• centrosome;
  • Chlamydia;
  • cytoskeleton;
  • micro- tubules


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Chlamydiae traffic along microtubules to the microtubule organizing center (MTOC) to establish an intracellular niche within the host cell. Trafficking to the MTOC is dynein dependent although the activating and cargo-linking function of the dynactin complex is supplanted by unknown chlamydial protein(s). We demonstrate that once localized to the MTOC, the chlamydial inclusion maintains a tight association with cellular centrosomes. This association is sustained through mitosis and leads to a significant increase in supernumerary centrosomes, abnormal spindle poles, and chromosomal segregation defects. Chlamydial infection thus can lead to chromosome instability in cells that recover from infection.

Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that is one of the leading causes of sexually transmitted disease and the most common cause of preventable blindness worldwide (1). In the USA, the incidence of new cases of chlamydial genital tract infection is approximately 4 million annually (2). These infections cause serious morbidity as they can lead to pelvic inflammatory disease, ectopic pregnancy, and tubal infertility (3).

Chlamydiae undergo a biphasic developmental cycle that includes an extracellular form called the elementary body (EB) that induces endocytosis by the host cell (4). The internalized chlamydiae remain within a membrane-bound vacuole termed an inclusion. Within the first few hours after endocytosis, EBs differentiate into metabolically active reticulate bodies (RBs) that multiply until differentiation back to EBs prior to lysis of the host cell. The nascent chlamydial inclusion acquires the host cell minus-end-directed motor protein, dynein, by a process that requires chlamydial protein synthesis and migrates along microtubule tracks to the microtubule organizing center (MTOC) (5). This centripetal migration is conserved in all chlamydial species, and the inhibition of dynein results in reduced development (6,7). The mechanism of dynein recruitment and activation is unique to chlamydiae in that the requirement for the cellular dynein-activating complex dynactin is circumvented by a chlamydial factor(s), thus bypassing the normal regulation of dynein activity (5).

Migration of the inclusion is a unidirectional process as the chlamydial inclusion remains in a perinuclear location associated with the MTOC throughout the developmental cycle. Microtubules are organized at the MTOC by centrosomes, which co-ordinate cellular architecture during interphase as well as the bipolar spindles for DNA segregation during mitosis (8). In this study, we show that the mature chlamydial inclusion exhibits a very close association with the centrosomes of the host cell and that maintenance of this interaction is, like migration, dependent on dynein in a dynactin-independent manner. This stable interaction leads to improper segregation of the centrosomes to the daughter cells and results in increased centrosome numbers per cell. The chlamydia-induced supernumerary centrosomes produce spindle defects in mitotic cells and ultimately cause chromosome instability.


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Association of centrosomes with the chlamydial inclusion

Because the chlamydial inclusion localizes to the MTOC, we sought to determine whether it also associates with the cell centrosomes. The centrosome organizes microtubules and is at the center of the MTOC. The centrosome, through its organization of the microtubules, organizes cellular architecture during interphase and is responsible for the bipolar spindles for DNA segregation during mitosis (8). To this end, we stained infected HeLa cells to observe the localization of the chlamydial inclusion in relation to the centrosomes and the microtubule network. Staining for both γ-tubulin (centrosomes) and β-tubulin (microtubule network) revealed that the centrosome is in proximity to the chlamydial inclusion during interphase (Figure 1A). This association is maintained throughout the cell cycle, as the centrosome is localized to the inclusion during both interphase and mitosis. During mitosis, the centrosomes at the spindle poles remain associated with the chlamydial inclusion, and frequently the centrosomes at each pole are both associated with chlamydial inclusions.


Figure 1. (A) Centrosomes associate with the chlamydial inclusion. Indirect immunofluorescence microscopy using antibodies to β-tubulin (blue), pericentrin (red), and Chlamydia trachomatis L2 (green) demonstrate that centrosomes (pericentrin, arrows) closely associate with the chlamydial inclusion in both interphase and mitotic cells. (B) Staining using antibodies to IncA or IncG (green), γ-tubulin (red), and DNA (blue) demonstrates the centrosomes (arrows) interact with inclusion membrane fibrils (arrow heads) after centrosomal partitioning during mitosis and after cytokinesis. Bar, 10 µm.

To visualize the relationship of the inclusion membrane to centrosomes, we stained infected cells for the chlamydial inclusion membrane. The inclusion membrane contains many chlamydial derived proteins termed Incs (9,10). We used antibodies (Abs) to two of these, IncA and IncG, to visualize the inclusion membrane in relation to the centrosomes (9,11). Staining of infected HeLa cells with Abs to IncA and IncG, DNA (Draq5) and centrosomes (γ-tubulin), reveals that the centrosomes are associated with the inclusion membrane. This association is not interrupted during centrosomal migration during the cell cycle. In late S or early G2-phase of the cycle, the centrosome is duplicated to produce two centrosomes per cell, which then migrate to opposite ends of the cell to organize the spindle poles during mitosis (12,13). The inclusion membrane and centrosomes remain associated throughout this process (Figure 1B, upper panel arrows) and even during cytokinesis when centrosomes have been segregated into daughter cells (Figure 1B, lower panel arrows). The centrosomes in uninfected daughter cells remain bound to a thin-membrane projection originating from the inclusion of the original cell (Figure 1B arrowhead). This dramatic staining pattern is seen in cells stained for either IncA or IncG and suggests that the association of centrosomes with the inclusion is so stable as to distort the inclusion membrane during cytokinesis.

Role of dynein in the association of centrosomes with the chlamydial inclusion

We previously demonstrated that trafficking of the nascent chlamydial inclusion to the MTOC was dependent on cytoplasmic dynein and that dynein remains associated with the mature chlamydial inclusion (5). To determine whether dynein is involved with the centrosomal interaction, we asked whether centrosomes would be released from the chlamydial inclusion by inhibiting dynein function. Dynein function can be inhibited by micro-injecting Abs to the dynein intermediate chain (DIC) directly into the cytosol of cells (5). HeLa cells infected with C. trachomatis serovar L2 for 10 h were micro-injected with the monoclonal Ab (mAb) dic74.1 directed against DIC. The cells were fixed 4 h after injection and stained for the presence of the injected Ab (anti-mouse IgG secondary), for centrosomes (anti-pericentrin), and DNA (Draq5) that stains both the nucleus and the chlamydiae. Confocal projection images were used to measure the distance in two-dimensional (2D) from the chlamydial inclusion to the centrosomes. In infected cells, greater than 95% of the centrosomes are observed within 1 µm of the chlamydial inclusion (data not shown). However, in dic74.1-injected cells, only about 25% of the centrosomes remained within 1 µm of the inclusion (Figure 2A). Micro-injection of a second Ab to DIC, dic70.1, yielded the same phenotype (data not shown). The disruption of the interaction was specific as micro-injection of an irrelevant control Ab to Rickettsia rickettsii had no effect on centrosomal localization, and greater than 95% of the centrosomes were within 1 µm of the inclusion (Figure 2A).


Figure 2. The centrosome inclusion interaction is dependent on dynein but independent of the dynein activating complex dynactin.(A) HeLa cells infected with Chlamydia trachomatis L2 for 12 h were micro-injected with the mAb DIC74.1 to inhibit the function of either dynein or an irrelevant antibody (Rickettsia rickettsii) and fixed 4 h post-injection. The cells were stained for the presence of the micro-injected antibody (green) as well as for γ-tubulin to highlight the centrosomes (red, arrows) and with Draq 5 to stain the chlamydial DNA (blue). The gray scale image shows the Draq 5 staining. The inclusions are circled in white to delineate them from the nucleus which also stains with Draq 5. The binned histograms show the distances between the host centrosomes and the chlamydial inclusions measured in confocal projections. (B) HeLa cells were transfected with GFP-p50 dynamitin or GFP for 8 h before being infected with C. trachomatis L2. Confocal micrographs show cells stained for Chlamydia (blue) and γ-tubulin to highlight the centrosomes (red, arrows) and GFP (green). The binned histograms represent distances between the centrosomes and the chlamydial inclusion after transfection with either pGFP-p50 dynamitin (to disrupt dynactin) or pGFP alone. Bar, 10 µm.

Although trafficking of the nascent inclusion to the MTOC is dynein dependent, it is uniquely independent of the dynein cargo binding and activator complex, dynactin (5). To determine whether the interaction of the centrosome with the chlamydial inclusion is similarly dynactin independent, we disrupted the dynactin complex by over-expression of p50 dynamitin to decouple the dynein-binding and cargo-anchoring functions of dynactin (14). HeLa cells were transfected with green fluorescent protein (GFP)-p50 dynamitin for 8 h before being infected with C. trachomatis. The cells were then fixed 16 h later. This allowed time for dynamitin to be expressed at high levels and for the inclusion to develop. A GFP-only expressing plasmid was used to control for the effect of transfection. The cells were fixed and stained for chlamydia and centrosomes (γ-tubulin). The distance of the centrosomes to the chlamydial inclusion was measured in the GFP positive cells (Figure 2B). In cells expressing GFP-p50-dynamitin greater than 75% of the centrosomes were within 1 µm of the inclusion. This was the same for GFP expressing cells alone (Figure 2B). These data indicate that the centrosome-inclusion interaction, like trafficking, is dynein dependent but dynactin independent thus suggesting a conserved mechanism.

Effect of chlamydial infection on the association of the centrosome with the nucleus

We next sought to determine whether the association of the chlamydial inclusion with centrosomes affected the juxtanuclear position of the centrosomes (15,16). In uninfected cells, greater than 95% of the centrosomes were within 5 µm of the nucleus with only a small minority greater than 10 µm from the nucleus (Figure 3). In C. trachomatis-infected cells at 24 h post-infection, greater than 75% of the centrosomes were within 5 µm of the nucleus and by 40 h post-infection and greater than 55% of the centrosomes were within 5 µm of the nucleus with a dramatic increase in those that were greater than 10 µm away (Figure 3). We next asked whether this was a specific event caused by the unique interaction of the chlamydial inclusion with the host centrosomes or whether it is a non-specific event resulting from steric interference with cellular organization attributable to the large size of the chlamydial inclusion. To address this question, we infected cells with another obligate intracellular bacterium, Coxiella burnetii. Coxiella burnetii infection also results in a large parasitophorous vacuole in the cytoplasm of infected cells (17). This vacuole, however, has not been reported to interact with centrosomes. HeLa cells were infected with C. burnetii for 5 days, as it is a slower growing organism, and staining was performed as above except the C. burnetii was stained using a Coxiella-specific Ab. Centrosome-nuclei distances were again measured and represented as binned histograms (Figure 3). The centrosomes in the C. burnetii-infected cells maintained a normal localization with 95% of the centrosomes being within 5 µm of the nucleus. The localization of the centrosomes in these cells was indistinguishable from uninfected cells (Figure 3). This suggests that the chlamydial inclusion uniquely leads to defects in normal centrosomal positioning.


Figure 3. Chlamydial infection disrupts the normal positioning of the centrosomes. HeLa cells were infected with Chlamydia trachomatis L2 for 24 or 40 h, Coxiella burnetii for 5 days, or mock infected. The organisms (red), nuclei (Draq 5, blue), and centrosomes (γ-tubulin, green, arrows) were observed in cells by fluorescence microscopy. The distances of the centrosomes (arrows) from the nucleus (as measured to the nearest edge) was measured in confocal micrographs, and the distributions are presented as binned histograms. Bar, 10 µm.

Induction of centrosomal supernumerary defects by C. trachomatis

Migration of centrosomes during mitosis is essential for the segregation of centrosomes into daughter cells to maintain normal centrosome numbers. Failed segregation of centrosomes can lead to supernumerary defects. We therefore assessed whether infection with C. trachomatis L2 resulted in an increase in cells containing greater than two centrosomes. HeLa cells were infected with L2 at a multiplicity of infection (MOI) of 5. At 40 h post-infection, the monolayers were fixed and stained to observe the Chlamydia (polyclonal anti-chlamydia), centrosomes (γ-tubulin), and nuclei (DNA dye Draq5) (Figure 4). Cells with greater than two centrosomes were considered abnormal (cells with arrows indicating the centrosomes). Uninfected HeLa cells have a background rate of cells with supernumerary centrosomes of 7.3 ± 2%. After infection with C. trachomatis L2 for 40 h, the percentage of cells with greater than two centrosomes rose to 60 ± 3. Again, to control for non-specific steric effects of a large inclusion, we infected HeLa cells with C. burnetii and after 5 days of infection cells with abnormal centrosome numbers remained the same as uninfected, 7.1 ± 2% (Figure 4). Infection with the common C. trachomatis sexually transmitted disease-associated serovars G or D resulted in a similar increase in the percentage of cells showing centrosomal defects to 54 ± 4 and 51 ± 3, respectively (Figure 4).


Figure 4. Chlamydial infection increases the number of centrosomes per cell. Representative confocal micrographs stained by indirect immunofluorescence for bacteria (Chlamydia or Coxiella, red), γ-tubulin (centrosomes, green), and Draq 5 (nuclei, blue) are shown. Centrosomes were counted in HeLa cells infected with Chlamydia trachomatis serovar L2, D, or G for 40 h, Coxiella burnetii for 5 days, or mock infected. The arrows indicate the centrosomes in cells with more than two centrosomes. Fifty microscopic fields were observed, and the percentage of cells with more than two centrosomes were calculated (graph). *p < 0.001 (Chi-square). Bar, 10 µm.

Supernumerary centrosomes can be caused by defects in cytokinesis, improper partitioning, or disregulation of the cell cycle. Chlamydiae have been reported to have no effect on the cell cycle as measured by bromodeoxyuridine labeling of DNA during S phase (18, 47). We repeated these studies and also found no cell cycle defects (data not shown). Chlamydial infection can, however, lead to defects in cytokinesis (18). We also detect defects in cytokinesis as many infected cells contain multiple nuclei and hypothesize that this may lead to the infected cells acquiring supernumerary defects. In addition, we measured the centrosome to nuclei ratio in infected and uninfected cells and found a definitive increase in centrosomes per nucleus. The ratio of centrosomes to nuclei in L2-infected cells is 2.59 ± 0.12 compared with 1.82 ± 0.12 for uninfected HeLa Cells. This suggests centrosome partitioning is also a contributing mechanism for these defects.

Because HeLa cells are derived from a cervical carcinoma and have a relatively high background of supernumerary centrosome defects, we asked whether abnormal centrosome numbers would be induced in a primary cell line by C. trachomatis infection. Primary human foreskin fibroblasts were infected with C. trachomatis L2 or C. burnetii and assayed for centrosomal number defects. The uninfected population had a 1.1 ± 1% rate of cells with greater than two centrosomes. This increased to 25.8 ± 3% at 24 h post-infection and 76.7 ± 6% by 48 h post-infection with C. trachomatis L2. Infection with C. burnetii for 5 days resulted in no significant change in centrosomal number defects (Figure 5). Therefore, the induction of centrosomal supernumerary occurs in both transformed and non-transformed cells.


Figure 5. Chlamydial infection of normal human fibroblasts also results in centrosome number defects. Representative fields of cultures stained by indirect immunofluorescence for bacteria (Chlamydia trachomatis L2 or Coxiella, red), γ-tubulin (centrosomes, green), and Draq 5 (nuclei, blue) are shown. The arrows highlight the centrosomes in cells with more than two centrosomes. Centrosomes were counted after infection with C. trachomatis serovar L2 for 24 h and 48 h, Coxiella burnetii for 5 days, or mock infected. Fifty microscopic fields were collected, and the percentage cells with more than two centrosomes were calculated (graph). *p < 0.001 (Chi-square). Bar, 10 µm.

Chromosome segregation defects in C. trachomatis-infected cells

To determine whether these extra centrosomes were functional or fragments of centrosomes, we looked to see whether spindle poles in infected mitotic cells also increased upon chlamydial infection. HeLa cells were infected with C. trachomatis L2 for 40 h and stained with Abs to Chlamydia (anti-chlamydia), centrosomes (γ-tubulin), and expressing GFP-β-tubulin (Figure 6A). Mitotic cells were scored as normal (two spindle poles per cell) or abnormal (more than two spindle poles per cell). An uninfected HeLa cell population, as supernumerary centrosome number would predict, contains about 6.6 ± 3% cells with more than two spindle poles (Figure 6A). Mitotic spindle defects were quantified for HeLa cells infected with C. trachomatis L2 for 40 h, and in this population 78 ± 6% of the mitotic cells had more than two spindle poles per cell. This is a dramatic increase over the uninfected population demonstrating that the extra centrosomes in the infected cell population were capable of organizing microtubule spindles for chromosome segregation (Figure 6A). Figure 6 shows two representative examples of this, one cell with four spindle poles and another cell with three spindle poles. Bipolar spindles are required for proper chromosome segregation. Because chlamydial infection leads to multipolar spindles during mitosis, we sought to determine whether these defects lead to chromosomal segregation defects. We therefore observed chlamydia-infected cells undergoing mitosis to look for gross defects in chromosome alignment during metaphase. Uninfected HeLa cells display chromosome congression defects in approximately 10% of the population, which is in agreement with the supernumerary centrosomes and multipolar mitotic cell numbers (Figure 6B). The percentage of cells with chromosome congression defects in HeLa cells infected with C. trachomatis L2 for 40 h increased to 55 ± 5 (Figure 6B). Shown are two representative examples of defective chromosome alignments in this infected cell population. The first shows a cell with two obvious spindles (β-tubulin staining), however, there is at least one chromosome (Draq 5 staining, arrows) that is not aligned at the metaphase plate (Figure 6B). The second example demonstrates metaphase with more than two spindle poles in a chlamydia-infected cell during metaphase. This cell contains three obvious spindles (β-tubulin staining) and highly disorganized DNA congression (Draq 5 staining) (Figure 6B).


Figure 6. Supernumerary centrosomes lead to multipolar spindles and DNA congression defects.(A) Representative confocal micrographs showing HeLa cells infected with Chlamydia trachomatis L2 for 40 h and stained with antibodies to Chlamydia (blue), γ-tubulin (red), and expressing GFP-β-tubulin (green). Cells with more than two spindle poles (arrows) were counted by microscopic observation (graph); n > 50, *p < 0.001 (chi-square). (B) Representative confocal micrographs of HeLa cells infected with C. trachomatis L2 for 40 h stained for Chlamydia (red), β-tubulin (green), and chromosomes (Draq 5, blue). The percentage of cell with chromosomal congression defects (misaligned chromosomes, arrows) observed during metaphase (graph); n > 50, *p < 0.001 (Chi-square). Bar, 10 µm.

Centrosome defects persist in a cell population previously infected with C. trachomatis

The chlamydial inclusion is inherited as a single organelle when a cell divides during infection, typically resulting in one infected and one uninfected daughter cell (18,19). To determine whether centrosomal defects induced by chlamydial infection persist in uninfected daughter cells, we ‘cured’ infected cells of chlamydia by treating with the prokaryotic protein synthesis inhibitor, rifampicin. HeLa cells were plated at a low density, infected with C. trachomatis at an MOI of 5 and incubated for 24 h to permit the development of large visible inclusions. Cultures were then treated with 50 µg/mL rifampicin and allowed to recover from the infection for 5 days. After 2 days in the presence of drug, the number of cells without inclusions increased, as the cells continued to divide. By 5 days after the drug treatment, there were few visible inclusions (data not shown). These cells were then plated on coverslips and fixed and stained for chlamydia (polyclonal anti-Chlamydia), centrosomes (γ-tubulin), and nuclei (DNA dye Draq 5). Cells with abnormal centrosome numbers were counted for both infected and control cultures that were treated with the drug but not infected. In the cured culture, 30 ± 3% of the population had supernumerary centrosomes and no visible chlamydial inclusions (Figure 7A). In contrast, only 8 ± 3% of the control culture population had more than two centrosomes (Figure 7A). These data demonstrate that the centrosomal defects induced by chlamydia during infection can be inherited by the uninfected daughter cells creating a population of cells that have increased rates of spindle defects and potential chromosome instability.


Figure 7. (A) HeLa cells cured of the chlamydial infection retain centrosomal defects and have increased rates of micronuclei formation. Representative confocal micrograph stained with antibodies to γ-tubulin (green), Chlamydia (blue), and with Draq 5 (nuclei, red). The centrosomes in cells with supernumerary defects are highlighted by the arrows. Percentage of cells in the cured and mock-treated population with more than two centrosomes per cell (graph). (B) The cured population was subjected to micronucleus assay and stained for DNA (Draq 5). The arrow indicates a representative micronucleus. The percentage of cells with micronuclei were calculated by counting more than 100 binucleated cells from three separate cured experiments and are represented by the graph. Bar, 10 µm.

We next asked whether this ‘cured’ population of cells had increased rates of chromosome instability. Chromosome instability can be measured by the micronucleus assay. Micronuclei are cytoplasmic chromatin-containing bodies formed when acentric chromosome fragments or chromosomes lag during anaphase and fail to become incorporated into daughter cell nuclei during cell division (20,21). Because supernumerary centrosomes lead to spindle abnormalities which in turn lead to micronucleus formation, the incidence of micronuclei serves as an index of chromosome instability. The ‘cured’ HeLa cells as above were plated on coverslips and assessed for the formation of micronuclei by the standard micronucleus assay (22). The mock-infected cells had a rate of 13 ± 4% cells with micronuclei. However, the ‘cured’ cell population had a rate of 33 ± 3% cells containing micronuclei indicating that, like the centrosome supernumerary defects, previously infected cells have increased rates of chromosome instability (Figure 7B).


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Chlamydiae modify the inclusion membrane very early in the developmental cycle to initiate trafficking to the perinuclear region of the host cell where they remain in close contact to the Golgi apparatus and accept sphingomyelin and cholesterol rich exocytic vesicles from the Golgi apparatus (23–25). The nascent chlamydial inclusions migrate toward the minus end of microtubules and aggregate at the MTOC utilizing the minus-end-directed microtubule motor, cytoplasmic dynein (5). Although this process requires the dynein motor complex, it is independent of the host cargo binding and dynein regulatory complex, dynactin (5). In this study, we show that centrosomes also closely associate with the chlamydial inclusion at the MTOC and that this interaction disrupts normal centrosomal positioning such that they no longer associate with the nucleus of the host cell. The association of the chlamydial inclusion membrane with centrosomes, like trafficking to the MTOC, is dependent on the activity of dynein but independent of the activities of dynactin. Thus it is possible that the same mechanisms that mediate the dynein dependent, early interactions of the inclusion with microtubules are stably maintained and lead to centrosomal number defects.

Abnormal centrosome numbers lead to the assembly of multipolar spindles that unequally distribute chromosomes to daughter cells resulting in genetic imbalances that precede and may contribute to cellular transformation (26). The rate of multipolar spindles and severe chromosomal alignment defects in C. trachomatis-infected cells was greatly increased compared with uninfected cells, thus indicating that the chlamydial induced extra centrosomes lead to mitotic defects. These centrosomal defects must be passed on to future cellular generations if chromosome instability and genetic imbalances are to occur. The normal chlamydial growth cycle ultimately leads to lysis of the host cell, but if a cell divides during infection, then the inclusion is typically retained by only one of the daughter cells (18,19). We demonstrated that this surviving population of cells can retain not only centrosome defects but also a greatly increased rate of chromosome instability. This suggests that these chlamydial induced defects can be maintained in a previously infected population, creating cells with an increased potential for genetic instability.

Centrosome supernumerary defects have been implicated in chromosome instability and loss of cell cycle control in early tumors and most aggressive carcinomas (12,27). Chromosomal instability develops at early stages of cervical neoplasia and can be detected even in premalignant lesions (28). Genomic instability can contribute to the rapid selection of clonal cell populations that are able to overcome the various environmental challenges that arise during carcinogenic progression (29). In this light, genomic instability has thus been considered as an enabling characteristic of tumor cells (30). We have shown that chlamydial infection can lead to genetic instability through the induction of centrosomal number defects. Chromosome instability caused by chlamydial infection may be an early event leading to the creation of a cellular population predisposed to transformation. This hypothesis is in good agreement with the observed long lag time between the association of chlamydial infection and cervical cancer, which is reported to be, on average, 5 years (31).

Chlamydial infections not only cause severe acute and chronic disease but have also been epidemiologically linked to a number of human cancers (31–37). Chlamydia has also been reported as a cofactor in increased cancer rates in patients also infected by human papilloma virus (HPV) (35). The high-risk HPV type 16 E6 and E7 oncoproteins can each induce abnormal centrosome numbers (38). However, extra centrosomes do not always lead to multipolarity as centrosomal clustering can prevent the formation of multipolar spindles in cells. Pre-cancerous cells need to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency and transformation to occur. The microtubule motor cytoplasmic dynein is a critical part of this coalescing machinery (39). The unique and dominant interaction of Chlamydia with the host dynein motor protein may be a confounding factor interfering with centrosomal clustering in HPV-infected cells.

The hypothesis that bacterial infections can contribute to cancer has endured for some time, but unlike viral induced cancers, specific oncogenes and molecular mechanisms have not been clearly established (40). There is a strong epidemiological link between chlamydial infection and cervical cancer (31,32,34,36,37). Our data describe a mechanism by which chlamydial infection, through the induction of abnormal centrosome numbers, may be a contributing factor in chromosome instability ultimately leading to transformation and tumor development.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References


Chlamydia trachomatis serovars L2 (LGV 434), D (UW-3/CX), or G (UW-524/CX) were grown in HeLa 229 cells, and EBs were purified by Renografin (Squibb) density gradient centrifugation as described (41). Coxiella burnetii (Nine Mile strain in phase II) was propagated in African green monkey kidney (Vero) fibroblasts (CCL-81; American Type Culture Collection) and purified by Renografin density gradient centrifugation, as previously described (42). Human foreskin (BJ)-derived normal human fibroblasts were obtained from ATCC (CRL-2522) and grown and infected in the same manner as the HeLa cells except the passage number was kept below 10.


All infections were carried out similarly unless otherwise noted. For chlamydial infection, cells were incubated with C. trachomatis EBs at a MOI of approximately 5 in Hanks' balanced salt solution (HBSS) (Invitrogen, Carlsbad, CA, USA) for 30 min at 4 °C. The inoculum was removed, and the coverslips were washed twice with HBSS plus 100 µg/mL heparin (Pharmacia, Peapack, NJ, USA) and once with HBSS without heparin. The HBSS was then replaced with RPMI media containing 10% FBS plus 10 µg/mL gentamicin. Infections were allowed to proceed for appropriate times. For C. burnetii infections, HeLa cells were incubated with purified organisms at an MOI of approximately 5 for 1 hour after which the inoculum was removed and the cells were washed once with HBSS. The HBSS was than replace with RPMI media containing 10% FBS without gentamicin.


HeLa cells were seeded on 12-mm glass coverslips in 24-well plates to obtain a monolayer of approximately 50% confluency. Transfections of plasmid constructs were performed using Lipofectamine 2000 (Invitrogen), as previously described (5). Expression vectors used were EGFP-C1, GFP-β-tubulin (BD Biosciences Clontech, Palo Alto, CA, USA) and GFP-dynamitin (43). Expression from the transfected vectors was allowed to proceed for 24–30 h before experimentation.


Micro-injection of Abs was performed using an automated micro-injection system as described previously (44). Micro-injected Abs used in these experiments were mouse mAb to dic74.1 (Covance, Richmond, CA, USA); mouse mAb to dic70.1 (Abcam, Cambridge, UK); and mouse mAb13-3 to Rickettsia rickettsii (45). Following injection, cells were washed once with RPMI plus 10% FBS, and fresh medium was added. About 10–15 min after injection, the coverslips were infected with C. trachomatis L2. The cells were fixed and permeablized with cold methanol. The injected Abs were detected using AlexaFluor 488-conjugated goat anti-mouse IgG secondary Abs.

Cell culture and microscopy

For fluorescent Ab staining, infected Hela cells on 25-mm number 1 borosilicate coverslips were fixed with cold methanol for 10 min. Abs used in these experiments were mouse mAb to γ-tubulin (Sigma, St. Louis, MO, USA) and mouse mAb to β-tubulin (Sigma). Polyclonal rabbit antisera to C. trachomatis L2, formalin-killed C. burnetii (46), and chlamydial inclusion membrane proteins IncA and IncG (11) were used. To visualize the primary Abs, we incubated the cells with the appropriate secondary Ab; AlexaFluor 488-conjugated IgG, AlexaFluor 567-conjugated IgG, or AlexaFluor 647-conjugated IgG (Molecular Probes, Eugene, OR, USA). To simultaneously visualize DNA, we stained cells with the DNA dye Draq 5 (Biostatus, Leicestershire, UK). Confocal images were acquired on a Perkin–Elmer UltraView spinning disk connected to a Nikon Eclipse TE2000-S microscope with a × 60, 1.4 numerical aperture oil-immersion objective, using a cascade-cooled CCD camera (Roper Scientific, Tempe, AZ, USA) under the control of Metamorph software (Universal Imaging, Downingtown, PA, USA). Projections were constructed using the ImageJ image software (written by Wayne Rasband at the U.S. National Institutes of Health and available at Morphometric measurements were also conducted using ImageJ software.

Micronucleus assay

The micronucleus assay was performed on the rifampicin cured and mock-infected cell population. For the rifampicin treatment, HeLa cells were seeded at 50% confluency and infected with C. trachomatis serovar L2 at a MOI of approximately 5. The infection was allowed to progress for 24 h before replacing the media with RPMI plus 10% FBS containing 50 µg/mL rifampicin. The cultures were monitored for 5 days and split and seeded onto coverslips for microscopy. The cells were plated on coverslips, allowed to adhere, and synchronized with RPMI media containing 10% FCS supplemented with 3 µg/mL aphidicolin (EMD Biosciences, Darmstadt, Germany) overnight. The media containing aphidicolin was removed and replaced with RPMI media with 10% FCS containing 5 mg/mL cytochalasin B (Sigma) to inhibit cytokinesis and incubated for 24 h. The cells were fixed in cold methanol and stained with the nuclear stain Draq 5 (Biostatus). Images were acquired using the spinning disk confocal microscope, and greater than 100 cells per condition containing two nuclei were evaluated for micronuclei. The entire experiment was repeated three times.


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

We thank J. Sager and T. Clark for technical assistance and Drs R. Heinzen, O. Steele-Mortimer, and T. Jewett for review of the manuscript. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
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