Nucleotide sequence of the papA gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli

190

205

SummaryUrinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a b-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5

Section: Sat Domains and Processingmentioning

confidence: 99%

Identification of Sat, an autotransporter toxin produced by uropathogenic Escherichia coli

Guyer¹,

Henderson

Nataro

et al. 2000

190

205

“…These fragments were produced by the method of Deininger (1983) as modified by fVlinton et al (1986) and subcloned into Sma l-cleaved dephosphorylated M13mp8 (Amersham), Recombinant DNA was transformed into E. coli DH5a and the transformed cells plated onto the M13 host strain, £ coli TG2. Single-stranded templates were prepared (Sanger et al, 1980) and sequenced by the dideoxy chain termination procedure (Sanger etal., 1977), as modified by Biggin et al (1983) but substituting the dGTP in all four dideoxynucleotide triphosphate solutions with an equivalent amount of 7-deaza-2'-dGTP (Boehringer Corporation Ltd, Lewes, England), The latter modification overcomes the compression problems associated with sequencing G-C rich DNA (fVlizusawa ef al., 1986), The sequence was determined for both strands and each nucleotide sequenced an average of 5,7 times. The sequence data were collated and analysed using an Apple II microcomputer with the Pascal operating system and editor, and the programs of Fritstensky ef al.…”

Section: Restriction Enzyme Mapping Subcloning and Dna Sequencingmentioning

confidence: 99%

Cloning and nucleotide sequence analysis of the serotype 2 fimbrial subunit gene of Bordetella pertussis

Livey

Duggleby

Robinson

1987

An oligonucleotide probe complementary to the beginning of the gene encoding the serotype 2(ST2) fimbrial subunit of Bordetella pertussis was synthesized and a cloned DNA fragment hybridizing with the probe identified and sequenced. Several lines of evidence indicate that an open reading frame with coding information for a polypeptide of 207 amino acids, including a 26-amino-acid signal sequence, is the ST2 gene. The protein deduced from the nucleotide sequence shows good agreement with the NH2-terminal amino acid sequence, amino acid composition and molecular weight of the purified fimbrial subunit. In addition, the proposed ST2 subunit is shown to have homology with other fimbrial subunits.

“…Although numerous studies have implicated P fimbnae in the pathogenesis of acute pyelonephritis (reviewed by Johnson, 1991) and the genetics of the pap operon which encodes P fimbriae are well understood (B^ga et al, 1984;Lindberg etal., 1987;Lund etal., 1987;Norgren et al, 1987;Normark etal., 1983;Rhen etal., 1985;van Die and Bergmans. 1984), we are unaware of any reports in which true isogenic P-fimbrial mutants of virulent clinical isolates have been constructed for testing in in vivo models of pyelonephritis.…”

Section: Introductionmentioning

confidence: 99%

Isogenic P‐fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of α Gal(1–4)β Gal binding in virulence of a wild‐type strain

Mobley

Jarvis

Elwood

et al. 1993

171

148

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were subcloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or alpha Gal(1-4)beta Gal-coated latex beads. CBA mice (N = 100) were challenged transurethrally with 10(5), 10(6), 10(7), or 10(9) cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.