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. 2013 May 7;8(5):e62706.
doi: 10.1371/journal.pone.0062706. Print 2013.

Mycoplasma pneumoniae CARDS toxin is internalized via clathrin-mediated endocytosis

Manickam Krishnan  1 , T R Kannan, Joel B Baseman
Affiliations

Mycoplasma pneumoniae CARDS toxin is internalized via clathrin-mediated endocytosis

Manickam Krishnan et al. PLoS One. .

Abstract

Bacterial toxins possess specific mechanisms of binding and uptake by mammalian cells. Mycoplasma pneumoniae CARDS (Community Acquired Respiratory Distress Syndrome) toxin is a 68 kDa protein, which demonstrates high binding affinity to human surfactant protein-A and exhibits specific biological activities including mono-ADP ribosylation and vacuolization. These properties lead to inflammatory processes in the airway and a range of cytopathologies including ciliostasis, loss of tissue integrity and injury, and cell death. However, the process by which CARDS toxin enters target cells is unknown. In this study, we show that CARDS toxin binds to mammalian cell surfaces and is internalized rapidly in a dose and time-dependent manner using a clathrin-mediated pathway, as indicated by inhibition of toxin internalization by monodansylcadaverine but not by methyl-β-cyclodextrin or filipin. Furthermore, the internalization of CARDS toxin was markedly inhibited in clathrin-depleted cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Binding and internalization of rCARDS toxin in HeLa cells.
A) rCARDS toxin localizes to HeLa cell surfaces. HeLa cell monolayers were treated with 10 µg/ml of rCARDS toxin for 1 h at 4°C, stained with rabbit polyclonal antibodies reactive against CARDS toxin (1∶1000) and counterstained with goat anti-rabbit IgG conjugated Alexa Fluor-633 antibodies (red). Cell nuclei were stained with DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride). Z series at 0.5 micrometer sections were obtained by combining a series of x-y scans taken along the z axis. B-D) rCARDS toxin internalization is time dependent. HeLa cells were treated with 10 µg/ml of rCARDS toxin for B) 15 min; C) 30 min; and D) 1 h at 37°C. Cells were processed as described above. White arrows point to internalized rCARDS toxin based upon horizontal and vertical z projections.
Figure 2
Figure 2. Binding kinetics of fluorescence labeled rCARDS toxin.
(A) rCARDS toxin binding to HeLa cells is concentration dependent. HeLa cells were treated with different concentrations (0.1 to 25 µg/well) of DyLight 649 fluorescence labeled rCARDS toxin for 1 h at 4°C. Cell monolayers were thoroughly washed with cold PBS buffer, and toxin binding was measured using fluorometer. (B) rCARDS toxin binding to HeLa cells is time dependent. HeLa cells were treated with 10 µg of DyLight 649 fluorescence-labeled rCARDS toxin for 15 min to 8 h at 4°C, and binding was analyzed as described above.
Figure 3
Figure 3. FACS analysis of rCARDS toxin association with HeLa cells.
A) HeLa cells were treated with 10 µg/ml of pacific blue-A fluorescence dye-coupled toxin (PBA-CARDS) at different time intervals (5 min to 8 h) at 4°C, washed and quantified by FACS. Cells treated with carrier solution served as controls. B) HeLa cells were treated with 10 µg/ml of rCARDS toxin for 30 min at 4°C, washed and incubated with rabbit polyclonal anti-CARDS toxin antibodies (1∶1000) and counterstained with goat anti-rabbit IgG-conjugated with Alexa Fluor 488 (1∶500) followed by FACS. The control shows HeLa cells with no toxin treatment but stained with primary and secondary antibodies.
Figure 4
Figure 4. Binding and internalization of biotin-labeled rCARDS toxin.
(A) Biotin-labeled rCARDS toxin binding to HeLa cells is time-dependent. HeLa cells were treated with 10 µg/ml of biotin-labeled rCARDS toxin for 15 min to 8 h at 4°C. After removing unbound toxin by washing, we analyzed binding using horseradish peroxidase-conjugated streptavidin. Cell-bound rCARDS toxin was measured at 450 nm using ELISA reader as described in Materials and Methods. (B) Internalization of rCARDS toxin in HeLa cells. HeLa cells were treated with 10 µg/ml of biotin-labeled rCARDS toxin for 30 min at 4°C and then incubated at 37°C for the indicated times after removing unbound toxin by washing. At specific time intervals, cells were fixed and surface-bound biotin was removed with MESNA. Internalized biotin-associated rCARDS toxin was quantified as described in Materials and Methods. Data are means ±SD of triplicate samples.
Figure 5
Figure 5. Clathrin-rCARDS toxin association.
(A) Small interfering RNA (siRNA) mediated gene silencing of clathrin heavy chain. HeLa cells were transfected with clathrin heavy chain targeted siRNA (C-siRNA) or scrambled siRNA (S-siRNA) as described in Materials and Methods. Twenty-four h after the second siRNA transfection, cells were lysed and clathrin expression was analyzed using anti-clathrin (1∶500) or anti-GAPDH (1∶2000) antibodies as detailed in Materials and Methods and described in this legend. (B) Quantification of clathrin heavy chain expression in C-siRNA- and S-siRNA-treated HeLa cells. Clathrin and GAPDH band intensities were estimated using KODAK 1D Image analysis software, and results are from three independent experiments. (C) Binding of rCARDS toxin in clathrin-depleted cells. S-siRNA-treated (open square) and C-siRNA-treated (open circle) HeLa cells were incubated with 10 µg/ml of rCARDS toxin for 1 h at 4°C, and toxin binding was analyzed as described in Materials and Methods. Data are means ±SD values from two triplicate experiments. (D) Internalization of rCARDS toxin in clathrin-depleted HeLa cells. S-siRNA-treated (white bar) and C-siRNA-treated (gray bar) HeLa cells were incubated with 10 µg/ml of Biotin-CARDS toxin for 1 h at 4°C and shifted to 37°C for 1 h. After removing surface-bound biotin with MESNA, internalized rCARDS toxin was measured as described in Materials and Methods. (E) Clathrin effect on rCARDS toxin-mediated vacuole formation. S-siRNA-treated (i) and C-siRNA-treated (ii) HeLa cells were incubated with 50 µg/ml of rCARDS toxin for 24 h at 37°C and analyzed under light microscopy for vacuole formation as described previously .
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References

    1. Baseman JB, Tully JG (1997) Mycoplasmas: sophisticated, reemerging, and burdened by their notoriety. Emerg Infect Dis 3: 21–32. - PMC - PubMed
    1. Waites KB, Talkington DF (2004) Mycoplasma pneumoniae and its role as a human pathogen. Clin Microbiol Rev 17: 697–728. - PMC - PubMed
    1. Sanchez-Vargas FM, Gomez-Duarte OG (2008) Mycoplasma pneumoniae-an emerging extra-pulmonary pathogen. Clin Microbiol Infect 14: 105–117. - PubMed
    1. Nisar N, Guleria R, Kumar S, Chand Chawla T, Ranjan Biswas N (2007) Mycoplasma pneumoniae and its role in asthma. Postgrad Med J 83: 100–104. - PMC - PubMed
    1. Gil JC, Cedillo RL, Mayagoitia BG, Paz MD (1993) Isolation of Mycoplasma pneumoniae from asthmatic patients. Ann Allergy 70: 23–25. - PubMed

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