Processive Dynamics of the Usher Assembly Platform During Uropathogenic Escherichia coli P Pilus Biogenesis

ABSTRACTUropathogenic Escherichia coli (UPEC) assemble hair-like surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili mediate the adherence of UPEC to the kidney epithelium, facilitating bacterial colonization and pyelonephritis1. P pili are assembled through the conserved chaperone-usher (CU) pathway2-4. In this pathway, a dedicated chaperone facilitates the folding of nascent pilus subunits in the periplasm and an integral outer membrane (OM) protein termed the usher provides the assembly platform and secretion channel for the pilus fiber. Much of the structural and functional understanding of the CU pathway has been gained through investigations of type 1 pili, which promote UPEC binding to the bladder epithelium and the development of cystitis5. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, as well as difficulties in isolating stable P pilus assembly intermediates from bacteria. Here, we have devised a method to circumvent these hindrances and determined cryo-EM structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates. These structures show processive steps in P pilus biogenesis, reveal differences between P and type 1 pili, and capture new conformational dynamics of the usher assembly machine.

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Processive dynamics of the usher assembly platform during uropathogenic Escherichia coli P pilus biogenesis

Nature Communications ◽

10.1038/s41467-021-25522-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Minge Du ◽

Zuanning Yuan ◽

Glenn T. Werneburg ◽

Nadine S. Henderson ◽

Hemil Chauhan ◽

...

Keyword(s):

Escherichia Coli ◽

Bacterial Colonization ◽

Conformational Dynamics ◽

Structural Heterogeneity ◽

Structural Basis ◽

Type 1 Pili ◽

Pilus Biogenesis ◽

Functional Understanding ◽

Pilus Assembly

AbstractUropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili facilitate bacterial colonization of the kidney and pyelonephritis. P pili are assembled through the conserved chaperone-usher pathway. Much of the structural and functional understanding of the chaperone-usher pathway has been gained through investigations of type 1 pili, which promote binding to the bladder and cystitis. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, and difficulties isolating stable P pilus assembly intermediates. Here, we circumvent these hindrances and determine cryo-electron microscopy structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates, revealing processive steps in P pilus biogenesis and capturing new conformational dynamics of the usher assembly machine.

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Periplasmic Peptidyl Prolyl cis-trans Isomerases Are Not Essential for Viability, but SurA Is Required for Pilus Biogenesis in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.22.7680-7686.2005 ◽

2005 ◽

Vol 187 (22) ◽

pp. 7680-7686 ◽

Cited By ~ 98

Author(s):

Sheryl S. Justice ◽

David A. Hunstad ◽

Jill Reiss Harper ◽

Amy R. Duguay ◽

Jerome S. Pinkner ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane Proteins ◽

E Coli ◽

Outer Membranes ◽

Type 1 Pili ◽

Pilus Biogenesis ◽

Quadruple Mutant ◽

Increased Susceptibility ◽

Pilus Assembly

ABSTRACT In Escherichia coli, FkpA, PpiA, PpiD, and SurA are the four known periplasmic cis-trans prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the cis and trans states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the surA single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of E. coli and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.

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Chaperone-Subunit-Usher Interactions Required for Donor Strand Exchange during Bacterial Pilus Assembly

Journal of Bacteriology ◽

10.1128/jb.185.9.2723-2730.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2723-2730 ◽

Cited By ~ 48

Author(s):

Michelle M. Barnhart ◽

Frederic G. Sauer ◽

Jerome S. Pinkner ◽

Scott J. Hultgren

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Strand Exchange ◽

Assembly Mechanism ◽

Type 1 Pili ◽

Pilus Biogenesis ◽

A Subunit ◽

Donor Strand Exchange ◽

Pilus Assembly

ABSTRACT The assembly of type 1 pili on the surface of uropathogenic Escherichia coli proceeds via the chaperone-usher pathway. Chaperone-subunit complexes interact with one another via a process termed donor strand complementation whereby the G1β strand of the chaperone completes the immunoglobulin (Ig) fold of the pilus subunit. Chaperone-subunit complexes are targeted to the usher, which forms a channel across the outer membrane through which pilus subunits are translocated and assembled into pili via a mechanism known as donor strand exchange. This is a mechanism whereby chaperone uncapping from a subunit is coupled with the simultaneous assembly of the subunit into the pilus fiber. Thus, in the pilus fiber, the N-terminal extension of every subunit completes the Ig fold of its neighboring subunit by occupying the same site previously occupied by the chaperone. Here, we investigated details of the donor strand exchange assembly mechanism. We discovered that the information necessary for targeting the FimC-FimH complex to the usher resides mainly in the FimH protein. This interaction is an initiating event in pilus biogenesis. We discovered that the ability of an incoming subunit (in a chaperone-subunit complex) to participate in donor strand exchange with the growing pilus depended on a previously unrecognized function of the chaperone. Furthermore, the donor strand exchange assembly mechanism between subunits was found to be necessary for subunit translocation across the outer membrane usher.

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

Journal of Bacteriology ◽

10.1128/jb.00366-16 ◽

2016 ◽

Vol 198 (17) ◽

pp. 2360-2369 ◽

Cited By ~ 1

Author(s):

Samema Sarowar ◽

Olivia J. Hu ◽

Glenn T. Werneburg ◽

David G. Thanassi ◽

Huilin Li

Keyword(s):

Escherichia Coli ◽

Oligomeric State ◽

Content Type ◽

Dimer Interface ◽

Adhesive Surface ◽

Type 1 Pili ◽

Pilus Biogenesis ◽

Multistep Process ◽

Pilus Assembly

ABSTRACTThe chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains ofEscherichia coliuse 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 β-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. UropathogenicE. colirelies 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.

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Human Urine Decreases Function and Expression of Type 1 Pili in Uropathogenic Escherichia coli

mBio ◽

10.1128/mbio.00820-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 32

Author(s):

Sarah E. Greene ◽

Michael E. Hibbing ◽

James Janetka ◽

Swaine L. Chen ◽

Scott J. Hultgren

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Human Urine ◽

Phase Variable ◽

Bladder Epithelium ◽

Content Type ◽

Type 1 Pili ◽

Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) is the primary cause of community-acquired urinary tract infections (UTIs). UPEC bind the bladder using type 1 pili, encoded by thefimoperon in nearly allE. coli. Assembled type 1 pili terminate in the FimH adhesin, which specifically binds to mannosylated glycoproteins on the bladder epithelium. Expression of type 1 pili is regulated in part by phase-variable inversion of the genomic element containing thefimSpromoter, resulting in phase ON (expressing) and OFF (nonexpressing) orientations. Type 1 pili are essential for virulence in murine models of UTI; however, studies of urine samples from human UTI patients demonstrate variable expression of type 1 pili. We provide insight into this paradox by showing that human urine specifically inhibits both expression and function of type 1 pili. Growth in urine induces thefimSphase OFF orientation, preventingfimexpression. Urine also contains inhibitors of FimH function, and this inhibition leads to a further bias infimSorientation toward the phase OFF state. The dual effect of urine onfimSregulation and FimH binding presents a potential barrier to type 1 pilus-mediated colonization and invasion of the bladder epithelium. However, FimH-mediated attachment to human bladder cells during growth in urine reverses these effects such thatfimexpression remains ON and/or turns ON. Interestingly, FimH inhibitors called mannosides also induce thefimSphase OFF orientation. Thus, the transduction of FimH protein attachment or inhibition into epigenetic regulation of type 1 pilus expression has important implications for the development of therapeutics targeting FimH function.IMPORTANCEUrinary tract infections (UTIs) are extremely common infections, frequently caused by uropathogenicEscherichia coli(UPEC), that are treated with antibiotics but often recur. Therefore, UTI treatment both is complicated by and contributes to bacterial antibiotic resistance. Thus, it is important to understand UTI pathogenesis to devise novel strategies and targets for prevention and treatment. Based on evidence from disease epidemiology and mouse models of infection, UPEC relies heavily on type 1 pili to attach to and invade the bladder epithelium during initial stages of UTI. Here, we demonstrate that the negative effect of planktonic growth in human urine on both the function and expression of type 1 pili is overcome by attachment to bladder epithelial cells, representing a strategy to subvert this alternative innate defense mechanism. Furthermore, this dually inhibitory action of urine is a mechanism shared with recently developed anti-type 1 pilus molecules, highlighting the idea that further development of antivirulence strategies targeting pili may be particularly effective for UPEC.

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