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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to the host cell cycle has also been investigated., At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.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.

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies., AuthorCount:8

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., AuthorCount:6

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., AuthorCount:6