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The pilus usher controls protein interactions via domain masking and is functional as an oligomer.

Werneburg GT, Henderson NS, Portnoy EB, Sarowar S, Hultgren SJ, Li H, Thanassi DG - Nat. Struct. Mol. Biol. (2015)

Bottom Line: Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD).We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher.We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, USA. [2] Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA.

ABSTRACT
The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

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Co-expression of FimDΔNΔplug andFimDΔC1ΔC2 ushers results in pilus assembly on thebacterial surface(a) Cartoon representations of theFimDΔC1ΔC2 (usher 1) andFimDΔNΔplug (usher 2) deletion mutants co-expressedin (c). The N, plug (P), C1, and C2 domains present in each usherconstruct are indicated. (b,c,d)whole-bacteria, negative-stain transmission EM of E. colistrain MM294ΔfimD expressing WT FimD (b),FimDΔNΔplug together withFimDΔC1ΔC2 (c), or vector only(d). The FimD expression plasmids used were as listed in Table 1. Scale bars = 500 nm.
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Figure 6: Co-expression of FimDΔNΔplug andFimDΔC1ΔC2 ushers results in pilus assembly on thebacterial surface(a) Cartoon representations of theFimDΔC1ΔC2 (usher 1) andFimDΔNΔplug (usher 2) deletion mutants co-expressedin (c). The N, plug (P), C1, and C2 domains present in each usherconstruct are indicated. (b,c,d)whole-bacteria, negative-stain transmission EM of E. colistrain MM294ΔfimD expressing WT FimD (b),FimDΔNΔplug together withFimDΔC1ΔC2 (c), or vector only(d). The FimD expression plasmids used were as listed in Table 1. Scale bars = 500 nm.

Mentions: The usher exists as an oligomer in the OM; however, only one usherprotomer is involved in secretion of the pilus fiber, and the function of theoligomer is not known20,23,30-32. Onepossibility is that the N domains of the nontranslocating ushers recruitchaperone-subunit complexes to the OM assembly platform, and these complexes arethen transferred to the C domains of the actively translocating usher. If true,then co-expression of FimDΔC1ΔC2 andFimDΔNΔplug usher mutants (N and C domainsavailable, respectively; Fig. 6a) shouldallow reconstitution of pilus biogenesis. Indeed, co-expression of theΔNΔplug and ΔC1ΔC2 FimD constructs resulted inassembly of functional type 1 pili, as measured by the HA assay (Table 1). Note that theΔNΔplug and ΔC1ΔC2 mutants did not assemble piliwhen expressed individually (Table 1).Pilus biogenesis on the bacterial surface by the strain co-expressingFimDΔC1ΔC2 and FimDΔNΔplugwas confirmed by electron microscopy (EM), which revealed levels of pilus fiberscomparable to the strain expressing WT FimD (Fig.6). Consistent with our finding that that the the plug domain masksthe C domains in the inactive usher, co-expression of a FimDΔNmutant (plug domain intact) with FimDΔC1ΔC2 did notresult in pilus assembly (Table 1). Inadditional experiments, we found that co-expression ofFimDΔNΔplug with aFimDΔplugΔC1ΔC2 mutant did not restore pilusassembly, and neither did co-expression of FimDΔN with aFimDΔplugΔC1ΔC2 mutant (Table 1). This indicates that a plug domainmust be present for successful complementation, but the plug cannot be locatedtogether with the C domains. These data demonstrate that individual ushermolecules are capable of functioning in trans for pilusbiogenesis in bacteria, and provide confirmation that the plug domain masks theC domains in the inactive usher.


The pilus usher controls protein interactions via domain masking and is functional as an oligomer.

Werneburg GT, Henderson NS, Portnoy EB, Sarowar S, Hultgren SJ, Li H, Thanassi DG - Nat. Struct. Mol. Biol. (2015)

Co-expression of FimDΔNΔplug andFimDΔC1ΔC2 ushers results in pilus assembly on thebacterial surface(a) Cartoon representations of theFimDΔC1ΔC2 (usher 1) andFimDΔNΔplug (usher 2) deletion mutants co-expressedin (c). The N, plug (P), C1, and C2 domains present in each usherconstruct are indicated. (b,c,d)whole-bacteria, negative-stain transmission EM of E. colistrain MM294ΔfimD expressing WT FimD (b),FimDΔNΔplug together withFimDΔC1ΔC2 (c), or vector only(d). The FimD expression plasmids used were as listed in Table 1. Scale bars = 500 nm.
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Figure 6: Co-expression of FimDΔNΔplug andFimDΔC1ΔC2 ushers results in pilus assembly on thebacterial surface(a) Cartoon representations of theFimDΔC1ΔC2 (usher 1) andFimDΔNΔplug (usher 2) deletion mutants co-expressedin (c). The N, plug (P), C1, and C2 domains present in each usherconstruct are indicated. (b,c,d)whole-bacteria, negative-stain transmission EM of E. colistrain MM294ΔfimD expressing WT FimD (b),FimDΔNΔplug together withFimDΔC1ΔC2 (c), or vector only(d). The FimD expression plasmids used were as listed in Table 1. Scale bars = 500 nm.
Mentions: The usher exists as an oligomer in the OM; however, only one usherprotomer is involved in secretion of the pilus fiber, and the function of theoligomer is not known20,23,30-32. Onepossibility is that the N domains of the nontranslocating ushers recruitchaperone-subunit complexes to the OM assembly platform, and these complexes arethen transferred to the C domains of the actively translocating usher. If true,then co-expression of FimDΔC1ΔC2 andFimDΔNΔplug usher mutants (N and C domainsavailable, respectively; Fig. 6a) shouldallow reconstitution of pilus biogenesis. Indeed, co-expression of theΔNΔplug and ΔC1ΔC2 FimD constructs resulted inassembly of functional type 1 pili, as measured by the HA assay (Table 1). Note that theΔNΔplug and ΔC1ΔC2 mutants did not assemble piliwhen expressed individually (Table 1).Pilus biogenesis on the bacterial surface by the strain co-expressingFimDΔC1ΔC2 and FimDΔNΔplugwas confirmed by electron microscopy (EM), which revealed levels of pilus fiberscomparable to the strain expressing WT FimD (Fig.6). Consistent with our finding that that the the plug domain masksthe C domains in the inactive usher, co-expression of a FimDΔNmutant (plug domain intact) with FimDΔC1ΔC2 did notresult in pilus assembly (Table 1). Inadditional experiments, we found that co-expression ofFimDΔNΔplug with aFimDΔplugΔC1ΔC2 mutant did not restore pilusassembly, and neither did co-expression of FimDΔN with aFimDΔplugΔC1ΔC2 mutant (Table 1). This indicates that a plug domainmust be present for successful complementation, but the plug cannot be locatedtogether with the C domains. These data demonstrate that individual ushermolecules are capable of functioning in trans for pilusbiogenesis in bacteria, and provide confirmation that the plug domain masks theC domains in the inactive usher.

Bottom Line: Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD).We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher.We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, USA. [2] Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA.

ABSTRACT
The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

Show MeSH
Related in: MedlinePlus