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Neisseria gonorrhoeae and Lactobacillus from initial adherence to effects on human cells
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Helena Aro)
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 [en]
Neisseria gonorrhoeae, Lactobacillus, adhesion, cell cycle, DNA damage, Neutrophils
National Category
Microbiology
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-75490ISBN: 978-91-7447-522-7 (print)OAI: oai:DiVA.org:su-75490DiVA: diva2:516906
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
List of papers
1. Adherence of clinically isolated lactobacilli to human cervical cells in competition with Neisseria gonorrhoeae
Open this publication in new window or tab >>Adherence of clinically isolated lactobacilli to human cervical cells in competition with Neisseria gonorrhoeae
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2008 (English)In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 10, no 12-13, 1325-1334 p.Article in journal (Refereed) Published
Abstract [en]

Lactobacilli are normal inhabitants of our microbiota and are known to protect against pathogens. Neisseria gonorrhoeae is a human specific pathogenic bacterium that colonises the urogenital tract where it causes gonorrhoea. In this study we analysed early interactions between lactobacilli and gonococci and investigated how they compete for adherence to human epithelial cervical cells. We show that lactobacilli adhere at various levels and that the number of adherent bacteria does not correlate to the level of protection against gonococcal infection. Protection against gonococcal adhesion varied between Lactobacillus species. Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus reuteri were capable of reducing gonococcal adherence while Lactobacillus rhamnosus was not. Lactobacillus strains of vaginal origin had the best capacity to remain attached to the host cell during gonococcal adherence. Further, we show that gonococci and lactobacilli interact with each other with resultant lactobacilli incorporation into the gonococcal microcolony. Hence, gonococci bind to colonised lactobacilli and this complex frequently detaches from the epithelial cell surface, resulting in reduced bacterial colonisation. Also, purified gonococcal pili are capable of removing adherent lactobacilli from the cell surface. Taken together, we reveal novel data regarding gonococcal and lactobacilli competition for adherence that will benefit future gonococcal prevention and treatments.

Keyword
Neisseria gonorrhoeae, Lactobacilli, Adherence, Pili
National Category
Microbiology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-75499 (URN)10.1016/j.micinf.2008.07.032 (DOI)18761100 (PubMedID)
Available from: 2012-04-20 Created: 2012-04-20 Last updated: 2017-12-07Bibliographically approved
2. 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
3. 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
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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.

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
4. Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression
Open this publication in new window or tab >>Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression
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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

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