Human bladder cells respond with <i>CYP24A1</i> and <i>CAMP</i> induction to 1,25D₃ and 25D₃.

<p>Normal bladder cells (HBEP, upper panel) and TERT-NHUC bladder cells (lower panel) were stimulated with 10⁻⁷ M 25D₃ or 10⁻⁸ M 1,25D₃ for 24 hours. Levels of gene-specific mRNA for <i>CYP24A1</i> (<b>A</b>, <b>B</b>), <i>CAMP</i> (<b>C</b>, <b>D</b>), <i>VDR</i> (<b>E</b>, <b>F</b>) and <i>CYP27B1</i> (<b>G</b>, <b>H</b>) in stimulated cells were determined and are presented as fold change compared to vector-treated control cells. Data presented are pooled from three independent experiments and shown as mean and standard deviation; ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05 (ANOVA with Dunnett's post-test).</p

25D₃ and cathelicidin in serum and bladder tissue of women during a three-month period of 25D₃ supplementation.

<p>Postmenopausal women were treated with 2000 units of oral 25D₃ daily for 12 weeks. Serum samples were collected before, at 6 and 12 weeks. Serum 25D₃ levels increased from a baseline median value of 68.5 nmol/L to 104.5 nmol/L at 6 weeks, <i>P</i><0.001 (Wilcoxon rank test), and 117 nmol/L at 12 weeks, <i>P</i><0.01 (Wilcoxon rank test). The dotted line denotes the recommended serum 25D₃ level of 75 nmol/L and median values are indicated (<b>A</b>). Despite treatment with 25D₃, serum cathelicidin levels did not increase (<b>B</b>). <i>E. coli</i>-infected bladder biopsies from women receiving 25D₃ supplementation responded with higher expression of <i>CAMP</i> mRNA, <i>P</i><0.05 (Mann-Whitney test) (<b>C</b>) and cathelicidin protein, <i>P</i> <0.05 (Mann-Whitney test) (<b>D</b>) compared to infected pieces prior to supplementation. Fluorescence immunohistochemistry showed increased cathelicidin protein expression in infected bladder biopsies. Tissue samples shown are from the same patient before (<b>E</b>) and after (<b>F</b>) 12 weeks of 25D₃ treatment. Cathelicidin is localized in the uroepithelium including the larger umbrella cells closest to the bladder lumen. Staining without primary antibody serves as negative control (<b>G</b>). Images are captured at ×63 magnification and depict an area corresponding to 119×119 µm. Cathelicidin is stained with AlexaFluor 594 (red), the cell nucleus with DAPI (blue).</p

1,25D₃ or 25D₃ in combination with <i>E. coli</i> increased cathelicidin protein in bladder epithelial cells.

<p>Production of cathelicidin increased in TERT-NHUC bladder cells after treatment with 1,25D₃ or 25D₃, and was further increased by infection with <i>E. coli</i> CFT073 (UPEC). Images are captured at ×63 magnification and depict an area corresponding to 119×119 µm (<b>A</b>). The control image (Neg control) is shown in the right upper panel. Fluorescence intensity was quantified and is presented as mean values with standard deviation; ***<i>P</i><0.001, *<i>P</i><0.05 (ANOVA with Dunnett's post-test) (<b>B</b>). An increase in cathelicidin protein was also recorded by flow cytometry in <i>E. coli</i>-infected TERT-NHUC bladder cells treated with 1,25D₃ or 25D₃. Grey is vector +<i>E. coli</i>, red is 1,25D₃ 10⁻⁸ M+<i>E. coli</i> and black is 25D₃ 10⁻⁷ M+<i>E. coli</i> (<b>C</b>).</p

Adhesion and immune induction by <i>E. coli</i> expressing or lacking curli and/or cellulose.

<p>(<b>A</b>) Adhesion to renal epithelial cells A498 was measured after 30 min. Curliated strains adhered significantly better to renal epithelial cells than strains lacking curli, independent of the expression of cellulose (^###<i>P</i><0.0001, <i>t</i>-test). Cellulose expression decreased the number of cell-associated bacteria in curliated (^***<i>P</i><0.0001, <i>t</i>-test) and non-curliated strains (^**<i>P</i> = 0.001, <i>t</i>-test). Results from three independent experiments in quadruplicates are shown as mean and standard deviation. Similar results were obtained for bladder epithelial cells (data not shown). (<b>B</b>) Induction of IL-8 was measured in culture supernatants of renal epithelial cells A498 stimulated with <i>E. coli</i> for 24 h. Curliated bacteria induced a significantly stronger IL-8 response than the mutants lacking curli in the presence (^##<i>P</i> = 0.001, <i>t</i>-test) and absence (^###<i>P</i><0.0001, <i>t</i>-test) of cellulose. In curliated bacteria, the expression of cellulose reduced IL-8 induction (^**<i>P</i> = 0.001, <i>t</i>-test). Results from three independent experiments in quadruplicate are shown as mean and standard deviation. Similar results were obtained for bladder epithelial cells (data not shown). (<b>C+D</b>) The phenotype of <i>E. coli</i> No. 12 could be restored by complementation of its mutants. Cellulose expression in strain B23 is inducible by aTc (left panels) and reduces adherence and IL-8 induction (^***<i>P</i><0.0001 and ^**<i>P</i> = 0.007, respectively, <i>t</i>-test). The curli subunits CsgA and CsgB are expressed from pWSK29-<i>csgBA</i> in strain WE11_1 (right panels) and increases adherence and IL-8 induction compared to WE11_1 carrying the vector pWSK29 only (^*<i>P</i> = 0.048 and ^**<i>P</i> = 0.003, respectively, <i>t</i>-test). Results in A498 cells are shown as mean and standard deviation. Data from three experiments in quadruplicate for adherence and from two experiments in triplicate for IL-8 induction are presented. (<b>E</b>) Mice were infected with the isogenic <i>E. coli</i> strains for 1 h. The curliated mutant were isolated from kidneys in significantly higher numbers than the double knockout (^#<i>P</i> = 0.026, Mann-Whitney U test). Individual values from <i>n</i> = 8–10 mice/group and medians are shown. (<b>F</b>) Levels of MIP-2 were measured in kidney tissue of infected mice after 16 h. In the absence of cellulose, the curliated mutant strain induced higher levels of MIP-2 compared to the non-curliated strain (^##<i>P</i> = 0.001, Mann-Whitney U test). Expression of cellulose reduced the induction of MIP-2 in the presence (^***<i>P</i><0.0001, Mann-Whitney U test) and absence of curli (^**<i>P</i> = 0.001, Mann-Whitney U test). Individual values from <i>n</i> = 5–10 mice/group and medians are shown.</p

LL-37 binds to recombinant polymerized CsgA and isolated wild-type curli.

<p>(<b>A</b>) Western blot analysis of supernatants after precipitation of LL-37 with curli. By adding polymeric CsgA (pol CsgA) or wild-type curli (wt curli) to a solution of 0.1 µM LL-37, the levels of LL-37 decreased in the supernatants after centrifugation. (<b>B</b>) Surface plasmon resonance. LL-37 exhibits a stronger association and lower dissociation rates to both polymeric (upper panel) and monomeric CsgA (lower panel) compared to the control peptides sLL-37 and VIP.</p

LL-37 prevents formation of biofilm by <i>E. coli</i> in vitro.

<p>(<b>A</b>) Different concentrations of LL-37 and the control peptides sLL-37 and VIP were added to the curli-expressing mutant. (<b>B</b>) At 2.5 µM, LL-37 caused more than 80% reduction of biofilm production, whereas the same concentration of the control peptides gave a reduction of only ∼10%. Mean and standard deviation from data of two separate experiments in triplicates are shown. The difference between LL-37 versus sLL-37 or VIP at 2.5 µM was statistically significant (^***<i>P</i> = 0.001, <i>t</i>-test). Similar results were obtained for the wild-type strain expressing both curli and cellulose (data not shown).</p

Oligos and plasmids used for mutant construction and complementation.

a<p>AmpR: resistance to ampicillin; Km^R: resistance to kanamycin.</p>b<p>Underlined sequences: restriction enzyme recognition sites; bold font: sequence for amplification of the pKD46 resistance/<i>km</i>RExTET cassette.</p

The monomeric form of CsgA remains stable in the presence of LL-37.

<p>(<b>A</b>) Monomeric CsgA was incubated for 20 h at 37°C without or with LL-37. When CsgA is incubated together with LL-37, the CsgA monomer is visible after SDS-PAGE, whereas the monomer is not detected in the absence of LL-37. When polymeric CsgA is treated with formic acid (FA), the CsgA monomer is detectable, excluding degradation of CsgA. The bands of ∼30 and ∼60 kDa are most likely the dimer and tetramer of CsgA, respectively. (<b>B</b>) The stability of the monomeric form of CsgA in the presence of LL-37 was also confirmed with CD spectroscopy after 60 h incubation. The CD spectrum reveals that CsgA alone exhibits a fiber-like structure with weak beta-sheet conformation and a decreased solubility. In contrast, CsgA incubated together with LL-37 displays an unstructured, random coil structure. The spectrum of LL-37 alone was subtracted from the spectrum of CsgA + LL-37.</p

Curli increase the resistance to the antimicrobial peptide LL-37.

<p>(<b>A</b>) Bladder epithelial cells T24 were infected with bacteria for 30 min and adherent bacteria were subjected to LIVE/DEAD staining. Curli and cellulose expression enhanced bacterial resistance to antimicrobial properties (^###<i>P</i><0.001 for curliated strains versus non-curliated strains, ^*<i>P</i> = 0.023 and ^**<i>P</i> = 0.003 for cellulose expressing strains with or without curli, <i>t</i>-test). Combined data from four experiments are shown. (<b>B+C</b>) Bacteria were exposed to conditioned medium of bladder epithelial cells T24 stimulated with phenylbutyrate to enhance LL-37 production. Curli expression enhanced bacterial survival over 30 min (^##<i>P</i> = 0.006, <i>t</i>-test) (<b>B</b>). Results from three experiments in triplicate are shown. Conditioned medium was incubated with neutralizing anti-LL-37-antibodies (nAb) or isotype control antibodies (Co) prior to bacterial inoculation. Neutralizing of LL-37 had no effect on viability of the curliated strain (left) but enhanced viability of the double knockout (right, ^*<i>P</i> = 0.047, <i>t</i>-test) (<b>C</b>). Results from four experiments in triplicate are shown. (<b>D–G</b>) The susceptibility to LL-37 and mCRAMP of <i>E. coli</i> strains expressing or lacking curli or cellulose was tested by the broth dilution method. The expression of curli increased the resistance to both LL-37 (<b>D+E</b>) and mCRAMP (<b>F+G</b>). A significant difference of bacterial growth was observed at 10 µM LL-37 between curliated and non-curliated strains (^###<i>P</i><0.001, <i>t</i>-test). The curliated strains were also significantly more resistant to 5 µM mCRAMP than bacteria not producing curli (^#<i>P</i><0.05, <i>t</i>-test). An increased resistance to both cathelicidins was not observed for cellulose. Mean and standard deviation from data of two separate experiments in triplicates are shown. The IC₅₀ is indicated by a broken line.</p