Chlamydia induces anchorage independence in 3T3 cells and detrimental cytological defects in an infection model

Abstract

Chlamydia are gram negative, obligate intracellular bacterial organisms with different species causing a multitude of infections in both humans and animals. Chlamydia trachomatis is the causative agent of the sexually transmitted infection (STI) Chlamydia, the most commonly acquired bacterial STI in the United States. Chlamydial infections have also been epidemiologically linked to cervical cancer in women co-infected with the human papillomavirus (HPV). We have previously shown chlamydial infection results in centrosome amplification and multipolar spindle formation leading to chromosomal instability. Many studies indicate that centrosome abnormalities, spindle defects, and chromosome segregation errors can lead to cell transformation. We hypothesize that the presence of these defects within infected dividing cells identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation. Here we demonstrate that infection with Chlamydia trachomatis is able to transform 3T3 cells in soft agar resulting in anchorage independence and increased colony formation. Additionally, we show for the first time Chlamydia infects actively replicating cells in vivo. Infection of mice with Chlamydia results in significantly increased cell proliferation within the cervix, and in evidence of cervical dysplasia. Confocal examination of these infected tissues also revealed elements of chlamydial induced chromosome instability. These results contribute to a growing body of data implicating a role for Chlamydia in cervical cancer development and suggest a possible molecular mechanism for this effect.

Figure 1. Chlamydia induces centrosome and spindle defects in replicating cells.

(A) End1/E6E7 cells were stained for centrosomes (green, top and bottom panel), mitotic spindles (green, middle panel), DNA (blue), and Chlamydia (red, infected panel). Uninfected cells had an average of 2.5±0.1 centrosomes/cell, and infected cells increased to an average of 3.5±0.1 centrosomes/cell, p<0.0001, N>150 (arrow). Uninfected End1s have an average of 21.2±4.6 percent multipolar spindles (arrow), and the infected cells to an average of 73.7±2.0 percent, p = 0.0005, N>150. The presence of multinucleated cells (stars) increased with infection from 6.7±0.8 to 36.6±3.3 percent, p = 0.0009, N>200. (B) COS-7 cells were treated and stained as above. Uninfected cells had 2.1±0.03 centrosomes/cell, and infection resulted in an increase to 2.8±0.2 centrosomes/cell, p = 0.0200, N>300. Uninfected COS-7s had 15.5±0.8 percent multipolar spindle formation, infected cells increased to 30.0±2.9 percent multipolar spindles, p = 0.0093, N>150. Uninfected cells were 13.9±1.9 percent multinucleated, and infected cells increased to 37.1±0.8 multinucleated, p = 0.0003, N>200. (C) Uninfected and infected 3T3 fibroblasts were evaluated as above. Uninfected 3T3 cells 2.2±0.1 centrosomes/cell, infected cells increased to 3.2±0.1 centrosomes/cell, p = 0.0014, N>300. Uninfected cells had 15.5±0.9 percent multipolar spindles, infected cells increased to 41.2±3.5 multipolar, p = 0.0021, N>150. Uninfected 3T3s were 2.9±0.01 percent multinucleated, and upon infection became 4.8±0.1 percent multinucleated, p<0.0001, N>200. Scale bars, 5 μm.

Figure 2. Chlamydial infection induces anchorage independence in 3T3 fibroblasts.

Mock-infected and infected 3T3s were cured of chlamydial infection and incubated for 4 weeks in soft agar. The cells were stained, and the colonies were enumerated and normalized to the 2500 cells initially plated. (A) The uninfected cells had an average of 1.7×10⁻⁴ ±3.1×10⁻⁵ colonies/cells plated, the infected cells an average of 1.5×10⁻³ ±2.6×10⁻⁴ colonies/cells plated. Cells treated with UV light for 1 minute had an average of 9.9×10⁻⁴ ±1.5×10⁻⁴ colonies/cells plated, while cells treated for 3 and 5 minutes had an average of 1.8×10⁻³ ±2.9×10⁻⁴ and 1.4×10⁻³ ±1.6×10⁻⁴ colonies/cells plated, respectively. 3T3 cells cured of an infection with the intracellular bacterial pathogen Coxiella burnetii had an average of 3.3×10⁻⁵ ±2.3×10⁻⁵ colonies/cells plated. N>72 wells, p<0.0001. (B) The images in the panels are examples of stained colonies from mock-infected and L2-infected cells after a 4 week incubation. The first column is a single well of a 6-well plate. The second column is a 10X magnification of the indicated area (dashed box).

Figure 3. Chlamydia infects actively replicating cells in vivo, and induces cell proliferation.

(A) A female FVB wild-type mouse infected with Chlamydia muridarum for 7 days, was treated with EdU (green) prior to sacrifice to detect cell proliferation, and formalin-fixed, paraffin-embedded sections (4 μm thick) were stained for Chlamydia (red) and DNA (blue). The sections were imaged by confocal microscopy. A chlamydial inclusion can be seen associated with an actively replicating EdU positive cell (arrow) within the cervical epithelium. (B) Female K14-HPV-E7 mice and their wild-type littermates were mock-infected and infected with Chlamydia muridarum for 7 days. The mice were treated with EdU, and the EdU positive cells per total cells present in multiple fields of view were counted to determine the rate of cell proliferation. Wild-type uninfected mice, N = 4, had an average of 3.0±0.5 percent cell proliferation, while the infected WT mice, N = 3, experienced a significant increase of cell proliferation at an average of 21.7±5.9 percent, p = 0.003. The uninfected E7 mice had an average of 5.7±1.0 percent cell proliferation, and the infected E7 mice had a significantly higher average of 19.7±2.6 percent, p = 0.0002, N = 4 mice/group. Scale bars, 5 μm.

Figure 4. Presence of Chlamydia muridarum induces CIN.

Infected and mock-infected groups of 3–4 K14-HPV-E7 mice and their wild-type littermates were sacrificed 7 days post-infection. H&E sections were evaluated by our pathologist (LJF) and each animal was given a score based on progression of cervical dysplasia; the scores were averaged for each animal. (A). The wild-type, mock-infected group received an average score of 1.3±0.3, N = 4, indicating these animals retained normal cervical epithelium after treatment. The wild-type infected group received an average score of 3.3±0.3, N = 3 representing a progression to CIN II, p = 0.0037. The K14-HPV-E7 mice followed a similar pattern with the uninfected group receiving a score of 1.8±0.5 indicating most of the animals had normal tissue, while the infected group received a score of 3.5±0.3 indicating these animals also progressed to CIN II, p = 0.0203, N = 4. (B) Uninfected tissue from a wild-type and K14-HPV-E7 mouse. (C) Infected tissue from the mice corresponds with epithelial projections into the stroma and contains cells that have increased nuclear:cytoplasm ratio (arrows). Scale bars, 50 μm.

Figure 5. Evidence of centrosome mislocalization and genetic instability in infected animals.

K14-HPV-E7 mice their wild-type littermates were infected on day 0 and reinfected on day 3, and the animals were examined for phenotypic evidence of precancerous characteristics within the cervix. (A) A 10 μm thick tissue section from a wild-type mouse was stained for centrosomes (green), Chlamydia (red), and DNA (blue). The arrow indicates the centrosome is localized to the chlamydial inclusion. (B) A K14-HPV-E7 tissue section was stained for E-Cadherin (green), Chlamydia (red), and DNA (blue). The stars denote two nuclei within the infected cell. The images in panel C are taken from wild-type tissue sections stained for E-cadherin (green), Chlamydia (red), and DNA (blue). The arrow indicates the formation of a micronuclei. Scale bars, 5 μm.