DNA hybridization analysis of the Pseudomonas aeruginosa elastase gene (lasB) from different clinical isolates
1Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Abstract:
Pseudomonas aeruginosa produces several extracellular virulence factors including elastase (which is encoded by lasB). Recently, we examined several clinical isolates of P. aeruginosa for the production of toxin A, elastase, exoenzyme S, and phospholipase C. Although the majority of the isolates produced a high level of elastase, a few isolates produced either very low or no detectable elastase. In this study, we tried to determine the presence of restriction site heterogeneity within lasB from these isolates and the possible correlation between such heterogeneity and the observed variation in elastase production. Chromosomal DNA from the isolates was digested with different restriction enzymes and examined by Southern blot hybridization experiments using two lasB probes. One lasB probe covers 636 bp of lasB structural gene while the other covers 240 bp of the lasB upstream region. Chromosomal DNA from P. aeruginosa PAO1 and PA103 was used as controls. Results indicate that chromosomal DNA from all isolates hybridized to both lasB probes. Depending on the restriction enzyme used for DNA digestion, lasB from 3 to 12% of the isolates showed different patterns of hybridization with the lasB structural gene probe. However, no difference in the hybridization pattern was seen with the lasB upstream probe. With the exception of one isolate, hybridization of genomic DNA from different isolates (with both probes) produced a single hybridization band. In that isolate, an additional hybridization band was detected. Immunoblotting experiments confirmed that elastase protein is not produced by 6 out of 67 isolates. However, lasB from four of these elastase-deficient strains showed no difference in the hybridization pattern with either lasB probe.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Pseudomonas aeruginosa elastase production varies. Restriction site heterogeneity in the lasB gene was observed in some clinical isolates, but did not consistently correlate with reduced elastase levels.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Pseudomonas aeruginosa is a significant opportunistic pathogen known for producing various extracellular virulence factors.
- Elastase, encoded by the lasB gene, is a key virulence factor, though its production can vary among clinical isolates.
- Previous observations noted a subset of P. aeruginosa isolates exhibiting significantly lower or undetectable elastase production.
Purpose of the Study:
- To investigate potential restriction site heterogeneity within the lasB gene in P. aeruginosa clinical isolates.
- To explore the correlation between observed lasB gene heterogeneity and variations in elastase production.
- To identify genetic differences contributing to differential elastase expression in P. aeruginosa.
Main Methods:
- Southern blot hybridization was employed using probes targeting the lasB structural gene and its upstream region.
- Chromosomal DNA from clinical isolates was digested with various restriction enzymes.
- Immunoblotting was used to confirm elastase protein production in elastase-deficient strains.
Main Results:
- Restriction site heterogeneity within the lasB structural gene was detected in 3-12% of isolates, varying with the restriction enzyme used.
- No significant hybridization pattern differences were observed in the lasB upstream region across isolates.
- While 6 out of 67 isolates were confirmed elastase-deficient via immunoblotting, lasB gene analysis showed no consistent heterogeneity in these specific strains.
Conclusions:
- Restriction site heterogeneity exists within the lasB gene of some P. aeruginosa clinical isolates.
- This genetic heterogeneity does not appear to be the sole or primary cause for the observed variations in elastase production.
- Further research is needed to elucidate the mechanisms underlying differential elastase expression in P. aeruginosa.
