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Mutations in E coli cistrons affecting adhesion to human cells do not abolish Pap pili fiber formation
Abstract:
A chromosomal DNA fragment which mediates Pap (pili associated with pyelonephritis) pili formation, mannose-resistant hemagglutination ( MRHA ) and binding to uroepithelial cells has been isolated from the uropathogenic Escherichia coli clinical isolate J96 , and genetically studied. Analysis of polypeptides expressed by the Pap DNA led to detection of a number of polypeptides ranging in mol. wt. from 13 000 to 81 000 daltons. The gene order and transcriptional orientation for four of the corresponding cistrons was: 13 000 ( papB ) 19 500 ( papA , structural gene for the Pap pilus subunit), 81 000 ( papC ) and 28 500 ( papD ). Analyses of a lacZ- papA gene fusion located a promoter upstream from papA within the cloned DNA. Transposon Tn5 insertions in any of these four cistrons decreased or eliminated Pap pili formation. A number of transposon Tn5 mutations were identified in a region distal to papD that expressed normal levels of the papA protein on the cell surface in the form of recognizable pili structures but did not agglutinate human erythrocytes or adhere to uroepithelial cells. This region expressed polypeptides of 15 000, 24 000, 26 000 and 35 000 daltons. This finding shows that Pap pili formation and binding properties can be genetically dissociated.
Insights
Genetic studies of uropathogenic Escherichia coli revealed that Pap pili formation and binding to human cells can be genetically separated. This dissociation impacts mannose-resistant hemagglutination and uroepithelial cell adherence.
Area of Science:
- Microbiology and Genetics
- Molecular Biology of Bacterial Pathogenesis
Background:
- Uropathogenic Escherichia coli (UPEC) utilizes Pap (pili associated with pyelonephritis) pili for adherence to host cells.
- Pap pili mediate mannose-resistant hemagglutination (MRHA) and binding to uroepithelial cells, crucial for urinary tract infections.
Purpose of the Study:
- To genetically characterize the DNA fragment responsible for Pap pili formation, MRHA, and uroepithelial cell binding in UPEC strain J96.
- To investigate the genetic basis for the dissociation of Pap pili formation and its functional properties (adherence and hemagglutination).
Main Methods:
- Isolation and genetic study of a chromosomal DNA fragment from UPEC J96 mediating Pap pili functions.
- Analysis of polypeptides expressed by the cloned Pap DNA, including gene mapping and transcriptional orientation.
- Construction of lacZ-papA gene fusion to identify the promoter region.
- Transposon Tn5 mutagenesis to identify genes essential for Pap pili formation and function.
Main Results:
- Identified four cistrons (papB, papA, papC, papD) involved in Pap pili formation, with defined gene order and transcriptional orientation.
- Demonstrated that mutations in these four cistrons significantly reduced or abolished Pap pili formation.
- Discovered a distinct genetic region distal to papD where mutations allowed Pap pili formation but impaired MRHA and uroepithelial cell adherence, indicating a functional dissociation.
Conclusions:
- Pap pili formation and their adhesive/hemagglutinative properties are controlled by distinct genetic elements within the Pap DNA.
- The study provides evidence for the genetic dissociation of Pap pili assembly and their functional roles in bacterial pathogenesis.