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Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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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.

Place, publisher, year, edition, pages
2013. Vol. 126, no 1, 339-347 p.
Keyword [en]
DNA damage, N. gonorrhoeae, VK2/E6E7
National Category
Microbiology
Research subject
Molecular Bioscience; Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-89534DOI: 10.1242/jcs.117721ISI: 000316460800032OAI: oai:DiVA.org:su-89534DiVA: diva2:619257
Note

AuthorCount:6;

Available from: 2013-05-02 Created: 2013-04-29 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Pathogenic Neisseria infections of human neutrophils and epithelial cells: focusing on host responses and immune evasion
Open this publication in new window or tab >>Pathogenic Neisseria infections of human neutrophils and epithelial cells: focusing on host responses and immune evasion
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
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:nbn:se:su:diva-92446 (URN)978-91-7447-725-2 (ISBN)
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
2. Neisseria gonorrhoeae and Lactobacillus from initial adherence to effects on human cells
Open this publication in new window or tab >>Neisseria gonorrhoeae and Lactobacillus from initial adherence to effects on human cells
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The causative agent of gonorrhoea Neisseria gonorrhoeae (gonococcus) colonises the urogenital tract epithelia. The vaginal tract microbiota of healthy females is dominated by Lactobacillus species. In paper I, the ability of lactobacilli to protect cervical cells against gonococcal adherence was investigated. The number of adhered lactobacilli did not correlate to the level of protection against gonococci. Instead, the protection was dependent on specific Lactobacillus isolates. Gonococci able to outmanoeuvre the normal microbiota colonise and may elicit an influx of neutrophils. In paper II the initial interaction between pathogens and neutrophils was investigated. N. gonorrhoeae was found to bind to the non-phagocytic rear (uropod) of the neutrophils. Results suggest that uropod binding is a trait specific of Neisseria species. By binding to the uropod bacteria could avoid the phagocytic front part of the neutrophils whilst being transported across the epithelial cell layer to new sites. Since gonorrhoea has been associated with cancer in several studies, effects of gonococcal colonisation on eukaryote genome integrity was investigated in paper III.  N. gonorrhoeae caused DNA stand breaks in vaginal epithelial cells and decreased the level of tumor protein p53. Infected cells showed increase of cyclin-dependent kinase inhibitors p21 and p27 along with reduced proliferation. The impact of lactobacilli colonisation on cervical cell proliferation was investigated in paper IV. Three out of four Lactobacillus isolates tested reduced cell proliferation. Decreased pH due to lactic acid production was found to be a contributing factor. However, vaginal isolated L. gasseri required a direct bacteria-cell interaction to affect cell cycle progression. Additional unknown factors also contributed as in the case of saliva isolated L. reuteri. In summary, this thesis investigates N. gonorrhoeae pathogenesis and the impact of Lactobacillus species in protection and colonisation.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2012. 54 p.
Keyword
Neisseria gonorrhoeae, Lactobacillus, adhesion, cell cycle, DNA damage, Neutrophils
National Category
Microbiology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-75490 (URN)978-91-7447-522-7 (ISBN)
Public defence
2012-05-25, lecture room G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, 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 3: Manuscript. Paper 4: Manuscript.

Available from: 2012-05-03 Created: 2012-04-20 Last updated: 2014-09-29Bibliographically approved
3. Host epithelium integrity in the female reproductive tract during Neisseria gonorrhoeae infection
Open this publication in new window or tab >>Host epithelium integrity in the female reproductive tract during Neisseria gonorrhoeae infection
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neisseria gonorrhoeae infections are asymptomatic in approximately fifty percent of the affected women. Left untreated, this can develop into a long-term inflammatory state with detrimental secondary complications such as ectopic pregnancy and sterility. Furthermore, studies have shown that N. gonorrhoeae infection may be a contributing factor to urogenital cancers.

We found that gonococcal infections induce DNA damage in human vaginal and cervical cells. As a consequence of the DNA damage the cell cycle progression is altered. Mitotic checkpoint genes and proteins important in regulation of metaphase were distorted. Partly because of this, the progression of mitosis was hampered. An additional contributing cause of the DNA damage and dysfunctional mitosis is the release of endogenous gonococcal restriction endonucleases.

Most N. gonorrhoeae infection studies are performed on cultured monolayers of cells derived from tumors.  In order to create a system which more resembled in vivo conditions and study N. gonorrhoeae infections, we developed a polarized epithelium of human non-tumorigenic vaginal VK2/E6E7 cells. In the search for an animal model for the human-restricted pathogen we evaluated the CD46 transgenic mouse model in the study of gonococcal infections.

In summary, this thesis aims to increase the understanding of the basic molecular function of how invasive gonococcal infections affect host cell cycle regulation, DNA integrity and potential predisposition to cellular malignancies in the epithelium of the female reproductive tract. Since the female reproductive tract is colonized with Lactobacillus, the impact of lactobacilli to the host cell cycle has also been investigated.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2014. 64 p.
Keyword
Female reproductive tract, Neisseria gonorrhoeae, VK2/E6E7 cells, DNA damage, cell cycle, mitosis, restriction endonucleases, infection models
National Category
Microbiology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-107713 (URN)978-91-7649-007-5 (ISBN)
Public defence
2014-11-07, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrheniusväg 12, 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 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2014-10-16 Created: 2014-09-24 Last updated: 2014-11-21Bibliographically approved

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