Article Figures & Data
Figures
- FIG. 1.
(A) Physical map indicating the genomic location of upaH in E. coli CFT073, UTI89, and MG1655. (B) Schematic illustration of the domain organization of UpaH from E. coli CFT073. Indicated are the signal peptide, the repeat region within the passenger domain, and the transmembrane β-domain. Numbers indicate amino acid positions of region boundaries. (C) Predicted structure of the β-domain of UpaH. Shown is a ribbon diagram of the structure of the β-domain (residues 2528 to 2845) obtained by homology modeling based on the crystal structure of the NalP β-domain (43), colored blue (N terminus) through green and yellow to red (C terminus). (D) Amino acid sequence of the repeats present in UpaH. Amino acids in red indicate ≤98% conservation; amino acids in blue indicate ≤55% conservation.
- FIG. 2.
(A) Western blot analysis of UpaH performed using whole-cell lysates prepared from E. coli MS427(pBAD) and MS427(pUpaH) grown in the presence of 0.1% arabinose. (B) Phase-contrast and immunofluorescence microscopy employing UpaH-specific antiserum against cells of E. coli strains MS427(pUpaH) (i) or MS427(pBAD) (ii) grown in the presence of arabinose. Overnight cultures were fixed and incubated with anti-UpaH serum followed by incubation with goat anti-rabbit IgG coupled to Alexa Fluor 488. (C) Immunogold electron microscopy of E. coli MS427(pUpaH) (i) and MS427(pBAD) (ii) grown in the presence of arabinose and labeled with anti-UpaH serum followed by protein A-gold (10 nm) conjugate. Gold particles were observed on the surface of E. coli MS427(pUpaH) but not MS427(pBAD).
- FIG. 3.
UpaH mediates biofilm formation. (A) Biofilm formation in polystyrene microtiter plates by E. coli MS427(pBAD) and MS427(pUpaH) following static growth in the absence (−) or presence (+) of 0.1% arabinose. Biofilm growth was quantified by solubilization of crystal violet-stained cells with ethanol-acetone and determination of the absorbance at 595 nm. Results represent averages of a minimum of 8 replicates ± SEM. Significant biofilm growth was observed for E. coli MS427(pUpaH) following induction of UpaH expression with arabinose (*, P < 0.001 as determined by analysis of variance [NOVA]). (B) Dynamic-flow-chamber assay examining biofilm formation of E. coli OS56(pBAD) and OS56(pUpaH) grown in the presence of 0.1% arabinose. Biofilm development was monitored by confocal scanning laser microscopy after 24 h. The images are representative horizontal sections collected within each biofilm and vertical sections (to the right of and below each larger panel, representing the yz plane and the xz plane, respectively) at the positions indicated by the white lines. E. coli MS427(pUpaH) produced a significant biofilm in this assay.
- FIG. 4.
(A) Western blot analysis of UpaH performed using whole-cell lysates prepared from E. coli CFT073, CFT073upaH, CFT073upaH(pBAD), and CFT073upaH(pUpaH). A band corresponding to UpaH was detected in E. coli CFT073 and CFT073upaH(pUpaH) but not in E. coli CFT073upaH and CFT073upaH(pBAD). (B) Western blot analysis of UpaH performed using whole-cell lysates prepared from E. coli M161, M161upaH, M357, and M357upaH. A band corresponding to UpaH was detected in E. coli M161 and M357 but not in E. coli M161upaH or M357upaH. (C) Phase-contrast (i) or immunofluorescence (ii) microscopy employing UpaH-specific antiserum against cells of E. coli CFT073, CFT073upaH, CFT073upaH(pUpaH), or CFT073PcLupaH. Overnight cultures were fixed and incubated with anti-UpaH serum, followed by incubation with goat anti-rabbit IgG coupled to Alexa Fluor 488. A positive reaction indicating surface localization of UpaH was detected only for CFT073upaH(pUpaH) and CFT073PcLupaH.
- FIG. 5.
(A) Biofilm formation in polystyrene microtiter plates by three sets of isogenic strains with respect to upaH: E. coli CFT073 and CFT073upaH; E. coli M161 and M161upaH; and E. coli M357 and M357upaH. Results represent averages of OD595 readings of at least 4 independent experiments ± SEM. There was no difference in the growth of wild-type and upaH mutant strains. (B) Dynamic-flow-chamber assay examining biofilm formation of E. coli CFT073 (i), E. coli CFT073upaH (ii), and E. coli CFT073PcLupaH (iii). Biofilm development was monitored by confocal scanning laser microscopy after 40 h. The images are representative horizontal sections collected within each biofilm and vertical sections (to the right of and below each larger panel, representing the yz plane and the xz plane, respectively) at the positions indicated by the white lines. (C) Three-dimensional image demonstrating the structure of the biofilm formed by E. coli CFT073 (i), E. coli CFT073upaH (ii), and E. coli CFT073PcLupaH (iii).
- FIG. 6.
(A) RT-PCR analysis of upaH transcription by CFT073 during colonization of the mouse urinary tract. Urine from six mice infected with CFT073 was collected and pooled at 18 h postinoculation and added directly into a 2× volume of RNAprotect (Qiagen). CFT073 cells were concentrated by centrifugation, and total RNA was extracted. Lane 1, upaH-specific PCR product (328 bp) obtained from genomic DNA (positive control); lane 2, 1-Kb Plus DNA ladder (Invitrogen); lane 3, PCR employing RNA prior to cDNA synthesis as a template (negative control); lane 4, upaH-specific PCR product (328 bp) obtained using the cDNA as the template. (B) Competitive mixed-infection experiment. C57BL/6 mice (n = 24) were challenged with a 1:1 mixture of E. coli CFT073cam and CFT073upaH. Total CFU were enumerated on selective medium to differentiate between CFT073cam (chloramphenicol resistant) and CFT073upaH (kanamycin resistant). Each symbol represents the total number of CFU for an individual mouse per 0.1 g of bladder tissue or the total number of CFU per ml urine. Lines connect values for the same mouse. Horizontal bars represent the median values. No significant colonization of the kidneys was observed.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s) Reference E. coli strains BL21 F−ompT hsdS(rB− mB−) dcm gal Stratagene CFT073 Wild-type UPEC isolate 38 CFT073cam CFT073lacZ::cam-gfp; Camr This study CFT073upaH CFT073upaH::kan; Kanr This study CFT073PcLupaH CFT073PcLupaH; constitutively expressed UpaH This study M161 Wild-type UPEC isolate This study M161upaH M161upaH::kan This study M357 Wild-type UPEC isolate This study M357upaH M357upaH::kan This study MS427 MG1655flu 46 OS56 MG1655flu; GFP+; Ampr 53 Plasmids pUpaHTruncated 747-bp portion of upaH gene from CFT073 was amplified with primers UpaHTF and UpaHTR, digested with NcoI-HindIII, and ligated into NcoI-HindIII-digested pBAD/Myc-HisB; Ampr This study pUpaH upaH gene from CFT073 was amplified with primers 520 and 521, digested with XhoI-HindIII, and ligated into XhoI-HindIII-digested pBAD/Myc-HisA-kan; Ampr Kanr This study pSG10 Chloramphenicol resistance gene from pKD3 inserted into HindIII site of gfp expression plasmid pKEN2 This study
