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. 2004 Jan;72(1):593-7.
doi: 10.1128/IAI.72.1.593-597.2004.

Autotransporter genes pic and tsh are associated with Escherichia coli strains that cause acute pyelonephritis and are expressed during urinary tract infection

Susan R Heimer  1 , David A Rasko, C Virginia Lockatell, David E Johnson, Harry L T Mobley
Affiliations

Autotransporter genes pic and tsh are associated with Escherichia coli strains that cause acute pyelonephritis and are expressed during urinary tract infection

Susan R Heimer et al. Infect Immun. 2004 Jan.

Abstract

We have identified two chromosomal open reading frames in uropathogenic Escherichia coli (UPEC) strain CFT073 which are highly homologous to serine protease autotransporters Pic and Tsh. Both cloned determinants were correlated with the presence of 105- to 110-kDa proteins in the culture supernatants. Furthermore, in cellular fractionation experiments, 30-kDa polypeptides were identified in the outer membrane; we speculated that these proteins are the beta-barrel portions of the autotransporter homologues. Furthermore, Pic-containing culture supernatants have serine protease activity. In reverse transcription-PCR analyses, the expression of the pic and tsh genes in E. coli CFT073 was higher in broth cultures grown at 37 degrees C than at 25 degrees C. Moreover, pic and tsh were expressed by bacteria isolated from urine of transurethrally infected mice. The tsh determinant was identified in 63% of our clinical UPEC strain isolates (n = 87) and in 33% of fecal strains (n = 27), whereas pic was present in 31% of the pyelonephritis (n = 67) and 7% of the fecal strains. There was no significant correlation between cystitis strains (n = 20) and the pic determinant VSports手机版. .

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Figures

FIG. 1.
FIG. 1.
Expression of autotransporter proteins from cloned pic and tsh determinants. Concentrated culture supernatants (A) and membrane fractions (B) prepared from E. coli BL21(DE3) pLysS transformed with either pBS SK+, pDRM005 (pic), or pDRM006 (tsh) were separated in SDS-10% (A) or -12% (B) PAGE and stained with Coomassie blue. The relative migrations of molecular mass standards are indicated at the left. Arrows denote protein bands that correspond to the predicted electrophoretic mobilities of Pic and Tsh proteins.
FIG. 2.
FIG. 2.
Protease activity of culture supernatants. Concentrated culture supernatants (2 μg) were incubated with a casein-Bodipy FL derivative (5 μg) at 37°C for 20 h in the absence (black bars) or presence (white bars) of 500 μM PMSF. Relative fluorescence units were measured at 535 nm using an excitation wavelength of 485 nm. Data represent triplicate measurements of three independent experiments. Asterisks denote significant differences in mean values of samples lacking and containing PMSF (unpaired t test, P < 0.005).
FIG. 3.
FIG. 3.
CBA mouse model of ascending UTI. (A to C) Ten female CBA mice were challenged transurethrally with CFT073 (∼1.0 × 109 CFU). Twenty-four hours postinoculation, tissue and urine samples were collected, homogenized, and quantitatively cultured on L agar. A median value of 1.8 × 106 CFU/ml of urine was isolated from infected animals, whereas only 90 CFU/ml of urine was obtained from uninfected animals. Bacteria isolated from pooled urine samples (n = 10) were analyzed by RT-PCR using three gene-specific primer pairs. Serially diluted RT-PCR products were separated in 2.0% agarose gel. (D) Female CBA mice were cochallenged transurethrally with CFT073 and CFT073Δpic (1:1) with 1.0 × 109 CFU per animal (n = 10). Six days postinoculation, tissue and urine samples were collected from each animal. Homogenized tissue and urine samples were quantitatively cultured on L agar. Values are the CFU per milliliter of urine or gram of tissue along with the median value of each group (horizontal line). (E) In a similar experiment, mice were independently challenged with CFT073 or CFT073Δpic with 1.0 × 109 CFU per animal (n = 10). Statistical differences between wild-type and mutant strains were determined by a one-tailed Wilcoxon matched pairs test (D) or Mann-Whitney test (E).
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References

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