Molecular characterization of UpaB and UpaC, two new autotransporter proteins of uropathogenic Escherichia coli CFT073

Abstract

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome-sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that the upaB and upaC AT-encoding genes are common in E. coli. The upaB and upaC genes were cloned and characterized in a recombinant E. coli K-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073, upaB is expressed while upaC is transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted its upaB (but not upaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of the upaB gene in CFT073 significantly reduced early colonization of the bladder.

Fig 1

(A) Western blot analysis of UpaB and UpaC performed using whole-cell lysates prepared from E. coli grown in the presence of 0.2% arabinose. (i) OS56(pUpaB), OS56(pBAD); (ii) OS56(pUpaC), OS56(pBAD). Lane M, Novex Sharp molecular weight marker. (B) Phase contrast (i), immunofluorescence (ii), and immunogold electron microscopy (iii) using UpaB-specific antiserum against cells of E. coli strains MS427(pUpaB) and MS427(pBAD) or UpaC antiserum against cells of E. coli strains MS427(pUpaC) or MS427(pBAD). Cells were grown in the presence of 0.2% arabinose. Overnight cultures were fixed and incubated with anti-UpaB or anti-UpaC serum, respectively, followed by incubation with goat anti-rabbit IgG coupled to Alexa Fluor 488 for immunofluorescence or protein A-gold (10 nm) conjugate. Gold particles were present on the surface of E. coli MS427(pUpaB) and MS427(pUpaC) but not on that of the MS427(pBAD) control strain.

Fig 2

Biofilm formation by E. coli OS56 cells harboring plasmids expressing UpaB and UpaC. The effect of AT expression on biofilm formation was assessed in E. coli OS56 (MG1655Δflu, Gfp+) cells containing the following plasmids pBAD, pUpaB, or pUpaC. All strains were grown in the presence of 0.2% arabinose to induce AT protein expression. Three assays were used: (A) static biofilm formation in polystyrene microtiter plates, (B) dynamic biofilm formation in a microfermentor system, and (C) dynamic biofilm formation using a flow chamber model. In the dynamic-flow-chamber biofilm development was monitored by confocal scanning laser microscopy after 15 h. The images are representative horizontal sections collected within each biofilm and vertical sections (to the right of and above each larger panel, representing the yz plane and the xz plane, respectively) at the positions indicated by the red and green lines. E. coli MS427(pUpaC) produced a biofilm with a significant increase in total biovolume, substratum coverage and mean thickness compared to the vector control strain at 15 h postinoculation (COMSTAT). In the microfermentor assay, UpaC also promoted significant biofilm growth compared to the vector control strain (unpaired t test with Welch′s correction P < 0.05).

Fig 3

UpaB mediates attachment to a range of ECM proteins in an ELISA-based binding assay. Black bars, E. coli MS427(pUpaB); hatched bars, E. coli MS427(pUpaC); white bars, E. coli MS427(pBAD). Results represent averages of results from 3 independent experiments + standard errors of the means (SEM). Mean absorbance readings were compared with negative-control readings MS427(pBAD).

Fig 4

The H-NS protein is a repressor of UpaC. (A) Western blot analysis of UpaB using whole-cell lysates; (B) Western blot analysis of UpaC using whole-cell lysates; (C) beta-galactosidase assay of upaB::lacZ-zeo and upaC::lacZ-zeo fusions; (D) curvature propensity plots showing predicted regions of curved DNA in the 250-bp upaC promoter. (E) Curved DNA PAGE gel of the amplified 250-bp upaC promoter at 4°C. Migration of this band is slightly retarded in the gel, suggesting that the DNA is curved. (F) Electrophoretic band shift of the amplified 250-bp upaC promoter and the bla promoter from digest pBR322 DNA. The pBR322 fragments not containing the bla promoter were not influenced by the increasing concentrations of (0.05, 0.1, 0.2, 0.5, 1, and 2 μM) H-NS. Images depict representative gels from at least two independent experiments.

Fig 5

(A) Phase-contrast (i) or immunofluorescence (ii) microscopy employing UpaB-specific antiserum against cells of E. coli strains as specified; (B) phase-contrast (i) or immunofluorescence (ii) microscopy using UpaC antiserum against cells of E. coli strains as specified. Strains were grown in the presence of 0.2% arabinose and labeled as induced where applicable. Overnight cultures were fixed and incubated with anti-UpaB or anti-UpaC serum followed by incubation with goat anti-rabbit IgG coupled to Alexa Fluor 488. A positive reaction indicating surface localization of UpaB or UpaC was detected only for the overexpressing strains.

Fig 6

Mouse UTI assays. (A) Competitive mixed-infection experiment employing a 1:1 mixture of E. coli CFT073^amp/CFT073upaB and E. coli CFT073^amp/CFT073upaC. Total CFU were enumerated from the bladders of infected mice on selective medium to differentiate between E. coli CFT073^amp/CFT073upaB and E. coli CFT073^amp/CFT073upaC. Each symbol represents the Log₁₀ fitness index calculated for each individual mouse, and the median is represented by a horizontal line. A Log₁₀ fitness index below 0 (shown by the dashed line) indicates that the upaB mutant is at a competitive disadvantage (P = 0.014). (B and C) Single-infection assay employing E. coli CFT073 and CFT073upaB. Data for individual mice are expressed as the Log₁₀ of total CFU per 0.1 g of bladder tissue at day 1 (B) and day 5 (C) postinfection. The number of mice used for each experiment (n) is indicated. The data represent a compilation of the results from two individual experiments. Medians are indicated by a solid line. E. coli CFT073 was recovered from the bladder of infected mice at day 1 postinfection in significantly higher numbers than CFT073upaB (P < 0.001). No statistical difference in the number of E. coli CFT073 and CFT073upaB was observed at day 5 postinfection.