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

Uropathogenic 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.

The Natural History and Composition of Urinary Catheter Biofilms: Early Uropathogen Colonization with Intraluminal and Distal Predominance.

PURPOSE: We sought to determine the composition and initiation site of bacterial biofilm on indwelling urinary catheters and to track biofilm progression with time. MATERIALS AND METHODS: Indwelling urinary catheters were collected from 2 tertiary care centers following removal from patients. Indwelling time was noted and catheters were de-identified. Catheters were sectioned, stained for biofilms and analyzed by spectrophotometry and visualization. Biofilm colonization patterns were analyzed using descriptive statistical analysis and bacterial composition was determined using next generation sequencing. RESULTS: We collected and analyzed a total of 33 catheters from 26 males and 7 females with indwelling time ranging from 15 minutes to 43 days. Biofilm colonization was consistently high on the region of the balloon for all indwelling times. After week 1 the distal third of the catheter had higher biofilm colonization than the proximal third (week 2 p=0.034). At all indwelling times the intraluminal surface of the catheter had greater biofilm colonization than the outer surface. Next generation sequencing detected potential uropathogenic bacteria in all 10 analyzed samples. CONCLUSIONS: The catheter balloon, its distal aspect and its lumen were the predominant locations of biofilm comprising uropathogenic bacteria. Strategies to prevent or treat biofilm should be targeted to these areas.

The small molecule nitazoxanide selectively disrupts BAM-mediated folding of the outer membrane usher protein.

Bacterial pathogens assemble adhesive surface structures termed pili or fimbriae to initiate and sustain infection of host tissues. Uropathogenic Escherichia coli, the primary causative agent of urinary tract infections, expresses type 1 and P pili required for colonization of the bladder and kidney, respectively. These pili are assembled by the conserved chaperone-usher (CU) pathway, in which a periplasmic chaperone works together with an outer membrane (OM) usher protein to build and secrete the pilus fiber. Previously, we found that the small molecule and antiparasitic drug nitazoxanide (NTZ) inhibits CU pathway-mediated pilus biogenesis in E. coli by specifically interfering with proper maturation of the usher protein in the OM. The usher is folded and inserted into the OM by the ß-barrel assembly machine (BAM) complex, which in E. coli comprises five proteins, BamA-E. Here, we show that sensitivity of the usher to NTZ is modulated by BAM expression levels and requires the BamB and BamE lipoproteins. Furthermore, a genetic screen for NTZ-resistant bacterial mutants isolated a mutation in the essential BamD lipoprotein. These findings suggest that NTZ selectively interferes with an usher-specific arm of the BAM complex, revealing new details of the usher folding pathway and BAM complex function. Evaluation of a set of NTZ derivatives identified compounds with increased potency and disclosed that NTZ's nitrothiazole ring is critical for usher inhibition. In summary, our findings indicate highly specific effects of NTZ on the usher folding pathway and have uncovered NTZ analogs that specifically decrease usher levels in the OM.

Effect of chaperone-adhesin complex on plug release by the PapC usher.

The P pilus of uropathogenic Escherichia coli is a multisubunit fiber assembled at the outer membrane in a defined sequence by a chaperone/usher secretion system, comprising a periplasmic chaperone and a beta-barrel outer membrane protein, the PapC usher. To gain insight into the pilus biogenesis mechanism, we used patch clamp electrophysiology to investigate the effect of the initiating adhesin subunit, as it is delivered to PapC in a complex with the chaperone. We show that the chaperone-adhesin complex facilitates opening of the PapC pore and appears to engage within the PapC lumen, in agreement with prior biochemical and structural data.

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

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.

Electrostatic networks control plug stabilization in the PapC usher.

The PapC usher, a ß-barrel pore in the outer membrane of uropathogenic Escherichia coli, is used for assembly of the P pilus, a key virulence factor in bacterial colonization of human kidney cells. Each PapC protein is composed of a 24-stranded ß-barrel channel, flanked by N- and C-terminal globular domains protruding into the periplasm, and occluded by a plug domain (PD). The PD is displaced from the channel towards the periplasm during pilus biogenesis, but the molecular mechanism for PD displacement remains unclear. Two structural features within the ß-barrel, an α-helix and ß5-6 hairpin loop, may play roles in controlling plug stabilization. Here we have tested clusters of residues at the interface of the plug, barrel, α-helix and hairpin, which participate in electrostatic networks. To assess the roles of these residues in plug stabilization, we used patch-clamp electrophysiology to compare the activity of wild-type and mutant PapC channels containing alanine substitutions at these sites. Mutations interrupting each of two salt bridge networks were relatively ineffective in disrupting plug stabilization. However, mutation of two pairs of arginines located at the inner and the outer surfaces of the PD resulted in an enhanced propensity for plug displacement. One arginine pair involved in a repulsive interaction between the linkers that tether the plug to the ß-barrel was particularly sensitive to mutation. These results suggest that plug displacement, which is necessary for pilus assembly and translocation, may require a weakening of key electrostatic interactions between the plug linkers, and the plug and the α-helix.

Allosteric signalling in the outer membrane translocation domain of PapC usher.

PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large ß-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a ß-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the ß-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue 'communities') within the translocation domain (especially around ß12-ß14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.

Molecular basis of usher pore gating in Escherichia coli pilus biogenesis.

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 ß-barrel translocation domain (TD), a ß-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 (ß12-13 loop) was found to facilitate pore opening. Mutation of the ß12-13 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.

Purification of the outer membrane usher protein and periplasmic chaperone-subunit complexes from the P and type 1 pilus systems.

Understanding molecular mechanisms of protein secretion by bacteria requires the purification of secretion machinery components and the isolation of complexes between the secretion machinery and substrate proteins. Here, we describe methods for the purification of proteins from the chaperone/usher pathway, which is a conserved secretion pathway dedicated to the assembly of polymeric surface fibers termed pili or fimbriae in gram-negative bacteria. Specifically, we describe the isolation of the PapC and FimD usher proteins from the bacterial outer membrane, and the purification of PapD-PapG and FimC-FimH chaperone--subunit complexes from the periplasm. These Pap and Fim proteins belong to the P and type 1 pilus systems of uropathogenic Escherichia coli, respectively.

Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine.

P pili are prototypical chaperone-usher pathway-assembled pili used by Gram-negative bacteria to adhere to host tissues. The PapC usher contains five functional domains: a transmembrane ß-barrel, a ß-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and two C-terminal (periplasmic) domains, CTD1 and CTD2. Here, we delineated usher domain interactions between themselves and with chaperone-subunit complexes and showed that overexpression of individual usher domains inhibits pilus assembly. Prior work revealed that the Plug domain occludes the pore of the transmembrane domain of a solitary usher, but the chaperone-adhesin-bound usher has its Plug displaced from the pore, adjacent to the NTD. We demonstrate an interaction between the NTD and Plug domains that suggests a biophysical basis for usher gating. Furthermore, we found that the NTD exhibits high-affinity binding to the chaperone-adhesin (PapDG) complex and low-affinity binding to the major tip subunit PapE (PapDE). We also demonstrate that CTD2 binds with lower affinity to all tested chaperone-subunit complexes except for the chaperone-terminator subunit (PapDH) and has a catalytic role in dissociating the NTD-PapDG complex, suggesting an interplay between recruitment to the NTD and transfer to CTD2 during pilus initiation. The Plug domain and the NTD-Plug complex bound all of the chaperone-subunit complexes tested including PapDH, suggesting that the Plug actively recruits chaperone-subunit complexes to the usher and is the sole recruiter of PapDH. Overall, our studies reveal the cooperative, active roles played by periplasmic domains of the usher to initiate, grow, and terminate a prototypical chaperone-usher pathway pilus.

Crystal structure of the FimD usher bound to its cognate FimC-FimH substrate.

Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyses their polymerization and mediates pilus translocation across the outer membrane. Here we report the crystal structure of the full-length FimD usher bound to the FimC-FimH chaperone-adhesin complex and that of the unbound form of the FimD translocation domain. The FimD-FimC-FimH structure shows FimH inserted inside the FimD 24-stranded ß-barrel translocation channel. FimC-FimH is held in place through interactions with the two carboxy-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a marked conformational change in the ß-barrel. The amino-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone-subunit complex. The FimD-FimC-FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.

Function of the usher N-terminus in catalysing pilus assembly.

The chaperone/usher (CU) pathway is a conserved bacterial secretion system that assembles adhesive fibres termed pili or fimbriae. Pilus biogenesis by the CU pathway requires a periplasmic chaperone and an outer membrane (OM) assembly platform termed the usher. The usher catalyses formation of subunit-subunit interactions to promote polymerization of the pilus fibre and provides the channel for fibre secretion. The mechanism by which the usher catalyses pilus assembly is not known. Using the P and type 1 pilus systems of uropathogenic Escherichia coli, we show that a conserved N-terminal disulphide region of the PapC and FimD ushers, as well as residue F4 of FimD, are required for the catalytic activity of the ushers. PapC disulphide loop mutants were able to bind PapDG chaperone-subunit complexes, but did not assemble PapG into pilus fibres. FimD disulphide loop and F4 mutants were able to bind chaperone-subunit complexes and initiate assembly of pilus fibres, but were defective for extending the pilus fibres, as measured using in vivo co-purification and in vitro pilus polymerization assays. These results suggest that the catalytic activity of PapC is required to initiate pilus biogenesis, whereas the catalytic activity of FimD is required for extension of the pilus fibre.

Modulating effects of the plug, helix, and N- and C-terminal domains on channel properties of the PapC usher.

The chaperone/usher system is one of the best characterized pathways for protein secretion and assembly of cell surface appendages in Gram-negative bacteria. In particular, this pathway is used for biogenesis of the P pilus, a key virulence factor used by uropathogenic Escherichia coli to adhere to the host urinary tract. The P pilus individual subunits bound to the periplasmic chaperone PapD are delivered to the outer membrane PapC usher, which serves as an assembly platform for subunit incorporation into the pilus and secretion of the pilus fiber to the cell surface. PapC forms a dimeric, twin pore complex, with each monomer composed of a 24-stranded transmembrane beta-barrel channel, an internal plug domain that occludes the channel, and globular N- and C-terminal domains that are located in the periplasm. Here we have used planar lipid bilayer electrophysiology to characterize the pore properties of wild type PapC and domain deletion mutants for the first time. The wild type pore is closed most of the time but displays frequent short-lived transitions to various open states. In comparison, PapC mutants containing deletions of the plug domain, an alpha-helix that caps the plug domain, or the N- and C-terminal domains form channels with higher open probability but still exhibiting dynamic behavior. Removal of the plug domain results in a channel with extremely large conductance. These observations suggest that the plug gates the usher channel closed and that the periplasmic domains and alpha-helix function to modulate the gating activity of the PapC twin pore.

Fiber formation across the bacterial outer membrane by the chaperone/usher pathway.

Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform--the usher--is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of gram-negative bacteria.

Topology of the outer membrane usher PapC determined by site-directed fluorescence labeling.

In contrast to typical membrane proteins that span the lipid bilayer via transmembrane alpha-helices, bacterial outer membrane proteins adopt a beta-barrel architecture composed of antiparallel transmembrane beta-strands. The topology of outer membrane proteins is difficult to predict accurately using computer algorithms, and topology mapping protocols commonly used for alpha-helical membrane proteins do not work for beta-barrel proteins. We present here the topology of the PapC usher, an outer membrane protein required for assembly and secretion of P pili by the chaperone/usher pathway in uropathogenic Escherichia coli. An initial attempt to map PapC topology by insertion of protease cleavage sites was largely unsuccessful due to lack of cleavage at most sites and the requirement to disrupt the outer membrane to identify periplasmic sites. We therefore adapted a site-directed fluorescence labeling technique to permit topology mapping of outer membrane proteins using small molecule probes in intact bacteria. Using this method, we demonstrated that PapC has the potential to encode up to 32 transmembrane beta-strands. Based on experimental evidence, we propose that the usher consists of an N-terminal beta-barrel domain comprised of 26 beta-strands and that a distinct C-terminal domain is not inserted into the membrane but is located instead within the lumen of the N-terminal beta-barrel similar to the plug domains encoded by the outer membrane iron-siderophore uptake proteins.

Blue Native electrophoresis to study mitochondrial and other protein complexes.

The biogenesis and maintenance of mitochondria relies on a sizable number of proteins. Many of these proteins are organized into complexes, which are located in the mitochondrial inner membrane. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a method for the isolation of intact protein complexes. Although it was initially used to study mitochondrial respiratory chain enzymes, it can also be applied to other protein complexes. The use of BN-PAGE has increased exponentially over the past few years and new applications have been developed. Here we review how to set up the basic system and outline modifications that can be applied to address specific research questions. Increasing the upper mass limit of complexes that can be separated by BN-PAGE can be achieved by using agarose instead of acrylamide. BN-PAGE can also be used to study assembly of mitochondrial protein complexes. Other applications include in-gel measurements of enzyme activity by histochemical staining and preparative native electrophoresis to isolate a protein complex. Finally, new ways of identifying protein spots in Blue Native gels using mass spectrometry are briefly discussed.

Mapping and identification of essential gene functions on the X chromosome of Drosophila.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.