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Pathogenic Neisseria infections of human neutrophils and epithelial cells: focusing on host responses and immune evasion
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Helena Aro)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

N. meningitidis and N. gonorrhoeae are obligate human pathogens that colonize mucosal surfaces and are often carried asymptomatically. These bacteria have developed adhesive structures that promote adherence to host cells and efficient colonization of new hosts. N. gonorrhoeae causes the sexually transmitted disease gonorrhea, which remains one of the most common sexually transmitted diseases, despite the availability of effective antibiotic treatments. N. meningitidis is frequently found in the nasopharynx of healthy individuals as a part of the normal microbiota. However, this bacterial species is a major cause of mortality when it causes septicemia and epidemic meningitidis. The design of vaccines conferring protection against multiple serogroups is difficult; this fact, combined with increased global resistance to antibiotics, emphasizes the need for a better understanding of the pathogenesis of these species. The aim of this thesis was to study the effects of bacterial adherence to human neutrophils (PMNs) and epithelial cells. The adherence of N. meningitidis and N. gonorrhoeae to primary PMNs was investigated in Paper I. Specific adherence of the bacteria to the PMN uropod was observed. By adhering to the uropod, the bacteria could avoid phagocytosis and use the migrating PMNs for transportation. The type IV pilus, which is a known bacterial adhesin, was found to promote uropod adherence. In Paper II, adherence of N. gonorrhoeae to non-polarized cervical and vaginal epithelial cells was found to cause DNA damage and delay cell cycle progression. Upregulation and nuclear localization of the cyclin-dependent kinase inhibitors p21 and p27 were observed, which could contribute to reduced cell proliferation. Interestingly, the levels of tumor suppressing protein 53 (TP53) were affected by bacterial colonization in a non-tumor cell line. In Paper III, colonization by Lactobacillus spp. was found to induce the accumulation of host cells in G1 phase and the upregulation of p21. The adherence of N. gonorrhoeae to polarized epithelial cells and the impact of PMN presence were investigated in Paper IV. N. gonorrhoeae adherence to polarized epithelial cells was significantly higher than adherence to non polarized cells. Cell culture medium containing degranulated products from stimulated PMNs was found to promote bacterial adherence. Finally, PMNs with bacteria adhered to the uropod were able to transport the bacteria through a polarized cell layer. In summary, this thesis investigates the impact of adhesion of pathogenic Neisseria spp. to host epithelial cells and PMNs. 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2013. , 59 p.
Keyword [en]
Neisseria meningitidis, Neisseria gonorrhoeae, Neutrophils, PMN, Epithelial cells, Polarized cells, Type IV pili, Adherence, Cell cycle, DNA damage, Lactobacillus
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-92446ISBN: 978-91-7447-725-2 (print)OAI: oai:DiVA.org:su-92446DiVA: diva2:639184
Public defence
2013-09-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2013-08-15 Created: 2013-08-05 Last updated: 2013-08-13Bibliographically approved
List of papers
1. Pathogenic Neisseria Hitchhike on the Uropod of Human Neutrophils
Open this publication in new window or tab >>Pathogenic Neisseria Hitchhike on the Uropod of Human Neutrophils
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, e24353- p.Article in journal (Refereed) Published
Abstract [en]

Polymorphonuclear neutrophils (PMNs) are important components of the human innate immune system and are rapidly recruited at the site of bacterial infection. Despite the effective phagocytic activity of PMNs, Neisseria gonorrhoeae infections are characterized by high survival within PMNs. We reveal a novel type IV pilus-mediated adherence of pathogenic Neisseria to the uropod (the rear) of polarized PMNs. The direct pilus-uropod interaction was visualized by scanning electron microscopy and total internal reflection fluorescence (TIRF) microscopy. We showed that N. meningitidis adhesion to the PMN uropod depended on both pilus-associated proteins PilC1 and PilC2, while N. gonorrhoeae adhesion did not. Bacterial adhesion elicited accumulation of the complement regulator CD46, but not I-domain-containing integrins, beneath the adherent bacterial microcolony. Electrographs and live-cell imaging of PMNs suggested that bacterial adherence to the uropod is followed by internalization into PMNs via the uropod. We also present data showing that pathogenic Neisseria can hitchhike on PMNs to hide from their phagocytic activity as well as to facilitate the spread of the pathogen through the epithelial cell layer. 

National Category
Microbiology
Research subject
Microbiology; Molecular Genetics
Identifiers
urn:nbn:se:su:diva-63644 (URN)10.1371/journal.pone.0024353 (DOI)000295173800021 ()
Available from: 2011-10-26 Created: 2011-10-26 Last updated: 2017-12-08Bibliographically approved
2. Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells
Open this publication in new window or tab >>Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells
Show others...
2013 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 126, no 1, 339-347 p.Article in journal (Refereed) Published
Abstract [en]

The constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (gamma H2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.

Keyword
DNA damage, N. gonorrhoeae, VK2/E6E7
National Category
Microbiology
Research subject
Molecular Bioscience; Molecular Genetics
Identifiers
urn:nbn:se:su:diva-89534 (URN)10.1242/jcs.117721 (DOI)000316460800032 ()
Note

AuthorCount:6;

Available from: 2013-05-02 Created: 2013-04-29 Last updated: 2017-12-06Bibliographically approved
3. Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression
Open this publication in new window or tab >>Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression
Show others...
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, e63592- p.Article in journal (Refereed) Published
Abstract [en]

We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

National Category
Microbiology
Research subject
Molecular Bioscience; Molecular Genetics
Identifiers
urn:nbn:se:su:diva-91528 (URN)10.1371/journal.pone.0063592 (DOI)000318852400046 ()
Note

AuthorCount:6;

Available from: 2013-07-01 Created: 2013-06-28 Last updated: 2017-12-06Bibliographically approved
4. Neutrophils increase adherence of Neisseria gonorrhoeae to epithelial cells and transport bacteria across a polarized epithelium
Open this publication in new window or tab >>Neutrophils increase adherence of Neisseria gonorrhoeae to epithelial cells and transport bacteria across a polarized epithelium
(English)Manuscript (preprint) (Other academic)
Keyword
Neutrophil, PMN, Neisseria gonorrhoeae, VK2/E6E7, Transwell, Polarized cells, Epithelial
National Category
Microbiology
Identifiers
urn:nbn:se:su:diva-92445 (URN)
Available from: 2013-08-05 Created: 2013-08-05 Last updated: 2013-08-06Bibliographically approved

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