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Department of Medical Biochemistry and Microbiology (IMBIM), Uppsala Biomedical Centre (BMC), Uppsala University, Uppsala, Sweden
1 Correspondence: Department of Medical Biochemistry and Microbiology, Box 582, Uppsala University, S-751 23 Uppsala, Sweden. E-mail: helena.aro{at}imbim.uu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: gonococcal infection • cyclins • cell cycle • cell cycle arrest
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. The program for cell growth and division consists of the four phases G1, S, G2, and M. The transition that occurs at the G1-checkpoint commits the cell to the proliferative cycle. If the conditions that signal this transition are not present, the cell enters a nonproliferative phase called G0.
Progression through the cell cycle is controlled by complexes of cyclins and cyclin-dependent kinases (Cdks) (3)
. Different cyclin/cyclin-dependent kinase complexes play unique roles in driving the cell toward the next phase, and specific regulation of the complex activity enables the cells to progress to the next phase only after completion of the previous phase. Following a predetermined sequence, cyclins steadily accumulate during interphase and are rapidly proteolyzed during late mitosis (4)
. Cyclin D1/Cdk4 or Cdk6 is the first cyclin complex to accumulate during G1, and is an important regulator of the activation from G0 to G1 and in the G1/S phase transition (5)
. In late G1, high levels of cyclin E/Cdk2 drive the cell through the G1/S transition and are degraded during G2 phase. During early S-phase, cyclin A/Cdk2 levels rise and are closely followed by an accumulation of cyclin B1/Cdk1. During late mitosis, cyclin A rapidly disappears in early prometaphase (3
, 6
7
8)
while cyclin B1 is rapidly degraded in metaphase (10)
. The cell employs numerous methods to regulate cyclin activity, including altering cyclin protein concentration, sub cellular localization of cyclins, rapid cyclin degradation, and multiple phosphorylations (9
, 10)
.
It is becoming increasingly apparent that the cell cycle also contributes to whether pathogenic bacteria may effectively grow and colonize within a host. Therefore, it is not surprising that many bacterial pathogens manipulate the host cell cycle to benefit bacterial attachment, survival and growth within the host. Indeed, alterations to the regulation of cell growth are important for the establishment of many bacterial infections. Microbial pathogens have developed a variety of strategies to manipulate host cell functions for their own benefit, including release of toxins that inhibit or activate cell cycle progression (2
, 11
, 12)
, association with host cell microtubules (13)
or blocking multiple checkpoints in the cell cycle (14)
. Pathogenic Escherichia coli is known to block the cell cycle during G2/M transition (15)
and Helicobacter pylori can block gastric cells in G1/S phase (16
17
18)
.
Neisseria gonorrhoeae (gonococcus) is an obligate human pathogen that causes the sexually transmitted disease gonorrhea. This Gram-negative bacterium primarily colonizes the mucosal surface of the male urethra and the female cervix but can also colonize the pharynx, the rectum and the conjunctiva of the eye. The bacteria can also cause chronic infection in humans. The initial attachment of the bacteria to the apical side of epithelial tissues is mediated by type IV pili (19
, 20)
, which are important for establishing infection (21)
. The pilus-mediated adhesion of N. gonorrhoeae to transformed epithelial cell lines has shown to be dependent on CD46, 
1 integrins and 2 integrins (22
23
24
25
26)
. In addition, gonococci express many virulence factors such as protein 1, porins, lipooligocaccharides, iron receptors, outer membrane vesicles, pilus biogenesis-associated proteins, which all may affect the host target cell during the initial infection process (for review, see ref. 27
).
In this study we have identified novel features describing N. gonorrhoeae pathogenesis. Upon contact with epithelial cells, the bacteria modify host cell cycle progression by down-regulating cyclin expression and arrest the host target cell in G1. We also characterized the adhesion of bacteria to cells in different cell cycle phases and found that the bacteria adhere equally well to interphase cells of different phases (G1, S, or G2) and do not adhere at all to mitotic cells. The bacteria show a preference for binding to cycling cells and not to cells situated in the nonproliferating G0 phase, which may provide an explanation for the cell and tissue tropism observed in chronic gonococcal infections.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and is referred to as MS11 P+ in the text. Piliated, Opa– phenotypes were distinguished by colony morphology under a binocular light microscope. The bacteria used did not express detectable levels of Opa as detected by SDS-PAGE of outer membrane preparations. MS11 P+ and N. meningitidis (FAM20) were grown on GCB (GC medium base; Difco, Detroit, MI, USA) agar plates containing Kellogg’s supplement (29)
at 37°C and 5% CO2 and were passaged every 18–20 h. MS11 P+ were heat killed at 95°C for 10 min. The E. coli strain DH5 were grown on LB agar plates (BD Biosciences, Bedford, MA, USA) at 37°C and 5% CO2.
Cell lines and growth conditions
HeLa (American Type Culture Collection, or ATCC CCL-2; Manassas, VA, USA) and ME-180 (ATCC HBT-33) cells were cultured in Dulbecco’s modified Eagles medium (DMEM) with glutamax (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS) (Sigma, St. Louis, MO, USA). hTERT-BJ cells (30)
(ATCC CRL-4001) are human telomerase immortalized fibroblasts derived from a human foreskin. They were cultured in minimal essential medium (MEM, Invitrogen) supplemented with 10% FCS for normal growth or 0.2% FCS during serum starvation. Cell lines were maintained at 37°C, 5% CO2 and occasionally grown in penicillin/streptomycin containing medium to prevent contamination. All experiments were performed with 10% or 0.2% FCS and without antibiotics.
Cell synchronization
HeLa cells were synchronized to the G1/S border by the addition of 2.5 mM thymidine (Sigma) for 24 h. The cells were then released from the thymidine block and washed three times in DMEM. Further incubation at 37°C for 4 h and 9 h resulted in cells arriving at S-phase and G2-phase respectively. Mitotic cells were obtained by adding nocodazol (100 µg/ml, Sigma) for 24 h. Mitotic cells were harvested by mitotic shake off, carefully shaking the dish until the mitotic cells were detached from the plastic. The cells were then gently washed in DMEM by centrifugation at 200 g for 10 min and placed on a coverslip.
Adhesion assays
It is very important to point out that all of the bacterial infection experiments were performed on nonconfluent (
40%) cells. Since tight confluence can induce a cell cycle arrest, we always confirmed that the cells were not confluent before and after each assay. Bacteria were resuspended in DMEM to a multiplicity of infection (M.O.I.) of 100. Bacteria were allowed to adhere for 60 min at 37°C, 5% CO2. The cells were then washed three times with PBS, then incubated in FCS supplemented DMEM for a total of 1–24 h. The cells were treated with 1% saponin for 5 min, serially diluted, spread onto GCB plates and incubated overnight at 37°C, 5% CO2. Colony-forming units were counted the next day. For fluorescent cell imaging assays, MS11 P+ bacteria (M.O.I.=100) were allowed to adhere to HeLa cells for 60 min. Unbound bacteria was washed away and the cells were fixed with 4% paraformaldehyde (Sigma) for 10 min. Cells were permeabilized for 10 min in 0.5% Triton X-100, 20 mM Tris HCl pH 7.4, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2. Adherent bacteria were detected with polyclonal antibodies against Neisseria (N0600–14, U.S. Biological, Swampscott, MA, USA; 1:500) and goat-anti-rabbit-IgG AlexaFluor-488 (5 µg/ml, Jackson Laboratories, Bar Harbor, ME, USA).
Flow cytometry assay
To measure DNA content, the cells were trypsinized and resuspended in cold PBS. Nonconfluent cells were washed in PBS and fixed in 4 ml of ice-cold ethanol for 10 min at 4°C. Cells were washed again, treated with RNase (40 µg/ml, Sigma), and stained with propidium iodide (40 µg/ml, BD Biosciences) for 30 min at 37°C. For cyclin B1 staining, the cells were permeabilized and fixed using a BD Cytofix/Cytoperm kit (BD Biosciences) as per the manufacturer’s instructions. Briefly, cells were fixed and permeabilized in 100 µl of BD Cytofix/Cytoperm solution (4°C, 20 min), washed twice in BD Perm/Wash solution and stored at 4°C until staining. All subsequent cell washes were performed with BD Perm/Wash solution. Cells were washed and resuspended in 50 µl of cyclin B1 (GNS1, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody (Ab) (4°C, 60 min), washed three times, and resuspended in 50 µl of chicken anti-mouse IgG AlexaFluor-488 (10 µg/ml, Invitrogen) Ab (4°C, 60 min). Cells were washed three times and analyzed immediately on a FACSort flow cytometer (BD Biosciences) and the resultant data was analyzed using Cell Quest Pro software (BD Biosciences). Each experiment was performed two or three times.
Western blot analysis
Infected or control cells were washed in PBS, then harvested in the plate by addition of sample buffer (87% glycerol, 10% SDS, 0.5 M Tris-HCl pH 6.8, 0.1% bromphenol blue) and immediately stored at –20°C until use. Samples were incubated at 95°C for 10 min and 5 µl was loaded onto a 12% Tris-HCl Ready gel (Bio-Rad, Hercules, CA, USA) and subjected to electrophoresis (200 V). After separation, proteins in the gel were transferred to PVDF membranes (Milli-Pore, Billerica, MA, USA) using semidry transfer system (Bio-Rad). The membranes were washed in water and blocked overnight at 4°C in 5% nonfat dry milk (Bio-Rad) diluted in PBS. The membranes were incubated with mouse antibodies to either cyclin B1 (GNS1, 0.4 µg/ml, Santa Cruz Biotechnology), cyclin D1 (AM29, 1.1 µg/ml, Zymed, San Francisco, CA, USA), or cyclin E (HE12, 2.6 µg/ml, Zymed), tubulin (T9028, 1 µg/ml, Sigma), or a rabbit polyclonal antibody (pAb) to CD46 (H294, 2 µg/ml, Santa Cruz Biotechnology) for 1 h. Incubation with secondary antibodies, goat anti-mouse IgG-HRP, or goat-anti-rabbit IgG-HRP (0.4 µg/ml Santa Cruz Biotechnology) was performed for 1 h. Immunoreactivity was visualized using an enhanced chemiluminescence (ECL) detection system (Perkin Elmer, Norwalk, CT, USA).
Fluorescence microscopy
Cells were cultured on hexametaphosphate-metasilicate-coated 13 mm coverslips (Menzel GmbH, Braunschweig, Germany) to 40% confluence. Cells were stained with cyclin B1 (GNS1, 0.4 µg/ml, Santa Cruz Biotechnology) Ab for 1 h. Nuclear DNA and bacterial DNA was stained and mounted with Vectashield mounting medium with 4',6'-diam idino-2-phenylidole (DAPI) (Vector Laboratories Inc., Burlingame, CA, USA). Images of the cells were captured using either a Zeiss LSM 510 confocal microscope or a Zeiss fluorescent microscope. Images were processed using ImageJ software (NIH, Bethesda, MD, USA) and exported to Adobe Photoshop. For the mitotic index assay, 300 cells were counted for each of the infection time points. The number of cells in metaphase, anaphase, or telophase (late mitosis) as detected by visual assessment of chromosome alignment were counted and expressed as a percentage of the total number of cells counted.
BrdU Cell proliferation assay
The quantification of bromodeoxyuride (BrdU) incorporation into S-phase cells was made using a BrdU ELISA kit (QIA58, Calbiochem, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, nonconfluent cells were infected with N. gonorrhoeae MS11 P+ for 3–24 h. During the last 2 h of infection, BrdU was added to the cells, allowing cells situated in S-phase to incorporate BrdU into their DNA. Cells were then fixed, permeabilized, washed, and stained with an Ab against BrdU for 60 min. A HRP conjugated secondary Ab was added for another 60 min followed by substrate solution. The level of absorbance from untreated cells was compared with the level of absorbance for infected cells for each separate time point. The level of incorporated BrdU was quantified from the absorbance at 450 nm, as measured by a spectrophotometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Synchronized HeLa cells were grown onto glass slides. MS11 P+ bacteria were allowed to adhere to nonconfluent cells for 60 min and unbound bacteria were washed away, cells were stained with DAPI and a pAb against Neisseria, then analyzed by fluorescent microscopy. Representative images of MS11 P+ adhesion to cells in G1, S, and G2 shown in Fig. 1B
(upper panels). To verify the cell cycle phase, synchronized cells were fixed with ethanol and the DNA was stained with propidium iodide (Fig. 1B
, lower panels). As shown, cells in G1 comprise of cells with primarily 2n DNA content, cells in G2 comprise of 4n DNA content, while cells in S-phase illustrate an intermediate 2n to 4n DNA content. Our data show the novel finding that bacteria were not able to adhere cells undergoing mitosis (Fig. 1A, C
). Fluorescent staining of bacterial adherence to mitotic cells in three of the mitotic phases (prophase, metaphase and anaphase) showed that bacteria do not adhere to any of these stages of mitosis (Fig. 1C
). This clearly shows that the bacteria preferentially adhere to cells in interphase for initial attachment to the host cell surface.
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We also analyzed the ability of N. gonorrhoeae to adhere to cells in a resting state of G0. We utilized the human hTERT-BJ, which will enter a resting phase (G0) on serum starvation (30)
. Although the cell line is not a natural host for gonococcal adhesion, high numbers of bacteria adhered to normal cycling hTERT cells, while almost no bacterial adhesion could be observed in noncycling G0 cells (Fig. 1D
). These data suggest that N. gonorrhoeae preferentially adheres to host cells undergoing an active cell cycle.
The expression of CD46 in G1, S, G2, and M cells
Since the bacteria were unable to adhere to cells in M phase, we then analyzed if there were any alterations in the expression of eukaryotic pilus receptors during the different cell cycle phases of HeLa cells. HeLa cells were synchronized as described above and samples were analyzed by Western blot. As a control for successful synchronization, the expression of cyclin B1(using Ab GNS1) and cyclin E (using Ab HE12) was performed. Cyclin B1 has been shown to be expressed at high levels in mitosis, before rapid degradation in prometaphase. We also note the expected high expression levels of cyclin E in G1, S, and G2 phase, and the lack of cyclin E expression in M phase (Fig. 2
). Expression of cellular receptors was determined using antibodies against CD46 (H-294), 1 integrin (R-164), and 2 integrin (N-19). The CD46 expression level was similar in G1, S, and G2 phase cells, but markedly up-regulated in mitotic cells (Fig. 2)
. These results indicate that CD46 is expressed during mitosis and that other factors influence the lack of bacterial adherence in mitotic cells. The expression of 1 integrin and 2 integrin in HeLa cells was below detection levels in our assay (data not shown).
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Infection with N. gonorrhoeae decreases cyclin B1 levels
As shown in Fig. 1
, we have established that the bacteria do not preferentially attach to cells of a particular interphase cell cycle phenotype during the first hour of infection. We further continued this study to determine whether gonococcal infection could influence components of the cell cycle machinery such as cyclins. Uninfected, unsynchronized HeLa cells or ME-180 cells express cyclins at different levels depending on the particular cell cycle phase of each individual cell (3)
. Both HeLa cells and ME-180 cells grown to nonconfluence were infected with gonococcal strain MS11 P+ for 60 min. Heat killed MS11 P+, nonadherent apathogenic E. coli (DH5) and adherent pathogenic N. meningitidis (FAM20) were used as controls. Unbound bacteria were washed away and the infection was allowed to continue for 3, 6, 18, or 24 h. For the 18 and 24 h time points, gentamicin was added after 6 h to prevent overgrowth of extracellular bacteria. Detection of -tubulin protein levels were also determined and used to normalize the protein loading per well. Untreated (UT) samples were normalized to contain 100% cyclin B1.
Western blot analysis of HeLa whole-cell lysates shows that these cells express significantly lower protein levels of cyclin B1 after 18 and 24 h of infection (Fig. 3
A). The same pattern of down-regulation of cyclin B1 was also seen in infected ME-180 cells (Fig. 3B
). Gonococcal-infected cells reduced cyclin B1 protein levels to 10% of normal UT cells. Bacteria that had been heat killed (HT), apathogenic E. coli, or pathogenic N. meningitidis were not able to induce any down-regulation of cyclin B1 within the 24 h of incubation (Fig. 3A, B
). Infected HeLa cells were also analyzed for cyclin B1 protein expression levels by immunofluorescence staining, and also clearly show that the cytoplasmic cyclin B1 levels are drastically reduced after 24 h of MS11 P+ gonococcal infection (Fig. 3C
).
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The expression of cyclin B1 in gonococcal-infected HeLa and ME-180 cells was further investigated in a flow cytometry assay after 3, 6, 18, and 24 h infection. The cyclin B1 expression in infected and control HeLa and ME-180 cells was quantified and expressed as the ratio of median fluorescence compared with background Ab fluorescence (the secondary Ab alone) (Fig. 3D
). HeLa cells infected with MS11 P+ for 18–24 h showed 3-fold reduced levels of cyclin B1 expression compared with 3 h infected cells, whereas 18–24 h MS11 P+-infected ME-180 cells showed a 2-fold reduction in cyclin B1 expression compared with 3 h infected cells (Fig. 3D
). Absence of treatment or control infections did not alter cyclin B1 expression after 24 h (Fig. 3D
), suggesting again that this observed down-regulatory effect of cyclin B1 is specific to viable N. gonorrhoeae.
Infection with N. gonorrhoeae decreases cyclin D1 levels in HeLa cells and cyclin E levels in HeLa and ME-180 cells
Again, both HeLa cells and ME-180 cells grown to nonconfluence were infected with gonococcal strain MS11 P+ for 60 min. Unbound bacteria were washed away and the infection was allowed to continue for 3, 6, 18, or 24 h. For the 18 and 24 h time points, gentamicin was again added after 6 h to prevent extracellular growth of the bacteria. Detection of -tubulin protein levels were also determined and used to normalize the protein loading per well. UT samples were normalized to contain 100% cyclin D1 or cyclin E expression. Western blot analysis of HeLa whole-cell lysates shows that these cells express significantly lower protein levels of cyclin D1 after 18 and 24 h of infection (Fig. 4
A). The same pattern of down-regulation of cyclin D1 was not seen in infected ME-180 cells (Fig. 4A
), with cyclin D1 expression levels being unaltered by gonococcal infection. cyclin E expression was down-regulated in both HeLa and ME-180 cells after 18 and 24 h of MS11 P+ infection (Fig. 4B
) to 30% of UT cells.
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Gonococcal infection arrests the cells in early G1 phase of the cell cycle
Since we had observed that gonococcal infection caused down-regulation of both G2 (cyclin B1) and G1 (cyclin E and cyclin D1) types of cyclins, we also wanted to monitor the effect of a gonococcal infection on cellular DNA content to examine whether gonococcal infection could influence cell cycle phase. We analyzed the DNA content of both infected and uninfected nonconfluent HeLa cells (1 and 24 h) by ethanol fixation and propidium iodide staining using flow cytometry. As shown in Fig. 5
A, the number of cells in the G2 peak (4n DNA) is greatly reduced after 24 h of infection, providing evidence that infected cells have arrested or are blocked in the G1 phase of the cell cycle. To further investigate the effect of gonococcal infection on the cell cycle, we enumerated the number of cells that were in late mitosis (metaphase, anaphase or telophase) in both MS11 P+-infected and untreated (UT) HeLa cells after 1 and 24 h. HeLa cells were grown on glass slides to 40% confluence, infected with MS11 P+ as described previously, stained with DAPI and viewed under a fluorescent microscope. About 300 cells were counted and the number of late mitotic cells were expressed as a percentage of total cells (Fig. 5B
). UT had a level of 1.5% of cells in late mitosis, whereas 24 h MS11 P+-infected cells resulted in an increase to 5.8% of cells in late mitosis (P<0.05). We hypothesize that the observed increase in number of late mitotic cells is a subpopulation of infected cells that have major difficulties completing mitosis due to less cyclin B1 levels in late G2 and/or are about to become arrested in G1 within a short period of time. These data, combined with cellular DNA content (Fig. 5A
) provide further evidence supporting our novel finding that gonococcal infection causes an arrest in the cell cycle at a late mitotic or early G1 phase. While counting the mitotic cells, we also observed that the phenotype of the 1 h and 24 h infected cell nuclei was different. In the 24 h infected cells, we observed an increased number of cells that appear not to have completed cytokinesis (Fig. 5C
). This morphology was not visualized after 1 h of infection (Fig. 5C
) or in uninfected cells (data not shown).
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We further analyzed the effect of the gonococcal infection on cell cycle activity by measuring the amount of cells in S-phase after 3–24 h of infection and UT controls. Infected HeLa cells (grown to 40% confluence) were treated with a pulse of BrdU during the final 2 h of infection to allow uptake of BrdU and thus quantification of cells situated in S-phase. After 24 h of MS11 P+ infection, the number of cells found in S-phase was reduced to 40% compared with UT control cells (Fig. 6
). This reduction was statistically significant (P<0.01). The small increase in S-phase cells after 6 h of infection was not significant (P>0.01). These data provide further clear evidence that gonococcal infection has the capacity to reduce cell growth and proliferation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our search to find a suitable model for investigation of both cell cycle analysis and N. gonorrhoeae infection we chose two human endocervical cell lines, ME-180 and HeLa. ME-180 cells are commonly used for gonococcal infection studies and exhibit high levels of bacterial adherence. However, ME-180 cells are not suitable for either thymidine synchronization or serum starvation to obtain cell populations in a specific cell cycle phase. On the other hand, HeLa cells are widely used and are well documented as a model for cell cycle research and can be easily synchronized into the different cell cycle phases. They have also been shown to be a good model for gonococcal infections (31
, 32)
. Thus, we have chosen to use both cell lines in our investigations.
During bacterial adhesion studies, we made the novel finding that gonococci prefer to adhere to cells that are situated in interphase (G1, S, or G2) and not to cells that are either in M or G0 (Fig. 1)
. Upon mitosis, the transcription and translation of proteins is greatly reduced, and it is most likely that cell surface proteins required for bacterial adherence are altered and that cellular signals are impaired. However, we found that CD46 expression is actually up-regulated during mitosis (Fig. 2)
. Despite at least one of the pilus receptors (CD46) being present on mitotic cells, a mitotic cell probably does not provide a suitable adhesion site for gonococci due to its lack of cell signaling and its changed morphology. In addition, it is likely that cells situated in a nongrowing G0 phase do not have a complete signal transduction system that is required for successful bacterial adherence and invasion under these experimental in vitro conditions.
The addition of gonococci to nonconfluent HeLa and ME-180 cells for 24 h greatly reduced cyclin B1 and cyclin E expression as detected using flow cytometry and/or Western blot analysis (Figs. 3
, 4)
. The lack of both cyclin B1 and cyclin E suggests that these 24 h infected cells are probably situated in early G1. Cyclin B is degraded during M-phase, and thus a cell that becomes arrested during early G1 would not be able to accumulate fresh levels of cyclin E and subsequently would not have the capacity to progress through the cell cycle. Thus, these data provide strong evidence supporting the notion that infected cells are arrested in early G1. Infection with gonococci also down-regulated expression of cyclin D in HeLa cells (Fig. 4)
. Examination of data from Western blot analysis of cyclin D levels revealed an interesting observation that cyclin D, however, was not reduced by gonococcal infection in ME-180 cells. The differing cyclin D1 expression levels in HeLa and ME-180 cells may be explained by the host cell response to the bacterial infection. Most likely, there is more cell damage in HeLa cells during the infection. Cyclin D is normally expressed in early G1, but if DNA damage has occurred, p53 accumulates in the cell and induces the p21-mediated inhibition of cyclin D/cyclin-dependent kinase (33)
.
The reduced expression of cyclins was dependent on viable N. gonorrhoeae, since pathogenic N. meningitidis, heat-killed gonococci or apathogenic E. coli did not alter the cyclin levels. Thus, we have established that a gonococcal infection will result in cells that lack adequate levels of cyclins. Without these important components of the cell cycle machinery, the cell will not be allowed to progress through the cell cycle and will be subsequently arrested in the early G1 phase.
DNA profiles of propidium iodide stained HeLa cells demonstrated that 24 h infected cells showed a significant reduction in the 4n DNA peak (G2) compared with the 1 h infected or uninfected cells (Fig. 5A
), thus suggesting an arrest or block of infected cells in G1. By counting the number of DAPI stained late mitotic cells (highly condensed chromatin) under a fluorescent microscope, we also detected an increased population of 24 h infected cells in metaphase, anaphase or telophase (Fig. 5B
). Initially we had expected to see a decrease in mitotic cells if the cells were arrested in G1. However, these data show that there is in fact an increased number of late mitotic cells after 24 h of infection. We suggest that these late mitotic cells also will eventually enter G1. However, for an unknown reason, it takes these cells a longer time to enter G1. One explanation may be that bacterial induced down-regulation of cyclin B1 influences the time spent in mitosis. In addition, phenotypic analysis of 24 h infected cell nuclei clearly identified a higher population of cells in cytokinesis/early G1. This population of cells appears to have failed to complete cytokinesis, or alternatively is blocked in a very early stage of G1 (Fig. 5C
). We suggest that the disregulated expression of cyclins in 24 h gonococcal-infected cells results in cells that fail to complete cyotkinesis. Finally, we analyzed the level of cells in S-phase after 3–24 h of infection. These data indicate that a significantly lower number of cells are in S-phase after 24 h of gonococcal infection (Fig. 6)
, again suggesting the cells have become "blocked" in the early G1 phase.
We believe that we have provided clear evidence that cells become arrested in an early G1 phase after 24 h of gonococcal infection. Bacteria can gain considerable advantage in arresting the cell cycle by causing cellular phenotypic changes in the host cell, possibly resulting in increased bacterial survival. Many studies show that infection with other Gram-negative bacteria causes an up-regulation of cyclins, thereby arresting the cells in G2/M and inhibiting cells re-entering G1, leading to high mitotic activity and malignancies (15
16
17)
. This is not the case for a gonococcal infection. Our novel finding is the first report that shows cyclin down-regulation and G1 arrest as a result of gonococcal infection. An arrest in G0/G1 has also been reported in Trypanosoma brucei infection, which is thought to account for the reduced cell responsiveness during chronic infections (34)
.
It is tempting to speculate that long-term asymptomatic gonococcal infections are beneficial to bacteria by arresting the target cells in a nonproliferating phase. The low metabolism in noncycling cells may reduce both the innate immune response (such as cytokine production), the intracellular lysosome killing, and impairment to the host epithelial barrier integrity (1)
, possibly resulting in prolonged local bacterial survival and existence. By inducing this arrest, the bacteria may block the cells ability to reach the important G1/S and G2/M checkpoints, which are essential for cellular growth. By not reaching these checkpoints, the cell will lose its ability to regulate normal cell cycle functions, possibly resulting in cellular malignancies or apoptosis. This novel finding, describing a cyclin down-regulation and consequent G1 arrest after infection will open up a new and interesting area in Neisserial pathogenesis. Future studies will rely on in vivo models to achieve a full understanding of the control of the cell cycle and its role in microbial pathogenesis. It will also be important to identify the molecular components of gonococci responsible for eliciting the cell cycle arrest in epithelial cells. Undoubtedly, our findings will prove useful to further dissect pathways involved in microbial pathogenesis and the control of mammalian cell cycle.
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ACKNOWLEDGMENTS |
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Received for publication August 25, 2006. Accepted for publication September 14, 2006.
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