Adaptive mutations in the signal peptide of the type 1 fimbrial adhesin of uropathogenic Escherichia coli - PubMed (original) (raw)

Comparative Study

. 2008 Aug 5;105(31):10937-42.

doi: 10.1073/pnas.0803158105. Epub 2008 Jul 29.

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Comparative Study

Adaptive mutations in the signal peptide of the type 1 fimbrial adhesin of uropathogenic Escherichia coli

Leah S Ronald et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Signal peptides (SPs) are critical for protein transport across cellular membranes, have a highly conserved structure, and are cleaved from the mature protein upon translocation. Here, we report that naturally occurring mutations in the SP of the adhesive, tip-associated subunit of type 1 fimbriae (FimH) are positively selected in uropathogenic Escherichia coli. On the one hand, these mutations have a detrimental effect, with reduced FimH transport across the inner membrane, fewer FimH and fimbriae expressed on the bacterial surface, and decreased bacterial adhesion under flow conditions. On the other hand, the fimbriae expressed by the mutants are significantly longer on average, with many fimbriae able to stretch to >20 microm in length. More surprisingly, the SP mutant bacteria display an increased ability to resist detachment from the surface upon a switch from high to low flow. This functional effect of longer fimbriae highlights the importance of the nonadhesive fimbrial rod for adhesive function. Also, whereas bacterial adhesion to bladder epithelial cells was preserved in most mutants, binding to and killing by human neutrophils was decreased, providing an additional reason the SP mutations are relatively common among uropathogenic strains. Thus, this study demonstrates how mutations in an SP, while decreasing transport function and not affecting the final structure of the translocated protein, can lead to functional gains of the extracellular organelles that incorporate the protein and overall adaptive changes in the organism's fitness.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Mutations in FimH SP and predicted effects. (A) fimH DNA tree indicating SP mutations. (B) Alignment of the four resulting SP variants, with SP regions denoted by N (amino terminus), H (hydrophobic core), and C (carboxyl terminus). (C) Average core hydrophobicity of FimH SP variants plotted on a graph depicting results from Doud et al. (3).

Fig. 2.

Fig. 2.

Mutations that decrease FimH SP function reduce fimbrial number and increase fimbrial length. (A) FimH SP transport activity measured by units of alkaline phosphatase activity. (B) Surface FimH measured by flow cytometry. (C and D) Average (C) number and (D) length of type 1 fimbriae measured on electron micrographs. (E and F) Uncoiled fimbrial length (E) and percent of fimbriae >20 μm (F) remaining attached to the AFM cantilever at the end of a pull. All error bars represent SEM. Double horizontal lines represent WT ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 3.

Fig. 3.

Static and dynamic bacterial adhesion to mannose-coated surfaces. Bacteria express FimH with different SPs or have no FimH. (A) Static binding by growth assay. Error bars represent SEM. (B) Surface accumulation of bacteria over 5 min at different shear stresses. Error bars represent the Poisson distribution 95% C.I. (C) Fraction of bacteria rolling on the surface relative to surface accumulated bacteria, which includes rolling and stationary. Error bars represent the binomial distribution 95% C.I. (not included where counts ≤1, which occurred only at the highest two shear levels). (D) Maintenance of adhesion after bacteria were bound to the surface statically, then subjected to high (2 Pa) and low (0.01 Pa) flow. Error bars at each second represent the SEM of three independent experiments. At the 90-s time point, a t test comparing WT and V-4E is significant (P = 0.015).

Fig. 4.

Fig. 4.

Interactions with bladder epithelial cells and human neutrophils. WT was additionally tested in the presence of soluble mannose (+Man). (A) Adhesion to T24 bladder epithelial cells. (B) Binding to neutrophils. (C) Killing by neutrophils. For each strain, fraction killed was measured as one minus the fraction of bacteria surviving incubation with neutrophils relative to a growth control, then the ratio to WT was taken. All error bars represent SEM. *, P < 0.05; **, P < 0.01.

References

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