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Preliminary characterization of Pseudomonas aeruginosa peptide chemotactins for polymorphonuclear leukocytes
P A Fontán1, C R Amura, V E García
1Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Paraguay, Argentina.
Abstract:
In a previous report, we showed that supernatants of Pseudomonas aeruginosa cultures exhibit chemotactic activity for polymorphonuclear leukocytes (PMNL). In this study, P. aeruginosa chemotactins were isolated, purified, and partially characterized. The organisms were cultured in Vogel-Bonner defined medium, and cultures were stopped in late log phase. Chemotactins withstood heating, remained unaltered after acid or alkali treatment in a pH range from 4 to 10, and resisted digestion by trypsin or carboxypeptidase, but chemotactic activity was decreased by 73% after incubation with pronase. Only 2% of the total chemotactic activity of culture supernatants could be extracted with chloroform. Chemotactins with molecular sizes less than 3 kDa constituted the largest contribution to the chemotactic activity of culture supernatants. Pretreatment of PMNL with 10(-5) M formylmethionyl-leucyl-phenylalanine (FMLP) inhibited chemotaxis towards FMLP and P. aeruginosa culture supernatants but not towards complement component C5a. In conclusion, the total chemotactic activity for PMNL of P. aeruginosa culture supernatants was due, almost exclusively, to chemotactins that have properties similar, if not identical, to those exhibited by formylmethionyl peptides.
Insights
Pseudomonas aeruginosa culture supernatants attract polymorphonuclear leukocytes (PMNL) due to potent chemotactins. These molecules are small, heat-stable, and resemble formylmethionyl peptides, suggesting a specific mechanism of action.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Pseudomonas aeruginosa is a pathogen known to elicit an immune response.
- Culture supernatants of P. aeruginosa have previously demonstrated chemotactic activity for polymorphonuclear leukocytes (PMNL).
Purpose of the Study:
- To isolate, purify, and characterize the chemotactic factors produced by P. aeruginosa.
- To elucidate the chemical properties and molecular size of these P. aeruginosa chemotactins.
Main Methods:
- Culturing P. aeruginosa in Vogel-Bonner defined medium.
- Assessing chemotactic activity after various treatments (heating, pH changes, enzymatic digestion, chloroform extraction).
- Determining molecular size distribution of active components.
- Investigating PMNL pre-treatment effects with formylmethionyl-leucyl-phenylalanine (FMLP).
Main Results:
- Chemotactins were heat-stable, resistant to pH extremes (4-10), and partially degraded by pronase but not trypsin or carboxypeptidase.
- Chloroform extraction yielded minimal activity (2%), indicating hydrophilicity.
- Components < 3 kDa were the primary contributors to chemotactic activity.
- FMLP pre-treatment inhibited chemotaxis to FMLP and P. aeruginosa supernatants, but not to C5a.
Conclusions:
- The chemotactic activity of P. aeruginosa supernatants for PMNL is predominantly caused by low molecular weight (< 3 kDa) factors.
- These chemotactins share properties with formylmethionyl peptides, suggesting a similar mechanism of action in PMNL recruitment.