxygen also plays also an important part in the biofilm formation of pathogenic Neisseria [157, 158]. The TCS NtrY/NtrX also regulates respiratory enzymes [159]. A transcriptomic analysis revealed that the deletion of the ntrX gene in gonococci changed the expression of 11 genes. Among the downregulated genes were aniA, norB, ccoP and ccR, which all encode for respiratory chain components [159]. The growth of the gonococcal ntrX mutant in aerobic and anaerobic conditions was not affected, but the number of dead bacteria within a biofilm was relatively high in the sub-stratum area, where the aniA expression has been shown to be upregulated [159, 160]. The signals that activate NtrY/NtrX have not been reported. The most studied TCS in meningococci is MisR/MisS (PhoP/PhoQ). The first reports suggested that MisR/MisS in meningococci, like its homologue in Salmonella typhimurium, is able to sense and adapt to changes in magnesium levels [161, 162]. These findings could not be repeated in a serogroup B strain, however, suggesting that this mechanism might vary between strains [163]. It is becoming more evident that MisR/MisS plays a

17 role in host-cell colonization [164, 165]. The TCS is upregulated upon host- cell contact and regulates the expression of 14 genes of the REP2 regulon that participate in the adaptation to growth on host cells [165-167]. MisR/MisS is required for full virulence in a murine model and is important in the regulation of genes involved in adhesion, capsule synthesis, iron acquisition, protein folding, and resistance to cationic antimicrobial peptides by affecting the LPS structure [165, 168-170]. In gonococci, MisR/MisS is also required for full virulence in a mouse model and plays a role in resistance to cationic antimicrobial peptides as it regulates genes associated with the maintenance of membrane integrity [171, 172]. The last and least understood TCS is PilR/PilS. In , the TCS is known to regulate the transcription of pilA, which encodes the major subunit of Tfp [173]. However, this is not the case in Neisseria, and it is suggested that PilR/PilS has evolved to become non- functional in N. gonorrhoeae [174]. No transcriptomic analysis of PilR/PilS has been reported in pathogenic Neisseria.

18 Chapter 3: N. meningitidis colonization of the nasopharyngeal epithelium

N. meningitidis is defined as an epicellular rather than extracellular bacterium since it can tightly adhere on the cell surfaces, induce host-cell membrane protrusions, and has the chance of an intracellular lifestyle [175]. Initial attachment and close contact to epithelial cells in the nasopharyngeal mucosa are crucial steps for meningococcal pathogenesis. Meningococci have evolved multiple strategies and virulence factors for efficient colonization [42].

3.1 The nasopharyngeal mucosa

The airway epithelium is the first line of defense, and pathogens encounter several challenges during colonization of the upper respiratory tract. The tightly connected epithelial surface of the nasopharynx is embedded in a mucous membrane. One of the most effective defense mechanisms against inhaled pathogens in the upper respiratory tract is mucociliary airway clearance. The membrane-bound mucus can trap particles, including pathogens, and clear them easily by frictional forces. Thus, tight adhesion to the epithelial surface and resistance to shear stress are prerequisites for bacterial colonization in the nasopharynx [176, 177]. The mucus not only entraps bacteria, it is supplemented with a number of compounds such as antimicrobial peptides, lysozyme, (ROS), and IgA antibodies [178]. Furthermore, bacterial components such as LPS, and surface proteins can activate pattern- recognition receptors expressed by epithelial cells. This activation leads to the production of pro-inflammatory effectors that activate and recruit professional phagocytes such as neutrophils and to the site of infection. The release of antimicrobial peptides by epithelial cells can also attract phagocytes [73, 179]. Examples of strategies utilized by meningococci to overcome host defenses in the mucosa are the expression of the polysaccharide capsule, LPS sialylation, IgA protease, ROS detoxification, downregulation of the complement system, and inhibition of

19 lysozyme maturation [32, 56, 81, 180-185]. In addition to the antimicrobial activity of the mucosa, bacteria that colonize the nasopharynx must be able to adapt to nutritional availability, microbiota competition, changes in temperature, and oxygen levels [178].

3.2 Initial adherence and microcolony formation

The initial adherence of encapsulated meningococci to host cells is mediated through Tfp and PilC adhesin [107, 140] (Figure 3). The surface-expressed complement receptor CD46 (cluster of differentiation 46) has been suggested as a target on epithelial cells, although attachment in a CD46-independent manner has also been reported [186-189]. Furthermore, meningococci can bind to transmembrane protein CD147 (cluster of differentiation 147), epithelial -activating factor receptor (PAFr), and β-adrenergic receptor on host cells in a Tfp-dependent manner [190-192]. Environmental temperature can influence the level of meningococcal adhesion. A decrease in temperature increases the expression of proteins that promote attachment to host cells [193]. After initial adherence, meningococci form PilX-dependent spherical aggregates called microcolonies on the cell surface [126, 194] (Figure 3). Meningococcal microcolonies were recently described as viscous structures that can readily adapt their shape to different environments [195]. PilW contributes to bacterial aggregation [99]. In addition, proteins that have not been associated with Tfp can negatively regulate aggregation. The surface- exposed Neisseria anti-aggregation factor A (NafA) is upregulated upon contact with host cells. Deletion of this gene in meningococci results in a hyper-aggregative and hyper-adhesive strain with alteration in the PilE protein levels [196]. Meningococcal adherence promotes rapid remodeling of the host-cell membrane and cortical plaque formation around the microcolonies, which protects them from shear stress [129] (Figure 3). Membrane protrusions form on the host cells as a result of the recruitment of molecular linkers of the ezrin/radixin/moesin family (ERM) and numerous membrane-associated proteins like CD44, EGFR and ICAM-1 underneath the microcolonies [197]. The presence of Tfp, PilT, either variant of PilC, and the minor pilin PilV is essential for the induction of the host-cell reorganization [102, 129, 197].

20 Interestingly, an 8-kb meningococcal disease-associated island has been discovered in invasive clonal complexes. The island encodes for a filamentous prophage called MDAΦ, which is secreted through the PilQ secretin [198, 199]. A role of MDAΦ prophage in the adherence to epithelial cells was recently established. The MDAΦ prophage does not exert adhesion itself but promotes bacterial aggregation by prophage bundling formation that contributes to shear-stress resistance [200]. Pathogenic Neisseria contain specific repeats called REP2 in the promoter of a group of genes [151]. Upon host-cell interaction, the activation of the TCS MisS/MisR affects the expression of 14 REP2-associated genes [165]. Among the genes upregulated are pilC1 and a transcriptional regulator crgA [113, 201]. The upregulation of pilC1 promotes stronger initial adhesion to the host cells [113]. However, in later stages of attachment, the role of CrgA as a gene regulator plays an important part in establishing intimate adhesion to host cells [201].

3.3 Microcolony dispersal and intimate adhesion

Following initial adhesion and several rounds of divisions, meningococci start to detach from microcolonies in a PilT-dependent manner and form a bacterial monolayer in close contact with host cells [102] (Figure 3). At this phase, membrane protrusions are no longer present on the host cell [102, 202]. REP2-associated phosphoglycerol transferase (PptB) is upregulated upon attachment to host cells. The addition of phosphoglycerol to serine 93 on PilE by PptB disrupts pili interactions and facilitates bacterial detachment from microcolonies [5]. In N. gonorrhoeae, the stability of the microcolony is affected by the amount of oxygen present. Depletion of oxygen and thus a lack of proton motive force induce microcolony dispersal independent of the host cells [203]. After microcolony detachment, meningococci depend on PilT-mediated retraction of Tfp to form close contact with host cells, and in this stage Tfp and the capsule are downregulated [62, 102, 204] (Figure 3). CrgA is thought to play a role in intimate adhesion through the downregulation of pilC1, pilE, and the capsule synthesis genes sia. This process reveals membrane proteins that are essential for intimate adhesion [69, 201]. However, another study failed to show CrgA-mediated upregulation of pilE,

21 pilC1 and sia genes [205]. Once in proximity to host cells, meningococci can adhere tightly on the epithelial surface or become internalized, which are both mediated by outer-membrane proteins Opa, Opc, and LPS [60, 89, 138]. Bacteria have been obtained inside cells, and the cell layer integrity is not damaged, which suggests that meningococci follow a transcellular route across the epithelial layer [204, 206, 207]. Moreover, the adhesins NadA, App, MspA, and NhhA play a role in intimate host-cell interaction at the epithelial surface [208-211].

Neisseria meningitidis

Host receptors

Figure 3. N. meningitidis colonization of the nasopharyngeal mucosa. Initial adherence of single bacteria or small aggregates is dependent on Tfp and the adhesin PilC. Once adhered, bacteria proliferate, form microcolonies, and induce cytoskeletal rearrangement of the host cell underneath. Later, PilT-mediated retraction of the Tfp and post-translational modifications of PilE promote the dispersal of bacteria from microcolonies. This facilitates transmission to a new host or transcellular invasion of the epithelial layer. Partially adapted from [5].

22 3.4 Biofilm formation

Bacteria can have two different lifestyles: single-cell planktonic bacteria and multicellular communities. Microbial communities called biofilms are defined as surface-adhered multicellular aggregates encased in an extracellular polymeric substances (EPS) matrix, which is mainly composed of polysaccharide, protein, and DNA [212]. The major advantages for bacteria in a biofilm are the ability to grow in a nutrient-deprived environment and protection from antimicrobials, host immunological responses, and physical stresses [212, 213]. These protective advantages of biofilms pose a great challenges in clinical treatment, especially in wound infections and the contamination of medical devices [214]. There is limited evidence of the existence of N. meningitidis biofilms in vivo. However, microcolonies have been detected by using immunohistochemistry in tonsillar tissue and in both brain and skin sections from patients with meningococcal disease [215-217]. Biofilm formation is the likely explanation for the capability of N. meningitidis to form long-term colonies in humans without the appearance of symptoms [217-219]. Numerous studies have shown the ability of N. meningitidis to form biofilm on surfaces and epithelial cells in vitro [63, 220-225]. The first and one of the most important steps in the formation of biofilm is the attachment to a surface. At first, the attachment is reversible and it can be facilitated by physical appendages like flagella, pili, and fimbriae. During this stage, the bacteria can form loose aggregates but are able to return back to single-planktonic bacteria [226]. The initial adherence of encapsulated meningococci to host cells is dependent on Tfp [140]. Nonpilated mutants deficient in pilE and pilQ can form a biofilm on a plastic surface in a static condition. However, the biofilm is flat and is not as thick compared to that of the wild-type strain. This suggests that the presence of the Tfp is not required, but its absence can alter the architecture of the biofilm [63, 222]. Nonaggregative pilX mutants also form flat biofilms [222]. It has been suggested that defects in the twitching motility rather than the absence of aggregates is responsible for the altered structure of the biofilms formed by mutants deficient in pilE and pilX [63, 222]. Although pili are not essential for biofilm formation, they can strengthen the resistance to shear forces [129]. In N. meningitidis, the presence of the polysaccharide capsule hinders

23 biofilm formation. The capsule both reduces the meningococcal surface hydrophobicity and hides surface adhesins, which are both highly important in biofilm formation. Most studies have therefore been performed using unencapsulated strains [63, 222, 227]. Biofilm formation on human airway epithelial cells by encapsulated strains have been reported, suggesting that this inhibition might not occur in vivo [220]. LPS can also negatively affect biofilm formation [63]. The progression from reversible to irreversible attachment is required for biofilm formation and depends on the production of EPS and surface proteins by the bacteria [226]. The EPS can constitute up to 90% of the biofilm, with bacteria comprising only 10%. However this can vary depending on the bacterial species involved [213]. Mature biofilms can be complex and contain channels that help in the distribution of nutrients throughout the biofilm. Environmental factors like carbon sources, shear- stress, microbial competition and temperature can influence the structure of the EPS matrix and therefore affect the maturation of the biofilm [226]. Pathogenic Neisseria do not produce extracellular [91]. However, extracellular DNA (eDNA) is known to be a major component in N. gonorrhoeae biofilms. Gonococcal eDNA can be released by outer- membrane blebs or through bacterial autolysis [160, 228-231]. Additionally, gonococci are able to secrete DNA actively through a Type IV secretion system encoded by the gonococcal genetic island (GGI) [232]. Only a few meningococcal strains contain GGI, and its role in pathogenesis is unclear and unlikely to contribute to DNA release. The GGI expressed by meningococci do not encode for peptidoglycanase, which is essential for the gonococcal DNA secretion [233, 234]. Like gonococci, frequently carried meningococcal strains are highly dependent on eDNA for biofilm formation. In the early stages of meningococcal biofilms, the release of eDNA by cell lysis is dependent on membrane-bound lytic transglycosylases A and B (MltA and MltB) and cytoplasmic N-acetylmutamyl-Lalanine amidase (AmpD) [221]. In later stages of meningococcal biofilms, the outer membrane phospholipase A (OMPLA) plays a role in eDNA release. eDNA both stabilizes the microcolonies and provides mechanical protection. eDNA- independent biofilm formation has also been reported in meningococci. Clonal complexes like ST8 and ST11 with low carrier rate but high

24 transmission rates are not dependent on eDNA. Consequently, the biofilm is formed more loosely and is sensitive to shear forces [221]. Based on these findings, the meningococcal biofilm formation can be identified by two different phenotypes. The stable settler type is dependent on eDNA and can colonize for long periods of time, while the loose spreader phenotype is independent of eDNA, it results in higher transmission rates, and it is more linked to invasive disease [221]. However, it is possible that eDNA- independent strains interact with eDNA-dependent strains to form more stable biofilms [235]. Various surface-exposed proteins have been shown to contribute to the formation of meningococcal biofilms by binding to eDNA, such as NhbA and autotransporters IgA protease, AutA, and AutB [224, 236, 237]. The autotransporter NalP governs the composition of the outer membrane and the secretome and can proteolytically cleave the eDNA-bound molecules and consequently reduce the level of biofilm formation [224]. Interestingly, alteration in the protein level of NalP and NhbA upon a reduction in temperature promotes meningococcal adhesion and biofilm formation [193]. The two-part secretion system HrpB-HrpA has been reported to play a role in meningococcal biofilm formation on both epithelial cells and collagen- coated glass. The adhesin HrpA is suggested to play a role in the maintenance of the biofilm structure [238]. Both transcriptomic and proteomic analysis have revealed that anaerobic respiration plays an important role in biofilm formation in pathogenic Neisseria [158, 239-241]. Neisseria relies on two central enzymes of the denitrification pathway for anaerobic respiration, AniA and NorB [242, 243]. FNR is mainly responsible for regulating aniA expression upon oxygen starvation. However, for full activation, aniA depends on the regulation of TCS NarQ/NarP in the presence of nitrite [152, 153]. The expression of norB is induced in the presence of NO [244]. The deletion of narP in meningococci results in defects in biofilm formation [157]. This has also been observed for mutants deficient in aniA and norB in gonococci [239]. Eventually, the EPS matrix can be degraded and individual bacteria or aggregates can detach from the biofilm and colonize new surfaces. Changes in the environment or limitations of nutritional availability within an expanding biofilm can result in biofilm dispersal, which allows bacteria to escape nutrient and oxygen starvation. Both physical and chemical factors

25 can be involved in biofilm dispersal, such as cell signaling molecules, EPS- degrading enzymes, NO, physical forces and oxygen and nutrient availability. Dispersal can also help in the remodeling of the biofilm architecture [245]. There is very little data on the dispersal of meningococcal biofilms. In N. gonorrhoeae, the expression of a thermonuclease Nuc involved in DNA digestion can play a role in biofilm dispersal and remodeling [246]. The polyamine spermine reduces the level of gonococcal biofilm formation but does not induce dispersal in pre-existing biofilms [247]. Furthermore, it has been shown that the expression of NagZ, a β-N-acetylglucosaminidase involved in peptidoglycan recycling, plays a role in gonococcal biofilm dispersal [248]. NO has also been shown to play an important role in gonococcal biofilm formation and dispersal. The accumulation of NO in NorB-deficient gonococci leads to biofilm dispersal. However, the timing and dosage of NO donation are important factors in the biofilm formation. In early stages, low concentrations of NO can induce dispersal, while in later stages when the anaerobic respiration has started, NO can support growth [158, 249]. Interestingly, Neisseria subflava and several oral pathogens can sense changes in temperature and disperse from biofilms towards higher temperature [250].

26 Chapter 4: Lactate and bacterial virulence

Sustained colonization and survival within the host require assimilation of nutrients essential for growth. Pathogens adapt to specific niches within the host that meet their nutritional requirements. Nutritional limitation is a challenge for bacteria in the nasopharyngeal mucosa. The host defense mechanism includes the restriction of nutrients such as sugars, iron, amino acids, and peptides [251-254]. The carbon energy sources of N. meningitidis are restricted to glucose, pyruvate, and lactate. These carbon sources are available in many sites of the body, including the saliva, blood circulation, and CSF, but at variable concentrations. Glucose is the main carbon source in the blood and CSF. Lactate is predominant in mucosal environments colonized by lactic bacteria, such as the nasopharynx. Moreover, lactate and pyruvate are the main carbon sources within phagocytic cells [255, 256]. Lactate utilization by meningococci is a major advantage that both supports long-term colonization in the mucosa and evasion of the host immune system [257].

4.1 Lactate utilization in pathogenic Neisseria

Lactate can be found in two stereoisomers: L-lactate and D-lactate. Humans can produce only L-lactate but are colonized by microorganisms that are able to produce both isomers [258]. Lactate permease (LctP) is needed to take up lactate in N. meningitidis [259, 260]. Once taken up, lactate is oxidized to pyruvate, which is catalyzed by lactate dehydrogenases (LDH). Three different LDHs have been identified in pathogenic Neisseria (Figure 4). One cytoplasmic D-LDH (LdhA), which is a NAD+ dependent, catalyzes the reversible conversion of pyruvate into D-lactate. Respiratory membrane- bound D-LDH (LdhD) and L-LDH (LldA) are restricted to the conversion of lactate to pyruvate [261-263]. The amount of energy produced from lactate depends on the availability of glucose. More energy is created from lactate in the presence of glucose as pyruvate is solely converted to acetyl-CoA, which is the first product in the citric acid cycle and a precursor of fatty acids. Less

27 energy is created in the absence of glucose as lactate is also required for gluconeogenesis to produce sugar, glycerol, and ethanolamine [256] (Figure 5). OM PP IM Lactate Pyruvate

L LctP L

LldA

P

Pyruvate pool P P

LdhD

D LdhA NADH

NAD+ EC CP

Figure 4. Lactate utilization in pathogenic Neisseria. OM, outer membrane; PP, periplasm; IM, inner membrane; EC, extracellular space; CP, cytoplasm; L, L- lactate; D, D-lactate; P, pyruvate; NAD+, oxidized nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide. Adapted from [263]. This illustration is not drawn to scale.

4.2 Effect of lactate on bacterial pathogenesis

A number of studies have examined the link between lactate metabolism and virulence in pathogenic Neisseria [256, 257] (Figure 5). The first indication was the ability of N. gonorrhoeae to use host-cell-derived L-lactate to increase metabolic rate and oxygen consumption to escape neutrophil oxygen-dependent killing [264]. In addition, lactate contributes to the serum resistance of N. gonorrhoeae by stimulating LPS sialylation [265]. The same stimulating effect has been observed with pyruvate [266]. The gluconeogenesis is turned off in presence of glucose. The carbon from lactate is incorporated only into fatty acid moieties, not the glycerol and ethanolamine of membrane lipids or the of LPS [267].

28 Glycolysis Glucose Gluconeogenesis (inhibited by glucose) Phosphatidyl Glycerols Glycerol-3-P Ethanolamines

Polysialic acid Sialic acid Phospho-enol-pyruvate capsule Resistance to serum killing CMP-NANA Pyruvate Lactate NAD+ NAD+ Escape from Sialated oxygen dependent LPS LPS Fatty acids Acetyl-CoA NADH bactericidal mechanism Ethanol O Citric acid 2 Acetate cycle Energy (ATP) Energy production and metabolic stimulation

Figure 5. Lactate metabolism and its effect on virulence in pathogenic Neisseria. Lactate is oxidized to pyruvate, which can either enter gluconeogenesis (red) or be converted to acetyl-CoA. Lactate can contribute to meningococcal virulence by promoting energy production and resistance to bactericidal mechanisms. Adapted and modified from [256] and [268].

Lactate plays a role in gonococcal intracellular survival within neutrophils and during microaerobic conditions in invasion and survival within cervical epithelial cells [263]. A lack of LctP impairs the ability of N. gonorrhoeae to colonize and survive in a mouse model [259]. A LctP-deficient meningococcal strain is more adhesive to epithelial cells than wild-type bacteria, but the long-term colonization of nasopharyngeal tissue is impaired [260]. Paper I has shown that lactate stimulates the microcolony dispersal of N. meningitidis. Interestingly, lactate-induced dispersal occurred in mutants that are unable to metabolically utilize lactate. Pyruvate, a downstream metabolite of lactate, did not affect the rate of microcolony dispersal. Consistent with previous findings, the stimulatory effect of lactate was not a result of oxygen depletion due to an increase in meningococcal oxygen consumption. Altogether, this suggests a mechanism that is independent of lactate metabolic utilization [269]. Paper II showed that the lactate-induced microcolony dispersal occurred in a cell-density-dependent manner and was

29 dependent on the PilT-mediated retraction of Tfp. Additionally, the activity of PptB and an optimal environmental temperature were required for bacteria to remain dispersed. Lactate influences the virulence of several pathogens, including colonizers of the upper respiratory tract. Lactate metabolism is important for the growth of H. influenza, Staphylococcus aureus and Streptococcus pneumonia and is required for full virulence in a mouse model [270-273]. Lactate-dependent serum resistance has been reported in H. influenza. However, another report was unable to repeat this finding, which makes it unclear whether lactate contributes to serum resistance [272, 273]. A lack of LDH activity in S. aureus and Enterococcus faecalis increases sensitivity to environmental stress [270, 274]. Furthermore, in Bacillus cereus, lactate affects both fermentative growth and enterotoxin gene expression [275]. LDH activity has been associated with microcolony and biofilm formation in several pathogens. In E. coli, the deletion of D-LDH and the resulting accumulation of intracellular acetyl-coA enhanced biofilm formation. Similar effects on biofilm formation were observed with other mutations in metabolic enzymes, resulting in acetyl-CoA accumulation. This suggests a role of the acetate intermediate in the biofilm formation of E. coli [276]. It was recently reported that L-LDH and D-LDH in P. aeruginosa enable cross-feeding of lactate between aerobic and anaerobic zones within colony biofilms [277]. Moreover, pyruvate conversion to lactate by D-LDH is important for the formation of microcolonies and biofilms in P. aeruginosa [278]. Nonetheless, the deletion of respiratory D-LDH in P. aeruginosa, enhances EPS production [277]. The expression of ezymes involved in pyruvate fermentation, the citric acid cycle, and glycolysis/glucogenesis is upregulated during the biofilm growth of pathogenic Neisseria, indicating the importance of energy metabolism [158, 239-241]. Paper III showed that meningococcal biofilm formation was promoted by the deletion of ldhA, which encodes cytoplasmic D-LDH. This occurred in an eDNA-dependent manner through increased autolysis.

30 Chapter 5: Polynucleotide phosphorylase

Bacterial pathogens encounter constant changes in the environment and must thus possess a mechanism to respond quickly. Transcription and translation are the major processes that contribute to successful protein synthesis. However, RNA turnover and processing also play also an important role in regulating rapid gene expression through ribonucleases (RNases). RNases can be divided into two groups: endonucleases, which cleave RNA internally, and exonucleases, which cleave RNA at the ends of chains in either the 3´-5´ or 5´-3´ direction. Among RNases, only a few can monitor and control the RNA level needed at certain circumstances [279]. Polynucleotide phosphorylase (PNPase) is a 3´-5´ phosphorolytic exoribonuclease that is important in controlling the expression of genes in response to environmental changes, including physiological and virulence processes, which occurs through different RNA degradation processes [280]. PNPase is a highly conserved multifunctional protein and found in most species apart from yeast and archaea [281].

5.1 The degradosome

In addition to acting alone, 10-20% of total E. coli PNPase can form a multienzyme complex called the degradosome, together with RNaseE, enolase, and RNA helicase B (RhlB). The degradosome plays an important role in mRNA decay [282, 283]. In Gram-negative bacteria, degradosome- mediated RNA degradation starts with an RNase E endonucleic cleavage that generates an unprotective 3´end. The free end attracts 3´ RNases like PNPase that degrade the RNA. However, exonucleases can only degrade single-stranded RNA but with the addition of a poly(A)tail by poly(A)polymerase, PNPase can degrade double-stranded RNA [284]. Regardless of the presence of RNase, the cooperation of RhlB RNA unwinding and PNPase allows for the degradation of RNA secondary structures [285].

31 In the C-terminal, RNase E contains a noncatalytic scaffold domain with RhlB, enolase, PNPase, and RNA binding sites, in addition to a membrane- anchoring sequence. The catalytic activity of RNase E is in the N-terminal [279, 286]. The membrane anchoring sequence is responsible for the localization of the degradosome through RNase E to the inner cytoplasmic membrane. Mutations in the sequence have a huge impact on the growth of E. coli [287]. The role of enolase in the degradosome is not fully established, but it has been suggested to participate in mRNA degradation by regulating glucose metabolism in E. coli [288]. Minor components, ribosomal proteins, chaperones, polyphosphate kinase, and poly(A)polymerase can interact with the degradosome and affect its configuration and activity [279]. Nonetheless, the localization and the amount of PNPase compared to other components of the degradosome in E. coli suggest that it mainly acts alone [289].

5.2 Polynucleotide phosphorylase structure and activity

Meningococcal PNPase shares 62.55% sequence identity with E. coli PNPase, which is the most widely studied among prokaryotes [290]. In E. coli, PNPase belongs to the PDX family of exoribonucleases, which are distinguished by a release of nucleoside 5´-disphosphates products during 3´- 5´ exonuclease activity [291]. Moreover, in a high concentration of nucleotide di-phosphate, PNPase can promote 3´ exonucleolytic RNA degradation by acting as an polyadenilating enzyme [292] (Figure 6A). PNPase contributes to both the stability and degradation of small RNA (sRNA) [293, 294]. In E. coli, the pnp gene is transcribed from two promoters, and with PNPase, it can regulate its own expression at the post- transcriptional level. After a RNase III cleavage in pnp mRNA, PNPase binds its 5´-untranslated region and targets it for degradation [295]. In E. coli, PNPase is organized into a ring-like homotrimer, where each monomer from the N-terminal contains two RNase-PH domains (PH1 and PH2). These domains are connected together by an α-helical domain and two RNA binding domains (KH and S1) (Figure 6B) [282, 296]. Once bound to KH and S1, the RNA is moved into the central channel and to the active catalytic site located mostly in PH2 domain [282]. PNPase activity requires Mg2+, and its binding site is located in the PH domain [296].

32

A Polymerization Degradation Pi NDPs Pi NDPs

AAGAAA

B

N RNase PH1 α-helical RNase PH2 KH S1 C

Figure 6: PNPase activity and structure. A. PNPase is a homotrimeric enzyme that functions as both an RNA polymerase and 3´- 5´ exonuclease. NDPs, nucleotide diphosphates; Pi, inorganic phosphate. Adapted from [297]. B. Linear illustration of the domains found in PNPase of the PDX family. Prokaryotic PNPase consists of two RNase PH domains connected by α-helical domain and two RNA binding domains, KH and S1. Adapted from [298].

5.3 Role of polynucleotide phosphorylase in pathogenic bacteria

PNPase exerts a pleiotropic effect on physiology and virulence in many pathogens through gene regulation, although it is not essential for bacterial survival [280]. PNPase plays a major role in adaptating to cold temperatures in many pathogenic bacteria, including E. coli, S. typhimurium, P. aeruginosa, Bacillus subtilis, , and several species within the genus Yersinia [299-306]. PNPase is needed for proper function of the type-three secretion system (TTSS) in , Yersinia pseudotuberculosis and P. aeruginosa. A lack of PNPase in Yersinia impairs the injection of virulence factors into host cells, while it reduces TTSS expression in P. aeruginosa. PNPase-deficient strains of Yersinia are more sensitive to killing and are less virulent in a mouse model [304, 305, 307, 308]. A loss of PNPase in E. coli O157:H7 completely abolishes the production of Shiga toxin, which is the main cause of death associated with the strain [309]. Higher intracellular accumulation of tetracyclin in PNPase-deficient strains of B. subtilis increased their sensitivity to the drug [306].

33 PNPase activity is also associated with colonization in a number of pathogens. In , PNPase negatively regulates the expression of Salmonella pathogenicity islands SPI, SPII, and the spv virulence gene cluster involved in invasion and intracellular survival. The inactivation of PNPase in S. enterica results in a strain that causes acute rather than chronic infections in an in vivo mouse model [301, 310]. Adhesion and invasion are reduced in C. jejuni lacking PNPase [311]. PNPase is also required for successful colonization and biofilm formation in S. typhimurium and E. coli [309, 312, 313]. A loss of PNPase affects the twitching motility in Dichelobacter nodosus and P. aeruginosa, as wells as the swimming motility in C. jejuni and E. coli [305, 311, 312, 314]. Paper IV characterized the role of PNPase in N. meningitidis. The deletion of PNPase resulted in a Tfp-mediated hyperaggregative and hyperadhesive growth-deficient mutant with reduced virulence in a mouse model. Additionally, the deletion affected expression of 469 genes, thus indicating an important role in meningococcal gene regulation [290].

34 PRESENT INVESTIGATION

Aims

The overall aim of the studies included in the thesis was to gain mechanistic insights into the interaction of bacterial, host and environmental factors involved in the colonization and virulence of N. meningitidis.

Specific aims:

• To describe bacterial and host-derived factors that influence the dispersal of N. meningitidis microcolonies (Papers I and II).

• To examine the importance of the lactate utilization of N. meningitidis in microcolony dispersal (Paper I)

• To investigate the effect of changes in environmental temperature on lactate-induced dispersal in N. meningitidis (Paper II).

• To evaluate the effect of deficiencies in lactate metabolism in N. meningitidis on aggregation and biofilm formation (Paper III).

• To establish the role of polynucleotide phosphorylate as a virulence factor in the pathogenesis of N. meningitidis in vitro and in vivo (Paper IV).

35 Results and discussion

Paper I

Despite the high carriage rate of meningococcus, colonization rarely results in an invasive disease [12]. The adhesion to the mucosal epithelium is a prerequisite for colonization and pathogenicity. The adherence of encapsulated meningococci is dependent on PilE and Tfp-associated proteins [6]. Initial adherence is followed by the formation of bacterial aggregates called microcolonies. Bacterial detachment from microcolonies enables close interaction with host cells, which can result in invasion and transmission through the epithelial layer [5, 6]. The purpose of this study was to understand and identify factors that influence the dispersal of meningococcal microcolonies. The interaction of N. meningitidis with the host cells has been suggested to be one of the factors involved in microcolony dispersal [5]. We examined the importance of host cells by live-cell imaging. In the presence of host cells, microcolonies dispersed rapidly in a synchronized manner. However, the dispersal was delayed in the absence of host cells. Interestingly, during our infection assays, we noticed that direct contact with host cells was not essential for rapid dispersal. Furthermore, the ΔpilC1 mutant, which is deficient in host cell adherence, was not affected in terms of the timing of microcolony dispersal. This further confirms previous observation that host- cell contact is not required. To study this, we examined whether compounds secreted from the host cells or meningococci might affect microcolony dispersal. We found that conditioned medium (CM) collected from infected and non-infected host cells was able to induce accelerated dispersal of pre-formed microcolonies in liquid. This demonstrated that a host cell-derived effector molecule stimulated the dispersal of meningococcal microcolonies. The CM did not have an inhibitory effect on microcolony formation, suggesting that bacteria must form microcolonies to respond to the compound in the CM.

36 In our model, the effector molecule accumulated at stimulatory levels within 1 h of incubation of uninfected cells. Further characterization suggested that the compound was heat stable, not a protein or nucleic acid, and smaller than 3 kDa. The effector molecule accumulated over time in the absence of infectious agent, which suggests that it might be a secreted host metabolite. Lactate is a metabolic byproduct secreted by human cells [315]. Since lactate plays a role in the metabolism and virulence of N. meningitidis [256, 257], we examined its effect on microcolony dispersal. Both L- and D- lactate were able to induce microcolony dispersal at millimolar concentrations. We observed that lactate-induced dispersion was a general mechanism for pathogenic Neisseria. Microcolonies formed by the meningococcal serogroup W strain JB515 and the gonococcal strain MS11 dispersed in a synchronized manner upon the addition of lactate. To examine the importance of lactate utilization, we constructed strains deficient in lactate permease (ΔlctP) and lactate dehydrogenases (ΔldhD, ΔldhA and ΔlldA). First, we examined the ability of the mutants to use lactate as the sole carbon source. The ΔlctP mutant was unable to grow on both isomers. The ΔldhD was unable to grow on D-lactate, and ΔlldA was unable to grow on L-lactate. Despite the inability to utilize D- or L-lactate as the sole carbon source, the mutants responded by accelerated dispersal in the same way as the wild-type. This finding indicates that lactate does not need to be metabolized to induce microcolony dispersal in meningococci. Surprisingly, the ΔlctP mutant microcolonies dispersed in all conditions tested, including the control medium. Lactate utilization by pathogenic Neisseria can stimulate metabolism, which leads to increased growth rate and oxygen consumption [256]. However, our results suggest that the stimulatory effect of lactate was not due to a change in the metabolic status. There was no change or a slight increase in the ATP concentration and NAD+/NADH ratio after the induction of dispersal, which indicated that there was no effect on the proton motive force. Furthermore, oxygen depletion did not affect microcolony dispersal in DMEM. Pyruvate has a similar stimulatory effect on meningococcal metabolism to that of lactate [266]. Nonetheless, the addition of pyruvate to microcolonies did not induce dispersal. We analyzed the mRNA level of genes linked to colonization to identify meningococcal virulence factors involved in the microcolony dispersal. The

37 gene expression was quantified by qPCR after treating microcolonies with CM. Significant downregulation was observed in genes that have previously been linked to microcolony dispersal and intimate adhesion (pilE, pilC, pilT, pptB and crgA) [5, 69, 102, 165, 166, 201]. However, no changes were observed when the protein levels of PilE, PilT, PilC, PilX and PilW were examined. In summary, our results demonstrate that the microcolony dispersal is independent of host-cell contact and instead depends on an effector molecule secreted by the host, which was identified as lactate. Lactate-mediated dispersal can occur in the millimolar range. The lactate-induced response is independent of lactate dehydrogenase activity and oxygen consumption. Our data reveal a potential role of lactate as a signaling molecule in the colonization of pathogenic Neisseria.

Paper II

Paper I shows that the environmental concentrations of lactate influence the dispersal of N. meningitidis microcolonies. However, the mechanisms behind the lactate-induced dispersal were not identified. The aim of this study was to gain insight into the mechanistic process and further identify meningococcal factors that contribute to the dispersal phenotype. Paper II summarizes our findings. Paper I revealed that lactate did not prevent microcolony formation, and instead, bacteria formed microcolonies and dispersed shortly afterwards. Therefore, we focused on understanding the role of meningococcal cell density in the lactate-mediated dispersal of microcolonies. We observed that lactate-induced dispersal occurred in a cell-density-dependent manner. Higher bacterial concentration in the presence of lactate correlated with earlier dispersal. The cell-density-mediated regulation of genes is called quorum sensing. Bacteria use quorum sensing (QS) systems to alter behavior in response to changes in cell density. Autoinducer-2 (AI-2) is the only known QS molecule that meningococci can produce, and its role is not completely understood [316, 317]. We therefore hypothesized that it contributed to density-dependent dispersal. However, the deletion of the lux gene, which is

38 involved in AI-2 production, did not affect the timing of microcolony dispersal. We also examined the role of TCS in microcolony dispersal. TCS are widely used by bacteria to respond and adapt to changes in the environment. Lactate can influence TCS activity in several pathogens, including E. coli and S. aureus [318-320]. Meningococci encode for four TCS [150, 151], and we observed that three of them do not contribute to lactate sensing and dispersal: NarP/NarQ, PilR/PilS and NtrY/NtrX. Furthermore, abnormal cell morphology was detected upon the deletion of ntrX, which encodes the regulator of the NtrY/NtrX system. This suggests a role of NtrX in bacterial cell division. Unfortunately, we were not successful in acquiring a misR deletion mutant. The deletion of misR in the serogroup C strain has been reported previously, resulting in severe growth defects, especially at low magnesium levels [161, 162, 170]. This might explain our difficulties during the transformation. However, the deletion of misR in serogroup B strain does not result in growth deficiencies [163], indicating that the effects are strain dependent. Previous studies have identified meningococcal factors that contribute to the transition from initial to intimate adhesion [5, 69, 102, 201, 202]. Post- translational modifications can fine-tune Tfp activity [321]. Upon adhesion to host cells, PptB is upregulated and adds phosphoglycerol to the PilE. This results in the blockage of pilus-pilus interaction and microcolony dispersal [5]. We were interested in examining whether PptB activity is involved in lactate-induced dispersal in liquid cultures as well. The deletion of pptB did not prevent dispersal, but we observed that detached bacteria did not remain dispersed and re-entered the aggregation phase shortly afterwards. This indicates that in our settings, PptB might be important for bacteria to remain in a dispersed state and thus facilitate transmission, which is consistent with previous findings [5]. During host-cell interactions, the detachment of bacteria from microcolonies and the transition to intimate adhesion require Tfp-retraction by the ATPase PilT [102]. We showed that PilT was essential for lactate- induced microcolony dispersal. This finding also suggests that pilus retraction rather than post-translational modifications are required for bacterial detachment. PilT retraction is known to require protein synthesis

39 [322]. By using spectinomycin treatment, we also showed that meningococcal microcolony dispersal was dependent on protein synthesis. An important factor that we had not addressed yet was the environmental temperature. The range of estimated temperature in the nasopharyngeal mucosa is 32-34°C [323]. We observed that microcolony dispersal was slower and less synchronized at 32°C than 37°C. Furthermore, dispersed bacteria reformed microcolonies afterwards. Environmental temperature can thus influence meningococcal microcolony dispersal. It is tempting to speculate that microcolony dispersal and subsequent intimate adhesion could therefore be associated with elevated host body temperature. To summarize, lactate-induced dispersal depends on bacterial concentration, although QS signaling is not involved. The dispersal occurs in a PilT-dependent manner, and PptB activity prevents reaggregation. Finally, our results suggest that environmental temperature might have a profound influence whether re-aggregation occurs upon dispersal.

Paper III

Biofilms are defined as multicellular aggregates that adhere to surfaces and embed in the extracellular matrix. The ability to form microcolonies can influence the architecture of biofilms in N. meningitidis [63, 222]. Paper I demonstrated that lactate induces meningococcal microcolony dispersal. Several studies have demonstrated the impact of lactate and pyruvate metabolism on biofilm formation in pathogenic bacteria [324-326]. The role of lactate metabolism in meningococcal biofilm formation is not yet clearly understood. As lactate influenced the group behavior of meningococci, the study focused on understanding the role of lactate metabolism in biofilm formation. Paper III revealed that the deletion of D-LDH ldhA (ΔldhA) in N. meningitidis enhances biofilm formation. Since the capsule inhibits biofilm formation in meningococci [63, 222], we constructed a ΔldhA/Δcap double mutant, which showed increased biofilm formation compared to the Δcap mutant. Furthermore, ldhA complementation reduced the level of biofilm formation to the wild-type levels. Meningococcal aggregation can affect the level and architecture of biofilms [63, 222]. We therefore examined aggregation ability of the ΔldhA

40 mutant. Interestingly, the ΔldhA mutant was more aggregative than the wild- type and complemented strain. However, no change was observed in the level of adhesion and the expression of the major PilE subunit. This is surprising since there is usually a correlation between aggregation and adhesion [126, 127, 196]. Furthermore, the ΔldhA mutant exhibited increased surface hydrophobicity. The polysaccharide capsule is hydrophilic and reduces biofilm formation [63, 222], so we speculated that there would be a change in the capsule level of the ΔldhA mutant. However, the amount of capsule remained unchanged between the strains. eDNA is the major component of the extracellular matrix in a meningococcal biofilm, although eDNA-independent biofilm formation has also been reported [221]. Furthermore, eDNA can increase the hydrophobicity of the bacterial cell surface [327]. This prompted us to investigate the role of eDNA in the biofilm formation of ΔldhA. Treatment with DNase I abolished the increased biofilm formation by the ΔldhA mutant. DNase I treatment did not affect the biofilm formation of the wild- type and Δcap. This supports the finding that FAM20, which is grouped in clonal complex ST11, does not depend on eDNA for biofilm formation [221]. Meningococcal DNA release occurs through autolysis [221]. The ΔldhA mutant exhibited increased cell lysis, which suggests a reason for the enhanced biofilm formation. Elevated levels of eDNA were detected in the culture supernatants of ΔldhA compared to the wild-type, which further supports the hypothesis. The role of eDNA in the enhanced aggregation of the ΔldhA mutant was also confirmed by DNase I treatment. In an attempt to identify additional virulence factors, we examined the expression of genes that were previously linked to the biofilm formation and autolysis of N. meningitidis. Gene expression was compared between the wild-type and ΔldhA during growth in log phase cultures and biofilms. We detected the upregulation of norB in both conditions. Furthermore, the expression of narP was upregulated in log phase cultures. Both are involved in respiration in a microaerobic environment. However, no changes were detected in the expression of aniA. There was no difference in the expression of siaD, pilE and nalP during growth in the log phase, but the expression was decreased in the ΔldhA mutant compared to the wild-type during biofilm growth. Additionally, a decrease was observed in the expression of the genes

41 mltA and ampD involved in meningococcal autolysis. This is surprising since we observed an increase in cell autolysis. Therefore, the mechanism behind ΔldhA autolysis remains to be determined. It is possible that a shift in metabolic pathways might influence the observed phenotype. Studies have shown changes in metabolic pathways during the biofilm formation of pathogenic Neisseria at the transcriptional and proteomic levels [158, 239-241]. This provides evidence that changes in metabolic status may influence the formation of multibacterial communities of N. meningitidis and is a subject for further research. Altogether, we report that a lack of ldhA in meningococci leads to increased autolysis and eDNA release, which promotes bacterial aggregation and biofilm formation.

Paper IV

Rapid responses to changes in the host environment are crucial for the efficient colonization of pathogenic bacteria. PNPase is a highly conserved 3´-5´exonuclease present in both eukaryotes and prokaryotes and plays an important role in RNA degradation and stability [281]. PNPase is well studied in pathogenic bacteria and plays an important role in the adaptation to sudden environmental changes by influencing physiology and virulence [280]. The aim of this project was to characterize the function of PNPase in N. meningitidis and to examine its role in virulence. The N. meningitidis gene NMC0710 codes for a protein that shares 62.55% identity with E. coli PNPase [290]. To verify the PNPase enzymatic activity of NMC0710, a his-tagged protein was purified from E. coli and the ability to degrade RNA was confirmed. The role of PNPase in N. meningitidis virulence has not been established. We constructed a PNPase deletion mutant (Δpnp), together with a complement strain. The growth of meningococci was severely affected upon the deletion of pnp. Furthermore, compared to the wild-type, the Δpnp mutant exhibited increased aggregation in liquid growth cultures, which was visualized at early time points in live- cell imaging and by sedimentation assay. There is a strong correlation between aggregation and adhesion in N. meningitidis [126, 196]. Therefore, the adherence of the Δpnp mutant to host epithelial cells was investigated. The mutant showed increased adherence, and PNPase was upregulated at both the mRNA and protein levels upon

42 adhesion to host epithelial cells. The fact that PNPase acts as an anti- aggregative factor and is upregulated upon adhesion to host cells suggests a role in the meningococcal transition from initial to intimate adhesion. In several bacteria, PNPase functions as an important regulator and influences the expression of metabolic and virulence genes [280]. The role of PNPase in meningococcal gene regulation was examined by performing a microarray analysis. Changes in gene expression in the Δpnp mutant compared to the wild-type were observed. The deletion of PNPase altered the expression of 469 genes (> 2-fold change in expression). Major changes were observed in genes that control metabolic pathways, which might explain the growth defects in the mutant. Similar effects on metabolism upon pnp deletion have been observed in E. coli [328, 329]. This demonstrates that PNPase plays a major role in the gene expression of N. meningitidis. To determine the causative factor for the increased aggregation and adherence, changes in the surface-exposed molecules were investigated. The transcriptional data revealed that the capsule biosynthesis genes were upregulated in the Δpnp mutant. However, no change was observed in the amount of capsule, surface hydrophobicity, and biofilm formation. Next, the relationship between enhanced aggregation in the Δpnp mutant and Tfp was investigated. Meningococci are highly dependent on Tfp to form aggregates and adhere to host cells [127, 140]. A double mutant deficient in PNPase and PilE was constructed and examined using live-cell microscopy. The double mutant was unable to form aggregates. These results demonstrate an important role of Tfp in the hyperaggregation of the Δpnp mutant. The role of Tfp in piliation and bundling was also examined. Western blot analysis showed an increased level of PilE in the Δpnp mutant, which might contribute to the hyperaggregation. Transmission electron microscopy revealed an increased number of pili bundles in the Δpnp mutant compared to the wild-type. In addition, microarray data showed an upregulation in genes involved in pilus assembly and stability (pilN, pilO, pilW). After confirming the role of PNPase in the virulence of N. meningitidis in vitro, the survival of the Δpnp mutant was examined in a mouse model. Mice were infected intraperitoneally with the wild-type or Δpnp mutant, and blood samples were collected after 2, 6, and 24 h. No change was detected in bacterial counts between the wild-type and the Δpnp mutant after 2 h of

43 infection. However, a significant decrease was detected in the counts of the Δpnp mutant compared to wild-type at 6 h post-infection. A similar decrease in the bacterial counts was observed after 24 h of infection. These data indicate that the deletion of PNPase attenuates the virulence of N. meningitidis in vivo. In conclusion, PNPase plays an important role in the gene expression of N. meningitidis that affects both growth and virulence. PNPase in meningococci negatively regulates aggregation and adhesion in vitro in a Tfp-dependent manner. Additionally, the lack of PNPase attenuates the virulence of N. meningitidis in a mouse model.

44 Future perspectives

“The important thing is not to stop questioning.” – Albert Einstein

Our investigation has raised questions that are addressed in this chapter. Paper I identified lactate as an effector molecule that drives microcolony dispersal in pathogenic Neisseria. We constructed deletion mutants in all four lactate metabolism genes and confirmed that this occurred independently of lactate utilization. The dispersal of ΔlctP microcolonies in the absence of lactate was unexpected. The role of LctP in meningococcal microcolony dispersal remains undetermined and is an interesting topic for further exploration. Because of its function as a lactate transporter, we speculate that the disruption of lactate export occurs and results in an accumulation of intracellular D-lactate produced by LdhA from pyruvate. The quantification of intracellular lactate could be useful for determining the accumulation of lactate in the ΔlctP mutant. Moreover, the construction of a ΔlctP/ΔldhA double mutant could give more information about the lactate accumulation in ΔlctP. The construction of an IPTG-inducible lctP strain would contribute to further investigation of the role of LctP in dispersal. The deletion of lctP results in dispersal in the absence of lactate. Thus we also consider the possibility of LctP as a negative regulator in dispersal. It would be interesting to examine the expression of lctP upon the addition of lactate. As we came across many questions during our work in Paper I, we continued the investigation and summarized the results in Paper II. The main focus was to identify meningococcal factors that might play a role in lactate-induced dispersal. It is evident that the bacteria within the microcolonies are responsive to a signal through an unknown mechanism. We observed that the dispersal depends on the bacterial concentration, but not the QS system AI-2. Further experiments suggest that this mechanism is not dependent on a compound secreted by meningococci unless it has very short half-life (unpublished data). Moreover, we have confirmed that the three TCS NarP/NarQ, PilR/PilS, and NtrY/NtrX do not play a role in lactate-induced dispersal. Additionally meningococci also possess the TCS MisR/MisS. We will continue to examine the role of MisR/MisS in

45 meningococcal dispersal. It would also be interesting to examine the observed phenotype upon deletion of ntrX. Paper II showed that the PilT-mediated retraction of the Tfp is essential for microcolony dispersal by using the ΔpilT mutant. Additionally, protein synthesis was required because lactate-induced dispersal was inhibited in microcolonies treated with spectinomycin. No changes were detected in the protein level of PilE upon the addition of CM using whole-cell lysate (Paper I), but the difference between extended and retracted Tfp was not investigated. Measurements of Tfp surface exposure are possible by performing a whole-cell ELISA, as described previously [132]. The differential expression of NhbA and NalP upon decreases in environmental temperature has been demonstrated to promote meningococcal aggregation [193]. Thus, it would be interesting to examine their role in the temperature-dependent reaggregation upon lactate-induced dispersal. The fact that reduced temperature influences microcolony dispersal and supports re-aggregation suggests that microcolony dispersal is determined by not only lactate concentration but also several environmental factors. The questions on environmental factors should be addressed, especially temperature, as well as the correlation between in vivo and in vitro effects. Paper III showed that the deletion of the D-LDH LdhA resulted in increased aggregation and biofilm formation. The biofilm mass was quantified using crystal violet staining in a static microtiter plate. Although this assay can be very useful, it has limitations such as a lack of fluid flow and nutrient depletion. Therefore, a secondary detection method using microfluidics could also be tested. The biofilm formation by the ΔldhA mutant was enhanced compared to wild-type as a result of increased eDNA release. However, we have not observed bacterial viability within the biofilm. An increase in EPS by lysis but not in bacterial number could be possible, and thus, it is important to measure viability. This could be achieved by LIVE/DEAD staining of the biofilms. Interestingly, we observed an increase in both aggregation and biofilm formation, yet we did not observe any differences in the adhesion of the ΔldhA mutant to host epithelial cells. The role of eDNA in meningococcal adhesion to host cells has not been well studied. It will be

46 interesting to examine whether the ΔldhA mutant also shows increased biofilm formation on host cells. Since we did not observe any differences in capsule expression and siaD mRNA level during log phase growth, it is plausible that elevated eDNA is the reason for the increased hydrophobicity in the ΔldhA mutant. It would be intriguing to explore the possibility of DNA binding to Tfp since they are the main surface components exposed in encapsulated meningococci. Other surface molecules are known to mediate aggregation and biofilm formation by DNA binding, such as the NhbA, autotransporters IgA protease, AutA and AutB [224, 225, 237]. NalP can cleave and release NhbA and autotransporters from the surface, resulting in reduced biofilm formation [224, 330, 331]. Interestingly, we observed the downregulation of nalP during ΔldhA biofilm formation. Therefore, the role of these surface proteins in DNA-mediated biofilm formation of the ΔldhA mutant could be interesting candidates for further investigation. A number of studies have observed a link between changes in metabolic status and biofilm formation [324-326, 332]. Therefore, the metabolic consequences of ldhA deletion must be assessed. The accumulation of acetate, pyruvate, and ethanol is known to occur upon deletion of ldhA in E. coli [333]. Moreover, the accumulation of the acetate intermediate acetyl- CoA promotes biofilm formation [276]. A shift in metabolic status might affect meningococcal autolysis. Changes in metabolic composition can also affect the pH level, which is an important factor in meningococcal autolysis. These metabolic processes will be interesting to explore. A detailed study based on the regulation of ldhA would be of great interest. It could be appropriate to examine the regulation of ldhA upon adhesion to host cells and in different environmental conditions, such as a microaerobic environment. Softberry promoter predictions identified two transcription factor binding sites for FNR and one for FarR [334]. FNR is an important regulator of the transition from aerobic to anaerobic growth [153]. The role of FarR as a transcription factor in meningococci is not completely understood. However, FarR expression is induced during growth in the late log phase and repressed by complement components [335]. This awaits further studies.

47 The role of PNPase in the virulence of N. meningitidis was investigated in Paper IV. It would be of interest to identify factors that influence PNPase expression or modulate its activity. Microarray analysis revealed that the deletion of pnp affected the regulation of numerous metabolic genes. It is becoming apparent that metabolites can bind to PNPase and modulate its activity, thus impacting gene regulation at the post-transcriptional level [336]. The TCA cycle metabolites citrate and ATP can directly bind PNPase and prevent its phosphorolytic and polymerization activity in E. coli [328, 329, 336]. Citrate binding to PNPase is believed to be a highly conserved process to regulate metabolism. Citrate binding residues have been identified in the amino acid sequence in prokaryotic and eukaryotic PNPases, as well as the archaeal exosome complex, thus being conserved across the three domains of life [336]. The meningococcal PNPase contains the mentioned binding residues in its amino acid sequence [336, 337]. It would be interesting to examine whether the activity of the meningococcal PNPase is under metabolic control. Furthermore, it would be intriguing to examine PNPase expression and responses to environmental stressors. Preliminary results indicate that meningococcal pnp is not induced at lower temperature (unpublished data). There are many unanswered questions about the activity, regulation and cooperation within the degradosome of N. meningitidis PNPase. Finally, it would be interesting to study the negative influence of PNPase on meningococcal aggregation and adhesion. The minor increase in the major Tfp subunit PilE is likely not the sole cause of the enhanced aggregation in the Δpnp mutant. Since there were no major changes in PilE level, it would be important to observe its localization. The number of surface-exposed Tfp could be examined by whole-cell ELISA. Alternatively, extracellular Tfp could also be sheared off and the remaining internal Tfp could be detected using SDS-PAGE. However, the results indicate that the Tfp bundling rather than the amount is the cause of the enhanced aggregation. Therefore, it would be extremely useful to perform mass spectrometry on purified Tfp from the Δpnp mutant and compare to the wild- type strain. This could give insight into the changes in post-translational modification patterns. Alterations in meningococcal glycosylation on PilE have been shown to affect both bacterial aggregation and adhesion to host cells [124].

48 This thesis has described several interesting findings on meningococcal colonization. We have observed both host and meningococcal factors that influence initial adhesion, microcolony formation, dispersal, and biofilm formation. At the same time, several exciting, provocative, interesting, and challenging questions were raised. Further studies in this field could yield fascinating results.

49 ACKNOWLEDGEMENTS

This thesis would never have been possible without the amazing people around me, which I am so grateful for. In particular, I would like to thank the following people:

I would like to express my greatest appreciation to my supervisor Ann-Beth Jonsson for this opportunity and introducing me to the fascinating field of Neisseria. I really appreciate the freedom you gave me during my PhD, trusting me to develop my projects on my own but still guided me to the right direction when needed. To summarize, Thank You!

My co-supervisor Klas Udekwu, for enjoyable conversations concerning science and life. Anders Nilsson, for joining my ISP committee and for your valuable comments.

Sushil Pathak, Hong Sjölinder, Helena Aro and all their group members, it was fun sharing lab space, ideas and friendship with you all. The members of KU, AN, KJ and AKL groups that joined our Monday Microbiology meetings for the nice discussions. Nadia and Leopold, from Department of Analytical Chemistry for the collaboration. I would like to offer my special thanks to everyone in F5 corridor. I think that the corridor FIKA was a great idea that should definitely continue. I would also like to thank the whole MBW staff.

I don´t think I would have survived without my F566 roomies: Hanna, you are such a kind person with an occasional sarcastic humor. I am so happy that you introduced my to Ethiopian culture, especially the amazing injera. I will miss having a doggy treat from you in the office! Gabriela, thank you for all the interesting and long-lasting discussions about running, exercise, cycling, travel and food. We shared so many interests and

50 it was so much fun sitting next to you. Pilar, the other member of the “healthy corner” club. I loved that we could talk forever about food. Also, the times during the preparation of the sampling for SEM imaging was one of the greatest times I had during my PhD! Even though we never used that data.

Raquel, for being a great friend and lab companion from my first day in ABJ group. We shared so many interest that I am starting to think that we actually come from the same neighborhood. Teaching with you was always so much fun! Sunil, thank you for taking the office candy storage to the next level. You are so kind, generous, selfless and helpful. I learned so much from you in science and life. Nele, you were the driving force in our group. Your hard work and positivity was very motivating for me. O. Sara E, I was lucky to start in ABJ group as a M.Sc. student under your guidance. I learned so many things from you. You were always so happy and positive. Your dancing and singing in the lab always cheered me up. Jakob, I really enjoyed working with you both in the PNPase and the Dispersal project. Also, thank you for the good times in Manchester during the IPNC. Miriam, I loved your enthusiasm for science and it was really inspirational to me. Mattias, for always being so kind, cheerful and for your tasty cakes. Manuel, for sharing the problem of identification of an “unknown” molecule and all the great lunch discussions. Fanglei, the master of cloning, for the help with making mutants. Xiao, my dumpling dealer. Linda W, for trying to teach me Swedish in Ashville and being constantly happy and cheerful person. Fredrik, Linda H, Sophia, Yeneneh, and Bea, thanks for fun memories and a good friendship. Big thanks to all the students we have had in ABJ group, especially Melanie who was under my supervision my last year. Thank you ALL for the great lunch discussions, afterworks, movie trips, movie making and potlucks.

I would like to thank all of my friends who have supported me along the way, especially during this year when I needed the most. I am so happy to have you all in my life. I am fortunate to have amazing friends in Iceland. Berglind og Maggi, María og Jan, Hildur, Thelma, Birna, Eyrún and Elsa (and your other

51 halves), our friendship means so much to me. My friends from my B.Sc. studies in Iceland, I love how we are all so different but still make such a good team. Special thanks to Þórdís, probably wouldn’t have finished my B.Sc. without you. My Icelandic-Stockholm family, for making the time in Stockholm awesome and “skál fyrir ABBA”.

Words cannot describe how grateful I am for my amazing family. It is a privilege to have such a big and close family. Bjarmó family (Ásgeir, Hildigunnur og fjölskylda), which I have known now for 10 years. Thank you for all the great memories that we have created. Hásteins family (Júlía og fjölskylda), for all the help and support, especially during this year. It means a lot to me. Amma Jóna og Afi Tryggvi, ég sakna ykkar svo mikið, takk fyrir hvatninguna í gegnum árin. Hóló family (Stína, Kári, Haffi, Erna og fjölskyldur), for being the best, for all the laughter, for all the greatest dinner parties and support through good and difficulties. Amma Alla og Afi Lilli, takk fyrir allar góðu stundirnar saman og takk fyrir allan stuðninginn.

I feel so rich and loved to have many siblings but it could be challenging at times especially when I was younger. Jóna Heiða, Kristín Erna, Þorleifur and Kolla, thank you all for the moral and emotional support during my studies and in life. My little nephew Ívan, for always being so happy and making everybody smile.

Ástin mín, Gunnar Steinn, thank you for all your patience during my studies. I could never have done this without your support, encouragement and love. Ég elska þig mest.

Mamma, you are the strongest woman I have ever known. Thank you for always loving me, being there for me, supporting me, my studies and our decision to move to Stockholm. Mamma þú ert best, ég elska þig.

This thesis is dedicated to my beloved father who passed away this year. He was my hero and the best role model I could ever imagine. Ég sakna þín óendanlega mikið. Ég mun alltaf geyma þig í hjarta mínu ♥

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