Glenn T. Werneburg

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.

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.

Effects of Focal Versus Total Cryotherapy and Minimum Tumor Temperature on Patient-Reported Quality of Life as Compared to Active Surveillance in Prostate Cancer Patients

OBJECTIVE To investigate the effects of focal (hemiablation) or total cryotherapy and minimum tumor temperature on patient-reported quality of life (QoL) in patients with prostate cancer. METHODS An Institutional Review Board-approved database was reviewed for patients who underwent cryotherapy or active surveillance (AS). QoL questionnaire responses were collected and scores were analyzed for differences between focal and total cryotherapy and between very cold (<-76°C) and moderate-cold (≥-76°C) minimum tumor temperatures. RESULTS A total of 197 patients responded to a total of 547 questionnaires. Focal and total cryotherapy patients had initially lower sexual function scores relative to AS (year 1 mean difference focal: -31.7, P <.001; total: -48.1, P <.001). Focal cryotherapy was associated with a more rapid improvement in sexual function. Both focal and total cryotherapy sexual function scores were not statistically significantly different from the AS cohort by postprocedural year 4. Very cold and moderate-cold temperatures led to initially lower sexual function scores relative to AS (year 1 very cold: -38.1, P <.001; moderate-cold: -30.7, P <.001). Moderate-cold temperature scores improved more rapidly than those of very cold temperature. Neither very cold nor moderate-cold temperatures had a statistically significant difference in sexual function scores relative to AS by postprocedural year 4. Urinary function and bowel habits were not significantly different between focal and total cryotherapy and between very cold and moderate-cold temperature groups. CONCLUSION Focal cryotherapy and moderate-cold (≥-76°C) temperature were associated with favorable sexual function relative to total cryotherapy and very cold temperature, respectively. No significant differences in urinary function or bowel habits were observed between groups.

The Escherichia coli P and type 1 pilus assembly chaperones PapD and FimC are monomeric in solution

UNLABELLED 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 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. IMPORTANCE Pili 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.

Electrostatic networks control plug stabilization in the PapC usher

Abstract The PapC usher, a β-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 β-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 β-barrel, an α-helix and β5-6 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 β-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.

Patient-reported quality of life progression in men with prostate cancer following primary cryotherapy, cyberknife, or active holistic surveillance

BackgroundTechnological advancements have led to the success of minimally invasive treatment modalities for prostate cancer such as CyberKnife and Cryotherapy. Here, we investigate patient-reported urinary function, bowel habits, and sexual function in patients following CyberKnife (CK) or Cryotherapy treatment, and compare them with active holistic surveillance (AHS) patients.MethodsAn IRB-approved institutional database was retrospectively reviewed for patients who underwent CK, Cryotherapy, or AHS. Quality of life (QoL) survey responses were collected every three months and the mean function scores were analyzed in yearly intervals over the 4 years post-treatment.Results279 patients (767 survey sets) were included in the study. There was no difference among groups in urinary function scores. The CyberKnife group had significantly lower bowel habit scores in the early years following treatment (year 2 mean difference: −5.4, P < 0.01) but returned to AHS level scores by year 4. Cryotherapy patients exhibited initially lower, but not statistically significant, bowel function scores, which then improved and approached those of AHS. Both CyberKnife (year 1 mean difference: −26.7, P < 0.001) and Cryotherapy groups (−35.4, P < 0.001) had early lower sexual function scores relative to AHS, but then gradually improved and were not significantly different from AHS by the third year post-treatment. A history of hormonal therapy was associated with a lower sexual function scores relative to those patients who did not receive hormones in both CyberKnife (−18.45, P < 0.01) and Cryotherapy patients (−14.6, P < 0.05).ConclusionsAfter initial lower bowel habits and sexual function scores, CyberKnife or Cryotherapy-treated patients had no significant difference in QoL relative to AHS patients. These results highlight the benefit of CyberKnife and Cryotherapy in the management of organ-confined prostate cancer.

MP23-17 THE CHARACTERISTICS AND PROGRESSION OF BACTERIAL BIOFILMS ON URINARY CATHETERS

INTRODUCTION AND OBJECTIVES: The majority of hospital-acquired urinary tract infections (UTIs) are catheter-associated UTIs (CAUTIs), which are associated with increased morbidity and mortality in patients, with 13,000 attributed deaths annually. CAUTIs are also associated with increased length of hospital stays and 0.40.5 billion USD in annual healthcare costs, as well as unnecessary antimicrobial use. The formation of biofilms (groups of bacterial cells that adhere to one another and to a fixed surface) on catheters is critical to the development and persistence of CAUTI, as biofilms function as both barriers to antibiotics and reservoirs of microbes. We sought to determine the natural history of biofilm formation on urinary catheters. In particular, we were interested in the starting location(s) of biofilm formation, and whether biofilms predominated proximally or distally on catheters, their timing and manner of progression, and whether catheter biofilm formation was predominantly extraluminal or intraluminal. METHODS: Foley catheters (n1⁄419) were collected from outpatient and inpatient clinics at a large university medical center from post-surgical patients at 1 to 28 days indwelling time. Each catheter was sectioned and stained, and biofilms were quantitated using spectrophotometry. RESULTS: Short-term catheters (indwelling <1 week) displayed predominant biofilm formation at the proximal (bladder-exposed) end, whereas long-term catheters (indwelling 3-4 weeks) displayed significant biofilm formation throughout all segments. Biofilm growth on short-term catheters was predominantly extra-luminal, whereas longterm catheters demonstrated significant extraand intra-luminal biofilm staining. CONCLUSIONS: The results of this preliminary study inform approaches to developing novel strategies to prevent and eradicate bacterial biofilms from urinary catheters. For example, this study suggests that catheter-coating techniques targeting the extraluminal surface of the proximal end of the urinary catheter may contribute to a delay of biofilm formation, and reduce the overall risk of CAUTI. Efforts are under way to further investigate biofilm progression with larger sample sizes, and to determine how a reduction in biofilm formation and progression may contribute to reduced CAUTI risk.