Processive dynamics of the usher assembly platform during uropathogenic Escherichia coli P pilus biogenesis.
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ABSTRACT:
SUBMITTER: Du M
PROVIDER: S-EPMC8410936 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane ?-barrel translocation domain (TD), a ?-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two C-terminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (?12-13 loop) was found to facilitate pore opening. Mutation of the ?12-13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.
| S-EPMC3870681 | biostudies-literature
Project description:Chaperone-usher pathway pili are extracellular proteinaceous fibres ubiquitously found on Gram-negative bacteria, and mediate host-pathogen interactions and biofilm formation critical in pathogenesis in numerous human diseases1. During pilus assembly, an outer membrane macromolecular machine called the usher catalyses pilus biogenesis from the individual subunits that are delivered as chaperone-subunit complexes in the periplasm. The usher orchestrates pilus assembly using all five functional domains: a 24-stranded transmembrane ?-barrel translocation domain, a ?-sandwich plug domain, an amino-terminal periplasmic domain and two carboxy-terminal periplasmic domains (CTD1 and CTD2)2-6. Despite extensive structural and functional characterization, the mechanism by which the usher is activated to initiate pilus biogenesis is unknown. Here, we present the crystal structure of the full-length PapC usher from Escherichia coli in complex with its cognate PapDG chaperone-subunit complex in a pre-activation state, elucidating molecular details of how the usher is specifically engaged by allosteric interactions with its substrate preceding activation and how the usher facilitates the transfer of subunits from the amino-terminal periplasmic domain to the CTDs during pilus assembly. This work elucidates the intricate workings of a molecular machine that catalyses chaperone-usher pathway pilus assembly and opens the door for the development of potent inhibitors to block pilus biogenesis.
| S-EPMC6258349 | biostudies-literature
Project description:BACKGROUND: The global spread of bacterial resistance has given rise to a growing interest in new anti-bacterial agents with a new strategy of action. Pilicides are derivatives of ring-fused 2-pyridones which block the formation of the pili/fimbriae crucial to bacterial pathogenesis. They impair by means of a chaperone-usher pathway conserved in the Gram-negative bacteria of adhesive structures biogenesis. Pili/fimbriae of this type belong to two subfamilies, FGS and FGL, which differ in the details of their assembly mechanism. The data published to date have shown that pilicides inhibit biogenesis of type 1 and P pili of the FGS type which are encoded by uropathogenic E. coli strains. RESULTS: We evaluated the anti-bacterial activity of literature pilicides as blockers of the assembly of a model example of FGL-type adhesive structures--the Dr fimbriae encoded by a dra gene cluster of uropathogenic Escherichia coli strains. In comparison to the strain grown without pilicide, the Dr? bacteria cultivated in the presence of the 3.5 mM concentration of pilicides resulted in a reduction of 75 to 87% in the adherence properties to CHO cells expressing Dr fimbrial DAF receptor protein. Using quantitative assays, we determined the amount of Dr fimbriae in the bacteria cultivated in the presence of 3.5 mM of pilicides to be reduced by 75 to 81%. The inhibition effect of pilicides is concentration dependent, which is a crucial property for their use as potential anti-bacterial agents. The data presented in this article indicate that pilicides in mM concentration effectively inhibit the adherence of Dr? bacteria to the host cells--the crucial, initial step in bacterial pathogenesis. CONCLUSIONS: Structural analysis of the DraB chaperone clearly showed it to be a model of the FGL subfamily of chaperones. This permits us to conclude that analyzed pilicides in mM concentration are effective inhibitors of the assembly of adhesins belonging to the Dr family, and more speculatively, of other FGL-type adhesive organelles. The presented data and those published so far permit to speculate that based on the conservation of chaperone-usher pathway in Gram-negative bacteria , the pilicides are potential anti-bacterial agents with activity against numerous pathogens, the virulence of which is dependent on the adhesive structures of the chaperone-usher type.
| S-EPMC3706281 | biostudies-literature
Project description:To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC).Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined.N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili.We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens.
| S-EPMC3956373 | biostudies-literature
Project description:P pili are important adhesive fibres that are assembled by the conserved chaperone-usher pathway. During pilus assembly, the subunits are incorporated into the growing fibre by the donor-strand exchange mechanism, whereby the beta-strand of the chaperone, which complements the incomplete immunoglobulin fold of each subunit, is displaced by the amino-terminal extension of an incoming subunit in a zip-in-zip-out exchange process that is initiated at the P5 pocket, an exposed hydrophobic pocket in the groove of the subunit. In vivo, termination of P pilus growth requires a specialized subunit, PapH. Here, we show that PapH is incorporated at the base of the growing pilus, where it is unable to undergo donor-strand exchange. This inability is not due to a stronger PapD-PapH interaction, but to a lack of a P5 initiator pocket in the PapH structure, suggesting that PapH terminates pilus growth because it is lacking the initiation point by which donor-strand exchange proceeds.
| S-EPMC1794691 | biostudies-literature
Project description:Up to eight different types of secretion systems, and several more subtypes, have been described in Gram-negative bacteria. Here, we focus on the diversity and assembly mechanism of one of the best-studied secretion systems, the widespread chaperone-usher pathway known to assemble and secrete adhesive surface structures, called pili or fimbriae, which play essential roles in targeting bacterial pathogens to the host.
| S-EPMC3297437 | biostudies-literature
Project description:A chemical synthesis platform with broad applications and flexibility was rationally designed to inhibit biogenesis of adhesive pili assembled by the chaperone-usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2-pyridones, termed pilicides, was evaluated in two different pilus biogenesis systems in uropathogenic Escherichia coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by approximately 90% in laboratory and clinical E. coli strains. The structure of the pilicide bound to the P pilus chaperone PapD revealed that the pilicide bound to the surface of the chaperone known to interact with the usher, the outer-membrane assembly platform where pili are assembled. Point mutations in the pilicide-binding site dramatically reduced pilus formation but did not block the ability of PapD to bind subunits and mediate their folding. Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone-subunit complexes to the usher. These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors.
| S-EPMC1693844 | biostudies-literature
Project description:Bacterial type 4a pili are dynamic surface filaments that promote bacterial adherence, motility, and macromolecular transport. Their genes are highly conserved among enterobacteria and their expression in enterohemorrhagic Escherichia coli (EHEC) promotes adhesion to intestinal epithelia and pro-inflammatory signaling. To define the molecular basis of EHEC pilus assembly, we determined the structure of the periplasmic domain of its major subunit PpdD (PpdDp), a prototype of an enterobacterial pilin subfamily containing two disulfide bonds. The structure of PpdDp, determined by NMR, was then docked into the density envelope of purified EHEC pili obtained by cryoelectron microscopy (cryo-EM). Cryo-EM reconstruction of EHEC pili at ∼8 Å resolution revealed extremely high pilus flexibility correlating with a large extended region of the pilin stem. Systematic mutagenesis combined with functional and interaction analyses identified charged residues essential for pilus assembly. Structural information on exposed regions and interfaces between EHEC pilins is relevant for vaccine and drug discovery.
| S-EPMC7003672 | biostudies-literature
Project description:Type IV pili are long fibers that are assembled by polymerization of a major pilin protein in the periplasm of a wide range of bacteria and archaea. They play crucial roles in pathogenesis, DNA transformation, and motility, and are capable of rapid retraction, generating powerful motor forces. PilN and PilO are integral inner membrane proteins that are essential for type IV pilus formation. Here, we show that PilN and PilO from Thermus thermophilus can be isolated as a complex with PilM, a cytoplasmic protein with structural similarities to the cytoskeletal protein MreB. The crystal structure of the periplasmic portion of PilN forms a homodimer with an extensive, conserved interaction interface. We conducted serial 3D reconstructions by electron microscopy of PilMN, PilMNO, and PilMNO bound to the major pilin protein PilA4, to chart the assembly of the inner membrane pilus biogenesis platform. PilN drives the dimerization of the PilMN complex with a stoichiometry of 2:2; binding of two PilO monomers then causes the PilN periplasmic domains to dissociate. Finally, two PilA4 monomers bind to the periplasmic domains of PilN and PilO, to generate a T-shaped complex that is primed for addition of the pilin to the nascent pilus fiber. Docking of structures for PilM, PilN, PilO, and PilA4 into the electron density maps of the transmembrane complexes was used to generate a sequence of molecular structures that chart the initial events in type IV pilus formation, and provide structural information on the early events in this important secretion process.
| S-EPMC3845092 | biostudies-literature
Project description:Type 1 pili are representative of a class of bacterial surface structures assembled by the conserved chaperone/usher pathway and used by uropathogenic Escherichia coli to attach to bladder cells during infection. The outer membrane assembly platform-the usher-is critical for the formation of pili, catalysing the polymerisation of pilus subunits and enabling the secretion of the nascent pilus. Despite extensive structural characterisation of the usher, a number of questions about its mechanism remain, notably its oligomerisation state, and how it orchestrates the ordered assembly of pilus subunits. We demonstrate here that the FimD usher is able to catalyse in vitro pilus assembly effectively in its monomeric form. Furthermore, by establishing the kinetics of usher-catalysed reactions between various pilus subunits, we establish a complete kinetic model of tip fibrillum assembly, able to account for the order of subunits in native type 1 pili.
| S-EPMC3650583 | biostudies-literature