Fibronectin is capable of activating macrophages for enhanced nonopsonic phagocytosis of Pseudomonas aeruginosa grown in vivo in rats or mice or in vitro on nutrient agar plates. In this study it was determined that while fibronectin was able to significantly increase phagocytosis of organisms grown in static broth, uptake of agitated bacteria could not be promoted. Agitated P. aeruginosa cultures were proven to lack surface pili expression, as assessed by electron microscopic studies. A pilus-deficient piLA::TnSOI mutant of P. aeruginosa PAO was constructed by gene replacement techniques. Phagocytosis of this mutant could not be enhanced by fibronectin regardless of growth conditions. Furthermore, 60 ,ug of exogenously added Pseudomonas pili per ml was capable of abrogating the enhanced phagocytosis of the wild-type strain observed with fibronectinstimulated macrophages. It is concluded that Pseudomonas pili were the bacterial ligands required for attachment to fibronectin-stimulated macrophages in the initial stages of nonopsonic phagocytosis.
In a previous investigation it was determined that Pseudomonas aeruginosa cells taken directly from a mouse in vivo growth system were significantly more susceptible to nonopsonic phagocytosis by macrophages than were similar cells after being washed in buffer (N.
Fibronectin is capable of enhancing uptake by macrophages of Pseudomonas aeruginosa grown in vivo in rats or mice or in vitro on nutrient agar plates. It was demonstrated that concentrations as low as 27 nM fibronectin produced significant enhancement of macrophage phagocytosis. Washing of fibronectin-treated macrophages did not prevent phagocytosis enhancement, but washing of fibronectin-treated bacteria did. The tetrapeptide arginine-glycine-aspartic acid-serine, which comprises the eucaryotic cell-binding domain of fibronectin, was also capable of promoting bacterial uptake, whereas the control tetrapeptide tetraglycine was not. Fibronectin caused depolarization of the mouse macrophage cell line P388D1, plasma membrane, as demonstrated by using a polarization-sensitive fluorescent probe. These data indicate that promotion by fibronectin of nonopsonic phagocytosis is mediated by the action of fibronectin on the macrophages.
Pseudomonas aeruginosa cytotoxin has been isolated previously from ceHl 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.
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