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Identification of Pseudomonas aeruginosa genes required for epithelial cell injury
P J Kang1, A R Hauser, G Apodaca
1Department of Medicine, University of California, San Francisco, 94143, USA.
Abstract:
We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa-induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pill, based on their resistance to phage PO4 and/or loss of twitching motility (twt-). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein-secretion apparatus appears to be involved in this process.
Insights
Researchers developed a genetic screen to identify non-cytotoxic Pseudomonas aeruginosa mutants. This screen revealed that bacterial pili and type III secretion are crucial for causing epithelial cell damage.
Area of Science:
- Microbiology
- Genetics
- Cell Biology
Background:
- Pseudomonas aeruginosa is a lung-virulent bacterium known to cause epithelial cell cytotoxicity.
- Understanding the genetic basis of this cytotoxicity is essential for developing targeted therapies.
Purpose of the Study:
- To develop a rapid and reproducible genetic screen for identifying factors involved in P. aeruginosa-induced cytotoxicity.
- To isolate and characterize non-cytotoxic mutants of a cytotoxic P. aeruginosa strain (PA103).
Main Methods:
- Development of a genetic screen using Madin-Darby canine kidney (MDCK) cells.
- Isolation of isogenic transposon-tagged mutants.
- Determination of transposon insertion sites via inverse PCR and DNA sequencing.
- Phenotypic analysis of mutants, including pili assessment, adherence, twitching motility, and cytotoxicity assays.
Main Results:
- Four classes of non-cytotoxic mutants were identified.
- Class 1 mutants showed defects in pili (based on phage PO4 resistance and/or loss of twitching motility).
- Class 2 mutants exhibited decreased adherence.
- Class 3 mutants displayed probable defects in the type III protein secretion system.
- Class 4 mutants had reduced, but not absent, cytotoxicity, including members from other classes.
Conclusions:
- Localized cytotoxicity by P. aeruginosa is a multi-step process requiring multiple components.
- Pili and other extracellular proteins are important for cytotoxicity.
- The type III protein secretion apparatus plays a significant role in P. aeruginosa-induced epithelial cell damage.