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pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes.

Beauregard KE, Lee KD, Collier RJ, Swanson JA - J. Exp. Med. (1997)

Bottom Line: Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol.These changes were not seen with LLO-deficient mutants.We conclude that acidic pH facilitates LLO activity in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow-derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9-6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.

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pH analysis of L. monocytogenes–containing vacuoles. Quantitative fluorescence microscopy was used to determine the fluorescence  intensities of vacuoles containing HPTS after infection with L. monocytogenes. A standard curve of pH to 440:405 nm intensity ratios was used to  calibrate the pH of the fluorescence intensities. These graphs are representative time courses from several experiments. Open symbols represent a  sudden loss of fluorescence from the vacuole and, therefore, the end of  the experiment. Traces that end in closed symbols are those that did not  show a sudden loss of fluorescence. These experiments were continued  until the fluorescence signal was lost through quenching and recycling of  the HPTS. (A) Phagosomes containing wild-type L. monocytogenes. Vacuoles showed sudden increases in pH followed by a decrease in the fluorescence signal as the HPTS was released to the cytoplasm. In some cases,  phagosomes reacidified and showed a secondary pH increase (squares). (B)  Control vacuoles in cells infected with wild-type L. monocytogenes. These  vacuoles acidified and occasionally showed evidence of perforation (squares).  (C) Cells were infected with DP-L2161 (diamond, phagosome; square,  control vacuole). This L. monocytogenes mutant lacks LLO because of an  hly deletion. (D) Cells were infected with wild-type L. monocytogenes in  the presence of 0.5 μM bafilomycin A1.
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Figure 4: pH analysis of L. monocytogenes–containing vacuoles. Quantitative fluorescence microscopy was used to determine the fluorescence intensities of vacuoles containing HPTS after infection with L. monocytogenes. A standard curve of pH to 440:405 nm intensity ratios was used to calibrate the pH of the fluorescence intensities. These graphs are representative time courses from several experiments. Open symbols represent a sudden loss of fluorescence from the vacuole and, therefore, the end of the experiment. Traces that end in closed symbols are those that did not show a sudden loss of fluorescence. These experiments were continued until the fluorescence signal was lost through quenching and recycling of the HPTS. (A) Phagosomes containing wild-type L. monocytogenes. Vacuoles showed sudden increases in pH followed by a decrease in the fluorescence signal as the HPTS was released to the cytoplasm. In some cases, phagosomes reacidified and showed a secondary pH increase (squares). (B) Control vacuoles in cells infected with wild-type L. monocytogenes. These vacuoles acidified and occasionally showed evidence of perforation (squares). (C) Cells were infected with DP-L2161 (diamond, phagosome; square, control vacuole). This L. monocytogenes mutant lacks LLO because of an hly deletion. (D) Cells were infected with wild-type L. monocytogenes in the presence of 0.5 μM bafilomycin A1.

Mentions: To calculate the pH of individual phagosomes, digital images were recorded at exc. 440 and 405 nm, and the intensity values from corresonding areas in the two images were divided to obtain ratio values (I440:I405). The ratios were calibrated using pH-clamped cells. Fig. 4, A and B, shows representative changes of pH for vacuoles in cells infected with wild-type L. monocytogenes. The pH of the bacterium-containing vacuoles decreased and then abruptly increased by 0.2–1.6 pH U. Increases in pH were observed in 24 out of 52 vacuoles containing L. monocytogenes (Table 1). The increases were rapid and usually accompanied by release of dye into the cytoplasm. Release of HPTS did not always follow the rise in pH. In fact, sometimes vacuoles reacidified (Fig. 4 A). This reacidification was often followed by another sudden increase in pH and more complete release of the HPTS into the cytosol.


pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes.

Beauregard KE, Lee KD, Collier RJ, Swanson JA - J. Exp. Med. (1997)

pH analysis of L. monocytogenes–containing vacuoles. Quantitative fluorescence microscopy was used to determine the fluorescence  intensities of vacuoles containing HPTS after infection with L. monocytogenes. A standard curve of pH to 440:405 nm intensity ratios was used to  calibrate the pH of the fluorescence intensities. These graphs are representative time courses from several experiments. Open symbols represent a  sudden loss of fluorescence from the vacuole and, therefore, the end of  the experiment. Traces that end in closed symbols are those that did not  show a sudden loss of fluorescence. These experiments were continued  until the fluorescence signal was lost through quenching and recycling of  the HPTS. (A) Phagosomes containing wild-type L. monocytogenes. Vacuoles showed sudden increases in pH followed by a decrease in the fluorescence signal as the HPTS was released to the cytoplasm. In some cases,  phagosomes reacidified and showed a secondary pH increase (squares). (B)  Control vacuoles in cells infected with wild-type L. monocytogenes. These  vacuoles acidified and occasionally showed evidence of perforation (squares).  (C) Cells were infected with DP-L2161 (diamond, phagosome; square,  control vacuole). This L. monocytogenes mutant lacks LLO because of an  hly deletion. (D) Cells were infected with wild-type L. monocytogenes in  the presence of 0.5 μM bafilomycin A1.
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Related In: Results  -  Collection

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Figure 4: pH analysis of L. monocytogenes–containing vacuoles. Quantitative fluorescence microscopy was used to determine the fluorescence intensities of vacuoles containing HPTS after infection with L. monocytogenes. A standard curve of pH to 440:405 nm intensity ratios was used to calibrate the pH of the fluorescence intensities. These graphs are representative time courses from several experiments. Open symbols represent a sudden loss of fluorescence from the vacuole and, therefore, the end of the experiment. Traces that end in closed symbols are those that did not show a sudden loss of fluorescence. These experiments were continued until the fluorescence signal was lost through quenching and recycling of the HPTS. (A) Phagosomes containing wild-type L. monocytogenes. Vacuoles showed sudden increases in pH followed by a decrease in the fluorescence signal as the HPTS was released to the cytoplasm. In some cases, phagosomes reacidified and showed a secondary pH increase (squares). (B) Control vacuoles in cells infected with wild-type L. monocytogenes. These vacuoles acidified and occasionally showed evidence of perforation (squares). (C) Cells were infected with DP-L2161 (diamond, phagosome; square, control vacuole). This L. monocytogenes mutant lacks LLO because of an hly deletion. (D) Cells were infected with wild-type L. monocytogenes in the presence of 0.5 μM bafilomycin A1.
Mentions: To calculate the pH of individual phagosomes, digital images were recorded at exc. 440 and 405 nm, and the intensity values from corresonding areas in the two images were divided to obtain ratio values (I440:I405). The ratios were calibrated using pH-clamped cells. Fig. 4, A and B, shows representative changes of pH for vacuoles in cells infected with wild-type L. monocytogenes. The pH of the bacterium-containing vacuoles decreased and then abruptly increased by 0.2–1.6 pH U. Increases in pH were observed in 24 out of 52 vacuoles containing L. monocytogenes (Table 1). The increases were rapid and usually accompanied by release of dye into the cytoplasm. Release of HPTS did not always follow the rise in pH. In fact, sometimes vacuoles reacidified (Fig. 4 A). This reacidification was often followed by another sudden increase in pH and more complete release of the HPTS into the cytosol.

Bottom Line: Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol.These changes were not seen with LLO-deficient mutants.We conclude that acidic pH facilitates LLO activity in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow-derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9-6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.

Show MeSH
Related in: MedlinePlus