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A nonhemolytic phospholipase C from Burkholderia cepacia
1Department of Microbiology, Miami University, Oxford, OH 45056, USA.
Abstract:
Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. Although several potential virulence factors-a protease, lipase, and two phospholipases C (one hemolytic and one nonhemolytic)-have been identified, only two, the protease and the lipase, have been described in detail. The goal of this study was to purify and characterize a nonhemolytic phospholipase C secreted by B. cepacia strain Pc224c. The enzyme was concentrated from culture supernatants and purified by polyacrylamide gel electrophoresis. The 54-kDa protein was stable in the presence of sodium dodecyl sulfate (up to 10%) and at 4 degrees, 22 degrees, and 37 degrees C; it was, however, inactivated at 100 degrees C. The enzyme bound to glass, chromatography matrices, and polyvinylidene difluoride and cellulose membranes, suggesting that it is hydrophobic. In a genetic approach, primers based on conserved sequences of a B. cepacia Pc69 hemolytic phospholipase C and both the Pseudomonas aeruginosa hemolytic and nonhemolytic proteins were designed to identify the Pc224c nonhemolytic phospholipase C gene. One polymerase chain reaction product was identified; it was sequenced and the sequence compared with sequences in the BLAST database. The best match was the Pseudomonas aeruginosa hemolytic phospholipase C. Ten additional B. cepacia strains were screened for the gene by Southern hybridization; five had the 4-kb band, suggesting that these strains have a similar form of the PLC gene. Nine of the ten strains reacted with the probe, suggesting that similar sequences were present, but in another form.
Insights
This study purified and characterized a nonhemolytic phospholipase C from Burkholderia cepacia, identifying its hydrophobic nature and genetic presence in other strains. This enzyme is a key virulence factor in cystic fibrosis lung infections.
Area of Science:
- Microbiology
- Biochemistry
- Genetics
Background:
- Burkholderia cepacia is an opportunistic pathogen causing severe lung infections in cystic fibrosis patients.
- Several virulence factors, including phospholipases C, have been identified, but detailed characterization is lacking for some.
- A nonhemolytic phospholipase C from B. cepacia strain Pc224c was targeted for purification and characterization.
Purpose of the Study:
- To purify and characterize the nonhemolytic phospholipase C secreted by B. cepacia strain Pc224c.
- To identify the gene encoding this enzyme and assess its prevalence in other B. cepacia strains.
Main Methods:
- Enzyme purification using polyacrylamide gel electrophoresis.
- Biochemical characterization of enzyme stability and properties (e.g., hydrophobicity).
- Genetic analysis involving PCR, DNA sequencing, BLAST database comparison, and Southern hybridization.
Main Results:
- A 54-kDa nonhemolytic phospholipase C was purified and found to be stable under various conditions but inactivated at 100°C.
- The enzyme exhibited hydrophobic properties, binding to various surfaces.
- A gene fragment was identified with sequence similarity to Pseudomonas aeruginosa hemolytic phospholipase C, and a similar gene was detected in five of ten additional B. cepacia strains.
Conclusions:
- The purified nonhemolytic phospholipase C is a distinct virulence factor of B. cepacia.
- The genetic analysis suggests the presence of a similar phospholipase C gene in other B. cepacia strains, potentially contributing to virulence.
- Further research is warranted to fully elucidate the role of this enzyme in B. cepacia pathogenesis.