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Human Cytomegalovirus Enhances A. actinomycetemcomitans Adherence to Cells

¹ Catholic University Leuven, Research Group for Microbial Adhesion, Department of Periodontology, Kapucijnenvoer 7, 3000 Leuven, Belgium;
² UCLA, School of Dentistry, 10833 Le Conte Avenue, Los Angeles, CA, USA;
³ Catholic University Leuven, Centre for Molecular Diagnostics, Herestraat 49, 3000 Leuven, Belgium;
⁴ University of Vermont, Department of Microbiology and Molecular Genetics, Stafford Hall, 95 Carrigan Drive, Burlington, VT, USA; and
⁵ Catholic University Leuven, Laboratory of Clinical Virology, Rega Institute for Medical Research, Minderbroedersstraat 10, 3000 Leuven, Belgium

View larger version (74K): [in this window] [in a new window]   Figure 1. hCMV antigen expression in epithelial cells. HeLa (A) and primary cells (B) were infected with hCMV for 48 hrs. Cells were fixed with ice-cold absolute methanol and blocked with 10% normal goat serum. To detect hCMV IE antigens, we incubated cells with primary mouse antibodies (1:80) recognizing IE (clone E13) (Argene, Varilhes, France), and reacted them with fluorescein-conjugated goat anti-mouse IgG antibodies (Dako, Heverlee, Belgium) at 1:200 dilution. Images were made with an Olympus IX70 confocal microscope. (1) Phase-contrast image, (2) fluorescent image, (3) overlap image showing bright IE-positive nuclei for some epithelial cells.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effect of hCMV concentration on adherence of Actinobacillus actinomycetemcomitans strains to HeLa cells. Cells were exposed to hCMV for 1 hr (MOI range: 10–200) and incubated for an additional 48 hrs. Uninfected cells served as controls. Data are expressed as the proportion of cell-associated bacteria relative to the numbers of bacteria added to the cells initially. Each datapoint represents the mean ± SEM for 4 independent experiments. *P < 0.05, hCMV-infected group vs. uninfected controls. #P < 0.05, Actinobacillus actinomycetemcomitans strain IDH1705 vs. strains JP2 and ATCC33384 at MOI 200.

View larger version (18K): [in this window] [in a new window]   Figure 3. Viral pre-infection time and bacterial adherence. (A) Effect of hCMV pre-infection time on adherence of Actinobacillus actinomycetemcomitans strains to HeLa cells. Cells were exposed for 1 hr to hCMV (MOI 100) and incubated for an additional 6, 12, 24, or 48 hrs, or were mock-infected for 48 hrs (squares). Uninfected cells served as controls. Data are expressed as the proportion of cell-associated bacteria relative to the numbers of bacteria added to the cells initially. Each datapoint represents the mean ± SEM for 12 independent experiments. *P < 0.05, hCMV-infected group vs. uninfected control; #P < 0.05, 48-hour hCMV-infected group vs. 48-hour mock-infected group; °P < 0.05, 48-hour vs. 24-hour hCMV-infected group. (B) Effect of hCMV pre-infection time on adherence of Actinobacillus actinomycetemcomitans strain JP2 to primary cells. Cells were exposed for 1 hr to hCMV or mock preparation (MOI 100) and incubated for an additional 6, 12, 24, or 48 hrs. Uninfected cells served as controls. Data are expressed as the proportion of cell-associated bacteria relative to the numbers of bacteria added to the cells initially. Each datapoint represents the mean ± SEM for 8 independent experiments. *P < 0.05, hCMV- or mock-infected group vs. uninfected control; #P < 0.05, hCMV vs. mock-infected group.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of IE-antigen expression on adherence of Actinobacillus actinomycetemcomitans strain JP2 to confluent monolayers. HEL and primary cells were exposed for 1 hr to hCMV or the mock preparation (MOI 100) and incubated for 96 hrs. Uninfected cells served as controls. Data are expressed as the proportion of cell-associated bacteria relative to the number of cell-associated bacteria on uninfected control cells (= 100%). Each datapoint represents the mean ± SEM for 6 independent experiments. *P < 0.05, hCMV- or mock-infected group vs. uninfected control; #P < 0.05, hCMV vs. mock-infected group.