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Influence of host and bacterial factors during Neisseria meningitidis colonization
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2018. , p. 75
Keywords [en]
Neisseria meningitidis, Colonization, Host-bacteria interactions, Lactate, Biofilms, Polynucleotide phosphorylase
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-161566ISBN: 978-91-7797-474-1 (print)ISBN: 978-91-7797-475-8 (electronic)OAI: oai:DiVA.org:su-161566DiVA, id: diva2:1259999
Public defence
2018-12-18, Vivi Täckholm-salen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Available from: 2018-11-23 Created: 2018-10-31 Last updated: 2019-10-10Bibliographically approved
List of papers
1. Host cell-derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal
Open this publication in new window or tab >>Host cell-derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal
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2017 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 13, no 4, article id e1006251Article in journal (Refereed) Published
Abstract [en]

The development of meningococcal disease, caused by the human pathogen Neisseria meningitidis, is preceded by the colonization of the epithelial layer in the nasopharynx. After initial adhesion to host cells meningococci form aggregates, through pilus-pilus interactions, termed microcolonies from which the bacteria later detach. Dispersal from microcolonies enables access to new colonization sites and facilitates the crossing of the cell barrier; however, this process is poorly understood. In this study, we used live-cell imaging to investigate the process of N. meningitidis microcolony dispersal. We show that direct contact with host cells is not required for microcolony dispersal, instead accumulation of a host-derived effector molecule induces microcolony dispersal. By using a host-cell free approach, we demonstrated that lactate, secreted from host cells, initiate rapid dispersal of microcolonies. Interestingly, metabolic utilization of lactate by the bacteria was not required for induction of dispersal, suggesting that lactate plays a role as a signaling molecule. Furthermore, Neisseria gonorrhoeae microcolony dispersal could also be induced by lactate. These findings reveal a role of host-secreted lactate in microcolony dispersal and virulence of pathogenic Neisseria.

National Category
Biological Sciences Cell and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-144725 (URN)10.1371/journal.ppat.1006251 (DOI)000402555700005 ()
Available from: 2017-07-20 Created: 2017-07-20 Last updated: 2019-01-21Bibliographically approved
2. Lactate-induced dispersal of Neisseria meningitidis microcolonies is mediated by changes in cell density and pilus retraction and is influenced by temperature change
Open this publication in new window or tab >>Lactate-induced dispersal of Neisseria meningitidis microcolonies is mediated by changes in cell density and pilus retraction and is influenced by temperature change
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-161562 (URN)
Available from: 2018-10-31 Created: 2018-10-31 Last updated: 2018-11-02Bibliographically approved
3. Deletion of D-Lactate Dehydrogenase A in Neisseria meningitidis Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release
Open this publication in new window or tab >>Deletion of D-Lactate Dehydrogenase A in Neisseria meningitidis Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release
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2019 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 422Article in journal (Refereed) Published
Abstract [en]

Neisseria meningitidis is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of N. meningitidis to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of N. meningitidis across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences N. meningitidis microcolony dispersal. Here, we examined the ability of N. meningitidis mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A (idhA) mutant had an increased level of biofilm formation. Deletion of IdhA increased the N. meningitidis cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the ldhA-delicient mutant, suggesting a critical role for eDNA. Compared to wild-type, the IdhA-deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the IdhA mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while norB, which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of IdhA in N. meningitidis promotes biofilm formation and aggregation through autolysis-mediated DNA release.

Keywords
Neisseria meningitidis, lactate dehydrogenase, eDNA, autolysis, biofilm
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-167499 (URN)10.3389/fmicb.2019.00422 (DOI)000460286700001 ()30891026 (PubMedID)
Available from: 2019-04-01 Created: 2019-04-01 Last updated: 2019-10-10Bibliographically approved
4. Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence
Open this publication in new window or tab >>Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence
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2016 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 84, no 5, p. 1501-1513Article in journal (Refereed) Published
Abstract [en]

Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase ( PNPase) is a 3'-5' exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis.

National Category
Biological Sciences Microbiology in the medical area
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-131999 (URN)10.1128/IAI.01463-15 (DOI)000377106600024 ()26930706 (PubMedID)
Available from: 2016-08-11 Created: 2016-07-05 Last updated: 2018-11-02Bibliographically approved

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