Influence of host and bacterial factors during Neisseria 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 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. 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 Polysaccharide capsule .................................................................................... 10 2.2 Lipopolysaccharide ........................................................................................... 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 ....................................................