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Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37.

Kai-Larsen Y, Lüthje P, Chromek M, Peters V, Wang X, Holm A, Kádas L, Hedlund KO, Johansson J, Chapman MR, Jacobson SH, Römling U, Agerberth B, Brauner A - PLoS Pathog. (2010)

Bottom Line: Our results suggest that curli and cellulose exhibit differential and complementary functions.Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys.Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms.

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LL-37 inhibits CsgA polymerization.(A) Monomeric CsgA was incubated without or with different concentrations of LL-37 (left). The CsgA monomers formed fibers that could be detected by the fiber-specific fluorescent dye Thioflavin (ThT). When bound to fibers, ThT gave rise to fluorescence that was detected by a Tecan plate reader. The fiber formation was inhibited by LL-37 in a dose-dependent manner and was completely inhibited at a molar ratio of 1∶1 (CsgA∶LL-37). As control peptides, VIP and sLL-37 were included (right). (B+C) Confocal images of polymerized CsgA stained with ThT. (B) The left image shows an overview, the right image a magnification of an aggregate of fibers. Scale bars are 250 µm (left) and 25 µm (right), respectively. (C) Fluorescent signals from CsgA incubated with LL-37, sLL-37, or VIP were quantified. Values were corrected for the contribution of the peptides themselves and are expressed in relation to the signal from CsgA alone. The inhibitory effect of LL-37 is significantly stronger than the effect of sLL-37 (** P = 0.007) or VIP (* P = 0.011). Combined results from two experiments are presented.
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ppat-1001010-g007: LL-37 inhibits CsgA polymerization.(A) Monomeric CsgA was incubated without or with different concentrations of LL-37 (left). The CsgA monomers formed fibers that could be detected by the fiber-specific fluorescent dye Thioflavin (ThT). When bound to fibers, ThT gave rise to fluorescence that was detected by a Tecan plate reader. The fiber formation was inhibited by LL-37 in a dose-dependent manner and was completely inhibited at a molar ratio of 1∶1 (CsgA∶LL-37). As control peptides, VIP and sLL-37 were included (right). (B+C) Confocal images of polymerized CsgA stained with ThT. (B) The left image shows an overview, the right image a magnification of an aggregate of fibers. Scale bars are 250 µm (left) and 25 µm (right), respectively. (C) Fluorescent signals from CsgA incubated with LL-37, sLL-37, or VIP were quantified. Values were corrected for the contribution of the peptides themselves and are expressed in relation to the signal from CsgA alone. The inhibitory effect of LL-37 is significantly stronger than the effect of sLL-37 (** P = 0.007) or VIP (* P = 0.011). Combined results from two experiments are presented.

Mentions: To explain a possible cause for the inhibition of biofilm formation by LL-37, the effect of LL-37 on curli formation was investigated. For this purpose, we utilized monomeric recombinant CsgA, the major subunit of curli, which spontaneously polymerizes [28]. CsgA polymerization was monitored with thioflavin T (ThT), a fluorescent dye that binds to polymerized, but not to monomeric CsgA. Our results demonstrated that CsgA polymerization started immediately after incubation at 37°C and reached a stationary phase after approximately 300 min (red line in Figure 7A). After prolonged incubation, the fluorescence declined, most likely due to degradation of ThT and/or precipitation of fibers [28]. The polymerization was inhibited by LL-37 in a dose-dependent manner, and at a molar ratio of 1∶1 (CsgA∶LL-37) fiber formation was completely inhibited (Figure 7A, left panel). The control peptides sLL-37 and VIP had a less pronounced effect on CsgA polymerization than LL-37 (Figure 7A, right panel).


Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37.

Kai-Larsen Y, Lüthje P, Chromek M, Peters V, Wang X, Holm A, Kádas L, Hedlund KO, Johansson J, Chapman MR, Jacobson SH, Römling U, Agerberth B, Brauner A - PLoS Pathog. (2010)

LL-37 inhibits CsgA polymerization.(A) Monomeric CsgA was incubated without or with different concentrations of LL-37 (left). The CsgA monomers formed fibers that could be detected by the fiber-specific fluorescent dye Thioflavin (ThT). When bound to fibers, ThT gave rise to fluorescence that was detected by a Tecan plate reader. The fiber formation was inhibited by LL-37 in a dose-dependent manner and was completely inhibited at a molar ratio of 1∶1 (CsgA∶LL-37). As control peptides, VIP and sLL-37 were included (right). (B+C) Confocal images of polymerized CsgA stained with ThT. (B) The left image shows an overview, the right image a magnification of an aggregate of fibers. Scale bars are 250 µm (left) and 25 µm (right), respectively. (C) Fluorescent signals from CsgA incubated with LL-37, sLL-37, or VIP were quantified. Values were corrected for the contribution of the peptides themselves and are expressed in relation to the signal from CsgA alone. The inhibitory effect of LL-37 is significantly stronger than the effect of sLL-37 (** P = 0.007) or VIP (* P = 0.011). Combined results from two experiments are presented.
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Related In: Results  -  Collection

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ppat-1001010-g007: LL-37 inhibits CsgA polymerization.(A) Monomeric CsgA was incubated without or with different concentrations of LL-37 (left). The CsgA monomers formed fibers that could be detected by the fiber-specific fluorescent dye Thioflavin (ThT). When bound to fibers, ThT gave rise to fluorescence that was detected by a Tecan plate reader. The fiber formation was inhibited by LL-37 in a dose-dependent manner and was completely inhibited at a molar ratio of 1∶1 (CsgA∶LL-37). As control peptides, VIP and sLL-37 were included (right). (B+C) Confocal images of polymerized CsgA stained with ThT. (B) The left image shows an overview, the right image a magnification of an aggregate of fibers. Scale bars are 250 µm (left) and 25 µm (right), respectively. (C) Fluorescent signals from CsgA incubated with LL-37, sLL-37, or VIP were quantified. Values were corrected for the contribution of the peptides themselves and are expressed in relation to the signal from CsgA alone. The inhibitory effect of LL-37 is significantly stronger than the effect of sLL-37 (** P = 0.007) or VIP (* P = 0.011). Combined results from two experiments are presented.
Mentions: To explain a possible cause for the inhibition of biofilm formation by LL-37, the effect of LL-37 on curli formation was investigated. For this purpose, we utilized monomeric recombinant CsgA, the major subunit of curli, which spontaneously polymerizes [28]. CsgA polymerization was monitored with thioflavin T (ThT), a fluorescent dye that binds to polymerized, but not to monomeric CsgA. Our results demonstrated that CsgA polymerization started immediately after incubation at 37°C and reached a stationary phase after approximately 300 min (red line in Figure 7A). After prolonged incubation, the fluorescence declined, most likely due to degradation of ThT and/or precipitation of fibers [28]. The polymerization was inhibited by LL-37 in a dose-dependent manner, and at a molar ratio of 1∶1 (CsgA∶LL-37) fiber formation was completely inhibited (Figure 7A, left panel). The control peptides sLL-37 and VIP had a less pronounced effect on CsgA polymerization than LL-37 (Figure 7A, right panel).

Bottom Line: Our results suggest that curli and cellulose exhibit differential and complementary functions.Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys.Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms.

Show MeSH
Related in: MedlinePlus