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Pathogenic Neisseria: Single cell motility, multicellular dynamics and antimicrobial susceptibility
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neisseria meningitidis and Neisseria gonorrhoeae can colonize humans without causing any symptoms. However, gonorrhea and invasive meningococcal disease are serious health concerns. An essential virulence factor for neisserial adhesion to host cells, twitching motility and microcolony formation/aggregation is the retractile type IV pili (Tfp). The scope of this thesis stretches from the motility of single Neisseria cells, via the multicellular dynamics of N. meningitidis microcolonies, to the bactericidal and endotoxin-inhibiting activity of a novel anti-meningococcal peptide. The Tfp machinery in pathogenic Neisseria is highly conserved. Nevertheless, our data demonstrate species-specific expression levels of the Tfp retraction ATPase PilT. By using live-cell microscopy and particle tracking together with visualization of pili, differences between N. gonorrhoeae and N. meningitidis were also observed in piliation and twitching motility speed. However, these differences could not be attributed to the contrasting PilT expressions per se (Paper I). The importance of PilT for pilus dynamics is well established in the literature while comprehensive knowledge of the paralog PilU is lacking. In Paper II, results suggest that PilU promotes timely formation of microcolonies. Furthermore, both PilU and PilT were required for full virulence of meningococci in vivo. The meningococcal response upon adhesion to host cells includes upregulation of the novel virulence factor Neisseria anti-aggregation factor A (NafA). Our data indicate that NafA limits microcolony formation by preventing excessive formation of Tfp bundles (Paper III). Microcolony dispersal is a prerequisite for close adhesion and mucosal invasion. Dispersal progressed rapidly on host cells and upon induction with host cell-conditioned medium (Paper IV). The dispersal phase was not altered in NafA-deficient meningococci. However, NafA may be important after microcolony dispersal on host cells for maintaining bacteria in a single cell state (Paper IV). In Paper V, a screen of cell-penetrating peptides for antimicrobial activity towards meningococci demonstrated that transportan-10 (TP10) exhibited rapid membrane-disruptive and bactericidal activity. TP10 also decreased bacteraemia levels in a murine model of meningococcal disease. Furthermore, TP10 reduced the proinflammatory effect of endotoxin on macrophages. Thus, TP10 displays two properties that may be utilized for the development of a peptide-based treatment against pathogens.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2014. , 82 p.
Keyword [en]
Neisseria, Type IV pili, Twitching motility, PilT, Microcolony dynamics, PilU, NafA, Antimicrobial peptide, Cell-penetrating peptide
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-108519ISBN: 978-91-7649-034-1 (print)OAI: oai:DiVA.org:su-108519DiVA: diva2:759725
Public defence
2014-12-05, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1 and 4: Manuscripts.

Available from: 2014-11-13 Created: 2014-10-29 Last updated: 2014-11-21Bibliographically approved
List of papers
1. Characterization of motility and piliation in pathogenic Neisseria
Open this publication in new window or tab >>Characterization of motility and piliation in pathogenic Neisseria
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
urn:nbn:se:su:diva-108511 (URN)
Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2014-10-31
2. Loss of Meningococcal PilU Delays Microcolony Formation and Attenuates Virulence In Vivo
Open this publication in new window or tab >>Loss of Meningococcal PilU Delays Microcolony Formation and Attenuates Virulence In Vivo
2012 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 80, no 7, 2538-2547 p.Article in journal (Refereed) Published
Abstract [en]

Neisseria meningitidis is a major cause of sepsis and bacterial meningitis worldwide. This bacterium expresses type IV pili (Tfp), which mediate important virulence traits such as the formation of bacterial aggregates, host cell adhesion, twitching motility, and DNA uptake. The meningococcal PilT protein is a hexameric ATPase that mediates pilus retraction. The PilU protein is produced from the pilT-pilU operon and shares a high degree of homology with PilT. The function of PilT in Tfp biology has been studied extensively, whereas the role of PilU remains poorly understood. Here we show that pilU mutants have delayed microcolony formation on host epithelial cells compared to the wild type, indicating that bacterium-bacterium interactions are affected. In normal human serum, the pilU mutant survived at a higher rate than that for wild-type bacteria. However, in a murine model of disease, mice infected with the pilT mutant demonstrated significantly reduced bacterial blood counts and survived at a higher rate than that for mice infected with the wild type. Infection of mice with the pilU mutant resulted in a trend of lower bacteremia, and still a significant increase in survival, than that of the wild type. In conclusion, these data suggest that PilU promotes timely microcolony formation and that both PilU and PilT are required for full bacterial virulence.

National Category
Immunology
Identifiers
urn:nbn:se:su:diva-80040 (URN)10.1128/IAI.06354-11 (DOI)000305599400030 ()
Note

AuthorCount:3;

Available from: 2012-09-12 Created: 2012-09-12 Last updated: 2017-12-07Bibliographically approved
3. NafA negatively controls Neisseria meningitidis piliation
Open this publication in new window or tab >>NafA negatively controls Neisseria meningitidis piliation
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 7, e21749- p.Article in journal (Refereed) Published
Abstract [en]

Bacterial auto-aggregation is a critical step during adhesion of N. meningitidis to host cells. The precise mechanisms and functions of bacterial auto-aggregation still remain to be fully elucidated. In this work, we characterize the role of a meningococcal hypothetical protein, NMB0995/NMC0982, and show that this protein, here denoted NafA, acts as an anti-aggregation factor. NafA was confirmed to be surface exposed and was found to be induced at a late stage of bacterial adherence to epithelial cells. A NafA deficient mutant was hyperpiliated and formed bundles of pili. Further, the mutant displayed increased adherence to epithelial cells when compared to the wild-type strain. In the absence of host cells, the NafA deficient mutant was more aggregative than the wild-type strain. The in vivo role of NafA in sepsis was studied in a murine model of meningococcal disease. Challenge with the NafA deficient mutant resulted in lower bacteremia levels and mortality when compared to the wild-type strain. The present study reveals that meningococcal NafA is an anti-aggregation factor with strong impact on the disease outcome. These data also suggest that appropriate bacterial auto-aggregation is controlled by both aggregation and anti-aggregation factors during Neisseria infection in vivo.

National Category
Microbiology
Identifiers
urn:nbn:se:su:diva-63671 (URN)10.1371/journal.pone.0021749 (DOI)000292293400029 ()21747953 (PubMedID)
Available from: 2011-10-26 Created: 2011-10-26 Last updated: 2017-12-08Bibliographically approved
4. A host cell-derived factor induces the dispersal of Neisseria meningitidis microcolonies
Open this publication in new window or tab >>A host cell-derived factor induces the dispersal of Neisseria meningitidis microcolonies
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
urn:nbn:se:su:diva-108515 (URN)
Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2014-10-31
5. Identification of Cell-Penetrating Peptides That Are Bactericidal to Neisseria meningitidis and Prevent Inflammatory Responses upon Infection
Open this publication in new window or tab >>Identification of Cell-Penetrating Peptides That Are Bactericidal to Neisseria meningitidis and Prevent Inflammatory Responses upon Infection
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2013 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 57, no 8, 3704-3712 p.Article in journal (Refereed) Published
Abstract [en]

Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:su:diva-92792 (URN)10.1128/AAC.00624-13 (DOI)000321761800031 ()
Note

AuthorCount:7;

Available from: 2013-08-23 Created: 2013-08-20 Last updated: 2017-12-06Bibliographically approved

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