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Pseudomonas aeruginosa cytotoxin: periplasmic localization and inhibition of macrophages
J L Kluftinger1, F Lutz, R E Hancock
1Department of Microbiology, University of British Columbia, Vancouver, Canada.
Abstract:
Pseudomonas aeruginosa cytotoxin has been isolated previously from cell autolysates. Both purified cytotoxin and periplasmic contents (osmotic shock fluid) cross-reacted on Western immunoblots with antibodies specific for cytotoxin. In addition, both preparations caused a significant reduction in antibody-mediated phagocytosis of P. aeruginosa M2 by mouse macrophage cell line P388D1. Phagocytosis was restored in each case on preincubation of cytotoxin or periplasmic contents with anti-cytotoxin serum. Both cytotoxin and periplasmic contents caused depolarization of the P388D1 cell membrane, as demonstrated with a polarization-sensitive fluorescent probe. Similar correlations were not observed for other P. aeruginosa cell fractions or for osmotic shock fluid from Escherichia coli C600. These data indicate that P. aeruginosa cytotoxin is localized in the periplasm and has the potential to inhibit macrophage-mediated phagocytosis, possibly by perturbing ion gradients across the macrophage plasma membrane.
Insights
Pseudomonas aeruginosa cytotoxin, found in the periplasm, inhibits macrophage phagocytosis by disrupting cell membranes. Antibodies can neutralize this effect, restoring normal immune cell function.
Area of Science:
- Microbiology
- Immunology
- Cell Biology
Background:
- Pseudomonas aeruginosa cytotoxin (PAC) previously isolated from cell autolysates.
- PAC's role in immune evasion and cellular mechanisms remained unclear.
Purpose of the Study:
- To determine the localization of PAC.
- To investigate PAC's effect on macrophage phagocytosis.
- To elucidate the cellular mechanism of PAC-induced inhibition.
Main Methods:
- Western immunoblotting using specific antibodies.
- Assessing antibody-mediated phagocytosis of P. aeruginosa M2 by P388D1 macrophages.
- Measuring P388D1 cell membrane potential using a fluorescent probe.
Main Results:
- Purified PAC and periplasmic contents cross-reacted with anti-cytotoxin antibodies.
- Both preparations significantly reduced phagocytosis, which was restored by anti-cytotoxin serum.
- PAC caused P388D1 cell membrane depolarization, unlike other bacterial fractions.
Conclusions:
- Pseudomonas aeruginosa cytotoxin is localized in the bacterial periplasm.
- PAC inhibits macrophage phagocytosis, likely by perturbing macrophage plasma membrane ion gradients.
- PAC represents a potential virulence factor contributing to P. aeruginosa immune evasion.