Demonstration of ECP on UPEC strains.

<p>(A) CFT073 displays peritrichous pili after growth in LB at 26°C with shaking and after incubation with pre-immune serum followed by negative staining and TEM observation. (B–D) Immunogold labeling of ECP (indicated by arrows) produced by F11 (B), CFT073 (C), and UTI89 (D) strains grown in LB at 26°C with anti-ECP antibodies and rabbit anti-IgG gold conjugate. (E) Magnification of labeled ECP on CFT073. Scale bars represent 500 nm. (F–H) Detection of ECP (green) on UPEC strains [F11 (F), CFT073 (G), UTI89 (H)] after 2 h of incubation with HeLa cells at 37°C and reacted with anti-ECP antibodies and Alexa-Fluor 488-conjugated secondary antibody. Cellular and bacterial DNA was stained with propidium iodide (red). Panel I is the negative control CFT073 incubated with preimmune serum. Images were taken with a 60× objective. The insets in panels F and G are high magnifications of the areas within the corresponding panels and show a fibrillar network (green) between bacteria formed by ECP.</p

Primers used in this study.

<p>Primers used in this study.</p

Role of ECP in adherence of UPEC to HeLa and HTB-4 cells.

<p>(A) Comparison of adherence levels of wild-type strain CFT073 and isogenic <i>ecpA</i> mutant to HeLa and HTB-4 cells at 37°C, as determined by plating out serial dilutions of adhering bacteria. (B) Comparison of invasion capabilities of HeLa and HTB-4 cells by the indicated UPEC strains as determined by the gentamycin-protection assay. For panels A and B standard deviations are represented by error bars and represent the average of all the results obtained from the 3 experiments performed. *, Statistically significant with respect to the wild-type strain (<i>p</i><0.05). (C) Giemsa staining of HeLa cell monolayers infected for 2 h with UPEC CFT073 strains showing reduction of adherence in the isogenic <i>ecpA</i> mutant. Images were taken with a 60× objective.</p

Optimal conditions for induction of ECP.

<p>Production of ECP in CFT073 bacteria grown in LB or DMEM at 26°C or 37°C was measured by flow cytometry using primary anti-EcpA antibodies and Alexa-Fluor 488 conjugate. In addition, levels of ECP production were determined in the presence of HeLa cells at 37°C. Standard deviations are represented by error bars and represent the average of all the results obtained from the 3 experiments performed.</p

Effect of D-mannose on type 1 pili-mediated adherence.

<p>Adherence of CFT073 and derivative strains to HeLa cells cultured in DMEM at 37°C was quantitated in the presence of 0, 0.5, and 1% D-mannose. Colony-forming-units (CFU) were obtained after plating out ten-fold dilutions of 1% TritonX-100-treated cells. Standard deviations are represented by error bars and represent the average of all the results obtained from the 3 experiments performed.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Role of ECP in biofilm formation.

<p>(A) Giemsa staining of biofilms of CFT073 and derivative strains showing a difference in the architecture of the biofilms formed after incubation of the bacteria for 24 h in DMEM at 37°C on glass coverslips contained in 24-well plates. (B) Immunofluorescence microscopy showing the presence of ECP in certain areas of the biofilms formed by CFT073 and CFT073 Δ<i>ecpA</i> (pMAT9). Images were taken with a 60× objective. (C) Quantification of biofilm formation by measuring Crystal Violet uptake. Standard deviations are represented by error bars and represent the average of all the results obtained from the 3 experiments performed. **, Statistically significant with respect to the wild-type strain (<i>p</i><0.01).</p

Analysis of derivative <i>ecpA</i> mutants.

<p>(A) Immunogold labeling of ECP produced by (a) CFT073, (b) CFT073 Δ<i>ecpA</i>, (c) CFT073 Δ<i>ecpA</i> (pMAT9), (d) F11, (e) F11 Δ<i>ecpA</i>, and (f) F11 Δ<i>ecpA</i> (pMAT9) with anti-ECP antibodies and rabbit anti-IgG gold conjugate. Scale bars represent 500 nm. (B) Immunofluorescence of UPEC strains with anti-ECP antibodies and rabbit anti-IgG and Alexa-Fluor 488-conjugated secondary antibody (green). Cellular and bacterial DNA was stained with propidium iodide (red). Images were taken with a 100× objective and magnified 5X. (C) Immunoblot showing EcpA production in CFT073, CFT073 Δ<i>ecpA</i> (pMAT9), F11 and F11 Δ<i>ecpA</i> (pMAT9), but not in the CFT073 Δ<i>ecpA</i> or F11 Δ<i>ecpA</i> mutants. DnaK detection was used as loading control of equal amounts of bacteria onto the gels. (D) Motility of UPEC strains in motility agar. Except for the CFT073 Δ<i>fliC</i> all other strains showed motility.</p