Related Experiment Videos
Proteus mirabilis MR/P fimbriae: molecular cloning, expression, and nucleotide sequence of the major fimbrial subunit
1Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore 21201.
Abstract:
Proteus mirabilis, a cause of serious urinary tract infection and acute pyelonephritis, produces several putative virulence determinants, among them, fimbriae. Principally, two fimbrial types are produced by this species: mannose-resistant/Proteus-like (MR/P) fimbriae and mannose-resistant/Klebsiella-like (MR/K) fimbriae. To isolate MR/P fimbrial gene sequences, a P. mirabilis cosmid library was screened by immunoblotting and by hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence of the isolated fimbrial polypeptide, ADQGHGTVKFVGSIIDAPCS. One clone, pMRP101, reacted strongly with a monoclonal antibody specific for MR/P fimbriae and with the DNA probe. This clone hemagglutinated both tannic acid-treated and untreated chicken erythrocytes with or without 50 mM D-mannose and was shown to be fimbriated by transmission electron microscopy. A 525-bp open reading frame, designated mrpA, predicted a 175-amino-acid polypeptide including a 23-amino-acid hydrophobic leader peptide. The unprocessed and processed polypeptides are predicted to be 17,909 and 15,689 Da, respectively. The N-terminal amino acid sequence of the processed fimbrial subunit exactly matched amino acid residues 24 to 43 predicted by the mrpA nucleotide sequence. The MrpA polypeptide shares 57% amino acid sequence identity with SmfA, the major fimbrial subunit of Serratia marcescens mannose-resistant fimbriae.
Insights
Researchers identified the gene sequence for mannose-resistant/Proteus-like (MR/P) fimbriae in Proteus mirabilis. This discovery advances understanding of MR/P fimbriae, key factors in urinary tract infections.
Area of Science:
- Microbiology
- Molecular Biology
Background:
- Proteus mirabilis causes severe urinary tract infections and pyelonephritis.
- Fimbriae are key virulence factors in P. mirabilis, with mannose-resistant/Proteus-like (MR/P) and mannose-resistant/Klebsiella-like (MR/K) types being prominent.
- Understanding fimbrial gene sequences is crucial for elucidating virulence mechanisms.
Purpose of the Study:
- To isolate and characterize the gene sequence encoding MR/P fimbriae in Proteus mirabilis.
- To identify the specific gene responsible for MR/P fimbrial production.
Main Methods:
- Screening of a P. mirabilis cosmid library using immunoblotting and DNA hybridization with a specific oligonucleotide probe.
- Characterization of positive clones through hemagglutination assays and transmission electron microscopy.
- Nucleotide sequencing and analysis of the identified open reading frame (mrpA).
Main Results:
- A P. mirabilis cosmid clone, pMRP101, was isolated, showing strong reactivity with MR/P fimbriae-specific antibodies and DNA probes.
- Clone pMRP101 demonstrated hemagglutination activity and was confirmed to be fimbriated via electron microscopy.
- A 525-bp open reading frame, mrpA, was identified, predicting a 175-amino-acid polypeptide (MprA) with a hydrophobic leader peptide.
- The N-terminal sequence of the processed MprA subunit precisely matched the mrpA gene sequence.
- MprA shares 57% amino acid identity with the major fimbrial subunit of Serratia marcescens.
Conclusions:
- The mrpA gene encodes the major subunit of MR/P fimbriae in Proteus mirabilis.
- This study successfully isolated and characterized the genetic basis for MR/P fimbriae production.
- The findings provide a foundation for further research into the role of MR/P fimbriae in P. mirabilis pathogenesis.