Porphyromonas gingivalis SerB-mediated dephosphorylation of host cell cofilin modulates invasion efficiency

Abstract

Porphyromonas gingivalis, a host-adapted opportunistic pathogen, produces a serine phosphatase, SerB, known to affect virulence, invasion and persistence within the host cell. SerB induces actin filament rearrangement in epithelial cells, but the mechanistic basis of this is not fully understood. Here we investigated the effects of SerB on the actin depolymerizing host protein cofilin. P. gingivalis infection resulted in the dephosphorylation of cofilin in gingival epithelial cells. In contrast, a SerB-deficient mutant of P. gingivalis was unable to cause cofilin dephosphorylation. The involvement of cofilin in P. gingivalis invasion was determined by quantitative image analysis of epithelial cells in which cofilin had been knocked down or knocked in with various cofilin constructs. siRNA-silencing of cofilin led to a significant decrease in numbers of intracellular P. gingivalis marked by an absence of actin colocalization. Transfection with wild-type cofilin or constitutively active cofilin both increased numbers of intracellular bacteria, while constitutively inactive cofilin abrogated invasion. Expression of LIM kinase resulted in reduced P. gingivalis invasion, an effect that was reversed by expression of constitutively active cofilin. These results show that P. gingivalis SerB activity induces dephosphorylation of cofilin, and that active cofilin is required for optimal invasion into gingival epithelial cells.

Figure 1. Western blot analysis of cofilin and p-cofilin levels in P. gingivalis–infected HIGK cells

Whole cell lysates of P. gingivalis 33277-infected, P. gingivalis ΔSerB-infected, or P. gingivalis ΔSerB+pserB-infected HIGK cells were examined by Western blotting with antibodies to cofilin (upper panel) or phospho(p)-cofilin (lower panel). Data are representative of three independent experiments.

Figure 2. Fluorescence analysis of cofilin and p-cofilin levels in P. gingivalis–infected HIGK cells

A) P. gingivalis 33277-infected or ΔSerB-infected HIGK cells were labeled with antibodies to cofilin (upper panel) or p-cofilin (lower panel) and analyzed by CSLM. Controls were uninfected HIGKs. Magnification x63 of cells at 10 min time point. B) Fluorescence intensity analysis of p-cofilin:cofilin in infected or control HIGK cells. Intensity values were obtained using Leica Confocal software (arbitrary units) to calculate ratios. Results are representative of two independent experiments (n=3 coverslips/group). Data are means and error bars indicate standard deviation. **, P <0.01; ***, P <0.001 by Tukey-Kramer Multiple Comparison test.

Figure 3. Internalization of P. gingivalis is inhibited by cofilin-silencing

HIGK cells were transfected with silence(si)-cofilin, non-silence(non-si), or scramble cofilin. Control was transfection agent only. A) Western blot analysis to confirm cofilin-silencing in HIGK cells. Whole cell lysates were examined by Western blotting with antibodies to cofilin. GAPDH was used as a loading control. B) Blots were analysed by scanning densiometry to determine relative cofilin levels. Data are representative of two independent experiments. C) Cofilin-silenced HIGK cells (or non-si/scramble controls) were labeled with cofilin antibodies (yellow) and analyzed by CSLM. Magnification x63. D) Cofilin-silenced HIGK cells (or non-silence/scramble controls) were infected with P. gingivalis for 10 min and analyzed by CSLM. P. gingivalis (green) was detected with specific antibodies, actin (red) was stained with TRITC-phalloidin, and nuclei (blue) stained with DAPI. Magnification x63. Results are representative of three independent assays. Data shown are maximum projections of z-stacks (10 slices/z stack, 3 coverslips/group). E) Invasion levels of P. gingivalis in HIGK cells following cofilin-silencing. Numbers of intracellular bacteria were counted throughout z-stacks (10 slices/stack; 3 coverslips/group). Results are representative of three independent assays. Data are means and error bars indicate standard deviations. **, P <0.01; ***, P <0.001 by Tukey-Kramer Multiple Comparison test.

Figure 3. Internalization of P. gingivalis is inhibited by cofilin-silencing

HIGK cells were transfected with silence(si)-cofilin, non-silence(non-si), or scramble cofilin. Control was transfection agent only. A) Western blot analysis to confirm cofilin-silencing in HIGK cells. Whole cell lysates were examined by Western blotting with antibodies to cofilin. GAPDH was used as a loading control. B) Blots were analysed by scanning densiometry to determine relative cofilin levels. Data are representative of two independent experiments. C) Cofilin-silenced HIGK cells (or non-si/scramble controls) were labeled with cofilin antibodies (yellow) and analyzed by CSLM. Magnification x63. D) Cofilin-silenced HIGK cells (or non-silence/scramble controls) were infected with P. gingivalis for 10 min and analyzed by CSLM. P. gingivalis (green) was detected with specific antibodies, actin (red) was stained with TRITC-phalloidin, and nuclei (blue) stained with DAPI. Magnification x63. Results are representative of three independent assays. Data shown are maximum projections of z-stacks (10 slices/z stack, 3 coverslips/group). E) Invasion levels of P. gingivalis in HIGK cells following cofilin-silencing. Numbers of intracellular bacteria were counted throughout z-stacks (10 slices/stack; 3 coverslips/group). Results are representative of three independent assays. Data are means and error bars indicate standard deviations. **, P <0.01; ***, P <0.001 by Tukey-Kramer Multiple Comparison test.

Figure 4. Expression of exogenous or constitutively active cofilin increases internalization of P. gingivalis

HIGK cells were transfected with exogenous cofilin, constitutively active cofilin (S3A), or constitutively inactive cofilin (S3E). Control was transfection agent only. A) Confirmation of an increase in cofilin expression in transfected cells. Cofilin-transfected HIGK cells (or control) were labeled with cofilin antibodies (yellow) and analyzed by CSLM. Magnification x63. B) Fluorescence intensity analysis of cofilin HIGK cells. Intensity values were obtained using Leica Confocal software (arbitrary units) to calculate cofilin fluorescence intensity. Results are representative of two independent experiments (n=3 coverslips/group). Data are means and error bars indicate standard deviations. *, P <0.05; **, P <0.01; ***, P <0.001 by unpaired t-test to control. C) Cofilin-transfected cells were infected with P. gingivalis for 10 min and analyzed by CSLM. P. gingivalis (green) was detected with specific antibodies, actin (red) was stained with TRITC-phalloidin, and nuclei (blue) stained with DAPI. Magnification x63. Results are representative of three independent assays. Data shown are maximum projections of z-stacks (10 slices/z stack, 3 coverslips/group). D) Invasion levels of P. gingivalis following cofilin-transfection. Numbers of intracellular bacteria were counted throughout z-stacks (10 slices/stack; 3 coverslips/group). Results are representative of three independent assays. Data are ratios of internalization in transfected-infected groups compared with non-transfected control-infected cells. *, P <0.05; **, P <0.01 by unpaired t-test to control.

Figure 4. Expression of exogenous or constitutively active cofilin increases internalization of P. gingivalis

HIGK cells were transfected with exogenous cofilin, constitutively active cofilin (S3A), or constitutively inactive cofilin (S3E). Control was transfection agent only. A) Confirmation of an increase in cofilin expression in transfected cells. Cofilin-transfected HIGK cells (or control) were labeled with cofilin antibodies (yellow) and analyzed by CSLM. Magnification x63. B) Fluorescence intensity analysis of cofilin HIGK cells. Intensity values were obtained using Leica Confocal software (arbitrary units) to calculate cofilin fluorescence intensity. Results are representative of two independent experiments (n=3 coverslips/group). Data are means and error bars indicate standard deviations. *, P <0.05; **, P <0.01; ***, P <0.001 by unpaired t-test to control. C) Cofilin-transfected cells were infected with P. gingivalis for 10 min and analyzed by CSLM. P. gingivalis (green) was detected with specific antibodies, actin (red) was stained with TRITC-phalloidin, and nuclei (blue) stained with DAPI. Magnification x63. Results are representative of three independent assays. Data shown are maximum projections of z-stacks (10 slices/z stack, 3 coverslips/group). D) Invasion levels of P. gingivalis following cofilin-transfection. Numbers of intracellular bacteria were counted throughout z-stacks (10 slices/stack; 3 coverslips/group). Results are representative of three independent assays. Data are ratios of internalization in transfected-infected groups compared with non-transfected control-infected cells. *, P <0.05; **, P <0.01 by unpaired t-test to control.

Figure 4. Expression of exogenous or constitutively active cofilin increases internalization of P. gingivalis

HIGK cells were transfected with exogenous cofilin, constitutively active cofilin (S3A), or constitutively inactive cofilin (S3E). Control was transfection agent only. A) Confirmation of an increase in cofilin expression in transfected cells. Cofilin-transfected HIGK cells (or control) were labeled with cofilin antibodies (yellow) and analyzed by CSLM. Magnification x63. B) Fluorescence intensity analysis of cofilin HIGK cells. Intensity values were obtained using Leica Confocal software (arbitrary units) to calculate cofilin fluorescence intensity. Results are representative of two independent experiments (n=3 coverslips/group). Data are means and error bars indicate standard deviations. *, P <0.05; **, P <0.01; ***, P <0.001 by unpaired t-test to control. C) Cofilin-transfected cells were infected with P. gingivalis for 10 min and analyzed by CSLM. P. gingivalis (green) was detected with specific antibodies, actin (red) was stained with TRITC-phalloidin, and nuclei (blue) stained with DAPI. Magnification x63. Results are representative of three independent assays. Data shown are maximum projections of z-stacks (10 slices/z stack, 3 coverslips/group). D) Invasion levels of P. gingivalis following cofilin-transfection. Numbers of intracellular bacteria were counted throughout z-stacks (10 slices/stack; 3 coverslips/group). Results are representative of three independent assays. Data are ratios of internalization in transfected-infected groups compared with non-transfected control-infected cells. *, P <0.05; **, P <0.01 by unpaired t-test to control.

Figure 5. Expression of LIMK-1 in HIGK cells reduces invasion levels of P. gingivalis

HIGK cells were transfected with LIMK-1 and/or S3A. Control was transfection agent only. A) Scanning densiometry of Western blots of LIMK-1 levels in transfected cells expressed as ratio to GAPDH. Data are representative of two independent experiments. B) LIMK-1/S3A-transfected HIGK cells were infected with P. gingivalis for 10 min and analyzed by CSLM. Control was transfection agent. P. gingivalis (green) was detected with specific antibodies, actin (red) was stained with TRITC-phalloidin, and nuclei (blue) stained with DAPI. Magnification x63. Results are representative of three independent assays. Data shown are maximum projections of z-stacks (10 slices/z stack, 3 coverslips/group). C) Invasion levels of P. gingivalis following LIMK-1/S3A transfection. Numbers of intracellular bacteria were counted throughout z-stacks (10 slices/stack; 3 coverslips/group). Results are representative of three independent assays. Data are means and error bars indicate standard deviations. *, P <0.05; **, P <0.01 by Tukey-Kramer Multiple Comparison test.