Structural basis of chaperone self-capping in P pilus biogenesis

Hung, Danielle L.; Pinkner, Jerome S.; Knight, Stefan D.; Hultgren, Scott J.

doi:10.1073/pnas.96.14.8178

Cited by 31 publications

(35 citation statements)

References 30 publications

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“…Periplasmic chaperones associated with processes such as pilus biogenesis (18), outer membrane biogenesis (2,5,9,39,48), and secretion (19), have been identified but an overall understanding of the periplasmic folding process and inner membrane protein biogenesis is still emerging. None of the classical cytoplasmic chaperones, such as members of the hsp 60, hsp 70, or hsp 100 family, is found in the periplasm, consistent with the fact that this compartment lacks the ATP required for their activity.…”

Section: Vol 186 2004 Role Of Tcph In V Cholerae Virulence Gene Exmentioning

confidence: 99%

TcpH Influences Virulence Gene Expression inVibrio choleraeby Inhibiting Degradation of the Transcription Activator TcpP

Beck¹,

Krukonis²,

DiRita

2004

J Bacteriol

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Expression of toxT, the transcription activator of cholera toxin and pilus production in Vibrio cholerae, is the consequence of a complex cascade of regulatory events that culminates in activation of the toxT promoter by TcpP and ToxR, two membrane-localized transcription factors. Both are encoded in operons with genes whose products, TcpH and ToxS, which are also membrane localized, are hypothesized to control their activity. In this study we analyzed the role of TcpH in controlling TcpP function. We show that a mutant of V. cholerae lacking TcpH expressed virtually undetectable levels of TcpP, although tcpP mRNA levels remain unaffected. A time course experiment showed that levels of TcpP, expressed from a plasmid, are dramatically reduced over time without co-overexpression of TcpH. By contrast, deletion of toxS did not affect ToxR protein levels. A fusion protein in which the TcpP periplasmic domain is replaced with that of ToxR remains stable, suggesting that the periplasmic domain of TcpP is the target for degradation of the protein. Placement of the periplasmic domain of TcpP on ToxR, an otherwise stable protein, results in instability, providing further evidence for the hypothesis that the periplasmic domain of TcpP is a target for degradation. Consistent with this interpretation is our finding that derivatives of TcpP lacking a periplasmic domain are more stable in V. cholerae than are derivatives in which the periplasmic domain has been truncated. This work identifies at least one role for the periplasmic domain of TcpP, i.e., to act as a target for a protein degradation pathway that regulates TcpP levels. It also provides a rationale for why the V. cholerae tcpH mutant strain is avirulent. We hypothesize that regulator degradation may be an important mechanism for regulating virulence gene expression in V. cholerae.

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Section: Vol 186 2004 Role Of Tcph In V Cholerae Virulence Gene Exmentioning

confidence: 99%

TcpH Influences Virulence Gene Expression inVibrio choleraeby Inhibiting Degradation of the Transcription Activator TcpP

Beck¹,

Krukonis²,

DiRita

2004

J Bacteriol

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“…During P pilus expression, some subunits leave the pathway (off-pathway) or fail to interact with the periplasmic chaperone, thereby forming in the periplasmic toxic misfolded aggregates that associate with the inner membrane (39,47). In laboratory strains of E. coli, the expression of all of the pap genes required for P pilus expression induces the Cpx envelope stress response, which in turn activates the expression of at least two genes that encode products required for efficient P pilus assembly (47).…”

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confidence: 99%

The Cpx Envelope Stress Response Affects Expression of the Type IV Bundle-Forming Pili of EnteropathogenicEscherichiacoli

2005

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The Cpx envelope stress response mediates adaptation to potentially lethal envelope stresses in Escherichia coli. The two-component regulatory system consisting of the sensor kinase CpxA and the response regulator CpxR senses and mediates adaptation to envelope insults believed to result in protein misfolding in this compartment. Recently, a role was demonstrated for the Cpx response in the biogenesis of P pili, attachment organelles expressed by uropathogenic E. coli. CpxA senses misfolded P pilus assembly intermediates and initiates increased expression of both assembly and regulatory factors required for P pilus elaboration. In this report, we demonstrate that the Cpx response is also involved in the expression of the type IV bundle-forming pili of enteropathogenic E. coli (EPEC). Bundle-forming pili were not elaborated from an exogenous promoter in E. coli laboratory strain MC4100 unless the Cpx pathway was constitutively activated. Further, an EPEC cpxR mutant synthesized diminished levels of bundle-forming pili and was significantly affected in adherence to epithelial cells. Since type IV bundle-forming pili are very different from chaperone-usher-type P pili in both form and biogenesis, our results suggest that the Cpx envelope stress response plays a general role in the expression of envelope-localized organelles with diverse structures and assembly pathways.

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“…However, so far the oligomeric state of apo PapD in solution has not been established. There have been three crystal structures solved for mutant versions of apo PapD (15,17), but no wild-type (WT) apo PapD structure has been reported. The first PapD crystal structure solved contained an E60D single mutation in the chaperone D1 (PDB entry 3DPA) (15) (Fig.…”

mentioning

confidence: 99%

“…There is a cleft between D1 and D2 of the chaperone that contains conserved hydrophobic residues that are critical to subunit binding (17). Targeted mutations in this interface severely reduce or abolish subunit binding (18) (Fig.…”

mentioning

confidence: 99%

The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution

Sarowar

Werneburg

et al. 2016

J Bacteriol

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The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains of Escherichia coli use this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in the periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 ␤-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function. IMPORTANCEPili are critical virulence factors for many bacterial pathogens. Uropathogenic E. coli relies on P and type 1 pili assembled by the chaperone/usher pathway to adhere to the urinary tract and establish infection. Studying pilus assembly is important for understanding mechanisms of protein secretion, as well as for identifying points for therapeutic intervention. Pilus biogenesis is a multistep process. This work investigates the oligomeric state of the pilus chaperone in the periplasm, which is important for understanding early assembly events. Our work unambiguously demonstrates that both PapD and FimC chaperones are monomeric in solution. We further demonstrate that the solution behavior of the FimH and PapGII adhesins differ, which may be related to functional differences between the two pilus systems.A n important step in the pathogenesis of uropathogenic Escherichia coli is the binding to host receptors within the urinary tract. This is accomplished by the recognition of sugar moieties on the surface of urothelial cells by lectins at the tip of elongated organelles, termed pili or fimbriae (1, 2). Specifically, uropathogenic E. coli expresses type 1 and P pili, which mediate binding to the bladder and kidney, respectively (3, 4). Type 1 and P pili are assembled by the chaperone/usher pathway, which is a conserved secretion system used to assemble a wide variety of pilus-type struct...

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Structural basis of chaperone self-capping in P pilus biogenesis

Cited by 31 publications

References 30 publications

TcpH Influences Virulence Gene Expression inVibrio choleraeby Inhibiting Degradation of the Transcription Activator TcpP

TcpH Influences Virulence Gene Expression inVibrio choleraeby Inhibiting Degradation of the Transcription Activator TcpP

The Cpx Envelope Stress Response Affects Expression of the Type IV Bundle-Forming Pili of EnteropathogenicEscherichiacoli

The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution

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