Dataset Information

Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry.

ABSTRACT: P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed "donor-strand exchange (DSE)" in which an N-terminal extension (Nte) of one subunit donates a beta-strand to an adjacent subunit, completing its Ig fold. Despite structural determination of the different subunits, the mechanism determining specificity of subunit ordering in pilus assembly remained unclear. Here, we have used noncovalent mass spectrometry to monitor DSE between all 30 possible pairs of P pilus subunits and their Ntes. We demonstrate a striking correlation between the natural order of subunits in pili and their ability to undergo DSE in vitro. The results reveal insights into the molecular mechanism by which subunit ordering during the assembly of this complex is achieved.

SUBMITTER: Rose RJ

PROVIDER: S-EPMC2525559 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry.

Rose Rebecca J RJ Verger Denis D Daviter Tina T Remaut Han H Paci Emanuele E Waksman Gabriel G Ashcroft Alison E AE Radford Sheena E SE

Proceedings of the National Academy of Sciences of the United States of America 20080826 35

P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed "donor-strand exchange (DSE)" in which an N-terminal extension (Nte) of one subunit donates a beta-strand to an adjacent subunit, completing its Ig fold. Des ...[more]

PMID: 18728178

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Project description:During pilus assembly within the Gram-positive bacterial envelope, membrane-bound sortase enzymes sequentially crosslink specific pilus protein monomers through their cell wall sorting signals (CWSS), starting with a designated tip pilin, followed by the shaft made of another pilin, ultimately anchoring the fiber base pilin to the cell wall. To date, the molecular determinants that govern pilus tip assembly and the underlying mechanism remain unknown. Here, we addressed this in the model organism Actinomyces oris. This oral microbe assembles a pathogenically important pilus (known as type 2 fimbria) whose shafts, made of FimA pilins, display one of two alternate tip pilins-FimB or the coaggregation factor CafA-that share a markedly similar CWSS. We demonstrate that swapping the CWSS of CafA with that of FimB produces a functional hybrid, which localizes at the pilus tip and mediates polymicrobial coaggregation, whereas alanine-substitution of the conserved FLIAG motif within the CWSS hampers these processes. Remarkably, swapping the CWSS of the normal cell wall-anchored glycoprotein GspA with that of CafA promotes the assembly of hybrid GspA at the FimA pilus tip. Finally, exchanging the CWSS of the Corynebacterium diphtheriae shaft pilin SpaA with that of CafA leads to the FLIAG motif-dependent localization of the heterologous pilus protein SpaA at the FimA pilus tip in A. oris. Evidently, the CWSS and the FLIAG motif of CafA are both necessary and sufficient for its destination to the cognate pilus tip specifically assembled by a designated sortase in the organism.ImportanceGram-positive pili, whose precursors harbor a cell wall sorting signal (CWSS) needed for sortase-mediated pilus assembly, typically comprise a pilus shaft and a tip adhesin. How a pilin becomes a pilus tip, nevertheless, remains undetermined. We demonstrate here in Actinomyces oris that the CWSS of the tip pilin CafA is necessary and sufficient to promote pilus tip assembly, and this functional assembly involves a conserved FLIAG motif within the CWSS. This is evidenced by the fact that an A. oris cell-wall anchored glycoprotein, GspA, or a heterologous shaft pilin from Corynebacterium diphtheriae, SpaA, engineered to have the CWSS of CafA in place of their CWSS, localizes at the pilus tip in a process that requires the FLIAG motif. Our findings provide the molecular basis for sortase-catalyzed pilus tip assembly that is very likely employed by other Gram-positive bacteria and potential bioengineering applications to display antigens at controlled surface distance.

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Dataset Information

Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry.

Publications

Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry.

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