Aggregatibacter actinomycetemcomitans cytolethal distending toxin modulates host phagocytic function

Abstract

Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. Human macrophages exposed to Aggregatibacter actinomycetemcomitans (Aa) Cdt respond through canonical and non-canonical inflammasome activation to stimulate cytokine release. The inflammatory response is dependent on PI3K signaling blockade via the toxin's phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity; converting PIP3 to phosphatidylinsoitol-3,4-diphosphate (PI3,4P2) thereby depleting PIP3 pools. Phosphoinositides, also play a critical role in phagosome trafficking, serving as binding domains for effector proteins during phagosome maturation and subsequent fusion with lysosomes. We now demonstrate that AaCdt manipulates the phosphoinositide (PI) pools of phagosome membranes and alters Rab5 association. Exposure of macrophages to AaCdt slowed phagosome maturation and decreased phago-lysosome formation, thereby compromising macrophage phagocytic function. Moreover, macrophages exposed to Cdt showed decreased bactericidal capacity leading to increase in Aggregatibacter actinomycetemcomitans survival. Thus, Cdt may contribute to increased susceptibility to bacterial infection. These studies uncover an underexplored aspect of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aa.

Keywords: Aggregatibacter actinomycetemcomitans; Cytolethal distending toxin; localized aggressive periodontitis; phagocytosis; phagosome maturation; phosphoinositide.

Figure 1

Specificity of Aa CdtB phosphatase activity. (A) Intracellular PI pools are modulated by CdtB. PI(3,4,5)P3, PI(3,4)P2 and PI(4,5)P2, levels were measured by ELISA in extracts prepared from THP1 macrophages treated with Cdt^WT (500 ng/ml) or Cdt^R117A (500 ng/ml) as in Methods. Data represent mean +/- STDEV (n=5) and compared using Student’s t-test, **p<0.05. (B) Visualization and localization of PI3,4P2 in THP1 macrophages treated with Cdt^WT (500 ng/ml) or Cdt^R117A (500 ng/ml). PI3,4P2 (Green) as indicated by white arrows, Nucleus (Blue). Quantification of PI(3,4)P2 florescence intensity presented as percent relative to control (untreated). Data represent mean +/- STDEV (five fields with average of 12 cells/field) and compared using Student’s t-test, **p<0.05. (C) Aa Cdt modulates PI3,4P2 intracellular pools in dose dependent manner. THP1 macrophages were treated with Cdt^WT (63ng/ml, 125ng/ml, 500ng/ml) and control (untreated) as in Methods. Visualization of PI3,4P2 level in dose dependent manner, PI3,4P2 (green, as indicated by white arrows), Nucleus (Blue). Quantification of PI(3,4)P2 fluorescence intensity presented as percent relative to the control (untreated). Results are expressed as percent of control (untreated), mean +/- STDEV (five fields with average of 12 cells/field) and compared using Student’s t-test. *p-value<0.05 vs. untreated control.

Figure 2

Cdt Phosphatase activity modulates host phagocytic activity. (A) Time lapse imaging of pHrodo™ in THP1 macrophages pre-treated with Cdt^WT or control (untreated) for 4 hours. pHrodo™ fluorescence (pseudo cyan colored from red) was measured 30 minutes over 3 hours (Top panel: Control (untreated), Bottom panel: Cdt^WT treated). Exemplar cells are outlined with white dotted boarder. Intensity measurements are plotted over the experimental time course in the corresponding graph. (B) pHrodo™ intensity at 180 minutes, using a 96 well plate format is shown as a function of increasing concentrations of Cdt^WT or Cdt^R117A as percent of control (untreated) as described in Methods. Data represent mean +/- STDEV (5 wells per individual experiment with 1x10⁶ cells/well) and compared using Student’s t-test. **p-value<0.005 Cdt^WT vs Cdt^R117A.

Figure 3

PI(3,4)P2 phagosome association increases with Cdt treatment. THP1 macrophages treated for 4 hours with Cdt^WT (A), Cdt^R117A (B), or control (untreated, C) prior to synchronized uptake of opsonized beads were fixed and stained for PI(3,4)P2 as in Methods. Green: PI(3,4)P2, Blue: nucleus, and Red: opsonized beads. (D) Quantified data represent average +/- STDEV of PI(3,4)P2 intensity in 2x2μm area (square) around the bead (10 beads for each for the conditions from 5 random fields) as a percent to control and compared using Student’s t-test. **p<0.05, vs Cdt^R117A.

Figure 4

Cdt treatment increases Rab5 phagosome association. THP1 macrophages treated for 4 hours with Cdt^WT (500ng/ml), Cdt^R117A (500ng/ml) or control (untreated) prior to synchronized uptake of opsonized beads and were fixed and stained for Rab5 and EEA1 as in Methods. Green: Rab5 (A) or EEA1 (B), Blue: nucleus, and Red: opsonized beads. Data represent mean +/- STDEV of Rab5 (A) or EEA1 (B) intensity in 2x2µm area (square) around the bead (10 beads for each for the conditions from 5 different fields) as percent relative to control (untreated) and compared using Student’s t-test. **p-value<0.05 Cdt^WT vs Cdt^R117A.

Figure 5

Cdt treatment does not alter levels of intracellular trafficking proteins, EEA1, Rab5, Rab7, and LAMP1. THP-1 macrophages treated with Cdt^WT, Cdt^R117A, or control (untreated). (A) Cells were challenged with opsonized latex beads for 30 minutes and lysates collected for western blot as in Methods. (B) Quantified data represent mean +/- STDEV of EEA1, Rab5, Rab7, and LAMP1 levels (n=3) as percent of control (untreated) and compared using Student’s t-test.

Figure 6

Cdt decreases phagosome maturation. (A) Time lapse imaging of DQ™-BSA in THP1 macrophages pre-treated with Cdt^WT or control (untreated) for 4 hours. DQ™-BSA fluorescence is indicated by white arrows every 30 minutes over 3 hours (Top panel: Untreated, Bottom panel: Cdt^WT). Intensity measurements are plotted over the same time course in the corresponding graph. (B) DQ™-BSA intensity at 180 minutes, using 96 well plate format is shown as a function of increasing concentrations of Cdt^WT or Cdt^R117A as percent of control (untreated) as in Methods. Data represent mean +/- STDEV (5 wells per individual experiment with 1x10⁶ cells/well) and compared using Student’s t-test. **p-value<0.005 Cdt^WT vs Cdt^R117A.

Figure 7

Cdt pretreatment enhances Aa survival. (A) THP1 macrophages were pre-treated with 500ng/ml Cdt^WT for 4 hours or control (untreated). Cells were subsequently incubated with D7S and D7SΔCdt for 0 and 12hours. Cells treated as in Methods and lysates were plated on agar plate and CFU units were determined. Data represent mean +/- STDEV (n=3) as percent of survived within 12 hours as described in Methods and compared using Student’s t-test **p<0.05 vs No Cdt. (B) Confirmation of Cdt production in D7S and loss of Cdt in D7SΔCdt strains. D7S and D7SΔCdt were plated on agar and then cultured by broth media. Western blot analysis was performed on bacterial lysates and positive control (purified Cdt) using mouse antibodies for Cdt subunits to confirm the mutations.

Figure 8

Overview of Cdt internalization and the effect on the phagosome. After the internalization, CdtB act as a phosphatase and convert the PI(3,4,5)P3 to PI(3,4)P2. Change in the PI pool leads to manipulation of phagosome maturation.