Francisella tularensis is a gram-negative, intracellular pathogen that causes the zoonotic disease tularemia. Due to its ease of dissemination and high lethality, F. tularensis is classified as a tier 1 select agent with potential for misuse as a bioweapon. The mechanisms by which Francisella replicates intracellularly and interacts with the host during infection are not well understood. Francisella produces spherical outer membrane vesicles (OMVs) and novel tubular extensions of its cell surface that are also released extracellularly. These OMV and outer membrane tubes (OMTs) contain Francisella virulence factors and are produced in response to amino acid starvation and during infection of macrophages. To investigate how the OMTs are formed, we used cryogenic electron tomography to examine the model Francisella spp., Francisella novicida, during in vitro culture and within the macrophage phagosome. OMT formation involved progressive alterations of the bacterial envelope, resulting in extensions of both the inner and outer membranes. A dynamic cytoplasmic structure was present at the base of the OMT that extended into the tubes during elongation, together with cytoplasmic material. OMT produced within the macrophage phagosome was associated with changes to the phagosomal membrane, suggesting a role in phagosomal escape. Consistent with this, using confocal microscopy, we observed co-localization of the Francisella type VI secretion system with the OMT, both within bacteria and in released tubular vesicles. These findings reveal the cellular transformations that occur during membrane tubulation by Francisella and provide insights into the function of membrane-derived structures during host-pathogen interactions. IMPORTANCE Francisella tularensis is an intracellular bacterial pathogen that causes the zoonotic disease tularemia. Following uptake by host cells, the bacteria rapidly escape the phagosome and replicate intracellularly. In previous studies, we found that Francisella produces tubular extensions of its cell surface in response to specific cues and during macrophage infection. In the present study, we used cryogenic electron tomography to examine tube formation by the model Francisella sp., F. novicida. This analysis revealed that tube formation involves extensive bacterial envelope alterations and a dynamic cytoplasmic organelle. Furthermore, tubes produced by bacteria within infected macrophages were associated with the breakdown of the phagosomal membrane. In addition, we found that the Francisella type VI secretion system, which is essential for phagosomal escape, co-localized with the bacterial tubes. These findings reveal the cellular transformations that occur during membrane tubulation by Francisella and suggest a role for the tubes in phagosomal escape.

Francisella spp. are Gram-negative, facultative intracellular pathogens. Francisella tularensis causes the human disease tularemia and is considered a biological threat agent due to its high infectivity and virulence. A central aspect of Francisella virulence is its ability to dampen host immune responses. We previously identified the outer membrane channel (OMC) protein TolC as a critical F. tularensis virulence factor required for suppression of apoptotic and proinflammatory responses during macrophage infection. TolC functions as part of multidrug efflux systems and the type I secretion pathway that exports bacterial effector proteins. In these systems, TolC forms tripartite complexes together with an inner membrane transporter and periplasmic membrane fusion protein (MFP). To advance understanding of TolC function in Francisella, we analyzed OMC and MFP homologs in Francisella novicida, a widely used model species that causes a tularemia-like disease in mice. In agreement with the previous F. tularensis studies, all three OMCs present in F. novicida contributed to multidrug resistance, but only TolC was important for suppressing macrophage cell death. In addition, we identified the EmrA1 MFP as important for resisting antimicrobial compounds and dampening host cell death. In contrast to results obtained with F. tularensis, the cell death triggered during infection with the F. novicida tolC and emrA1 mutants was dominated by pyroptosis rather than apoptosis. These data expand our understanding of TolC function in Francisella and underscore both conserved and differential aspects of F. novicida and F. tularensis. IMPORTANCE Francisella tularensis is a Gram-negative intracellular bacterial pathogen and causative agent of tularemia. We previously identified the outer membrane channel protein TolC as contributing to antimicrobial resistance and subversion of host responses by F. tularensis. To advance understanding of TolC function in Francisella and to identify components that might work together with TolC, we took advantage of a transposon mutant library in F. novicida, a model species that causes a tularemia-like disease in mice. Our findings identify TolC and the membrane fusion protein EmrA1 as important for both antimicrobial resistance and suppression of macrophage cell death. This study also revealed differences in cell death pathways triggered by F. novicida versus F. tularensis infection that may relate to differences in virulence.

With the introduction of siliconized artificial membranes, various artificial feeding systems (AFS) for hard ticks (Ixodidae) have been developed over the last decades. Most AFS utilize similar core components but employ diverse approaches, materials, and experimental conditions. Published work describes different combinations of the core components without experimental optimizations for the artificial feeding of different tick species. Amblyomma americanum L., (Acari: Ixodidae) (lone star tick) is a known vector and reservoir for diverse tick-borne pathogens, such as Rickettsia amblyommatis and Ehrlichia chaffeensis. Ongoing environmental changes have supported the expansion of A. americanum into new habitats, contributing to increased tick-borne diseases in endemic areas. However, a significant knowledge gap exists in understanding the underlying mechanisms involved in A. americanum interactions with tick-borne pathogens. Here, we performed a systematic analysis and developed an optimized AFS for nymphal lone star ticks. Our results demonstrate that Goldbeater's membranes, rabbit hair, hair extract, and adult lone star ticks significantly improved the attachment rate of nymphal ticks, whereas tick frass and frass extract did not. With the optimized conditions, we achieved an attachment rate of 46 +/- 3% and a success rate of 100% (i.e., one or more attached ticks) in each feeding experiment for nymphal lone star ticks. When fed on sheep blood spiked with R. amblyommatis, both nymphal and adult lone star ticks acquired and maintained R. amblyommatis, demonstrating the feasibility of studying A. americanum-pathogen interactions using AFS. Our study can serve as a roadmap to optimize and improve AFS for other medically relevant tick species.

Francisella tularensis is a zoonotic pathogen and the causative agent of tularemia. F. tularensis replicates to high levels within the cytosol of macrophages and other host cells while subverting the host response to infection. Critical to the success of F. tularensis is its ability to delay macrophage apoptosis to maintain its intracellular replicative niche. However, the host-signaling pathway(s) modulated by F. tularensis to delay apoptosis are poorly characterized. The outer membrane channel protein TolC is required for F. tularensis virulence and its ability to suppress apoptosis and cytokine expression during infection of macrophages. We took advantage of the F. tularensis ∆tolC mutant phenotype to identify host pathways that are important for activating macrophage apoptosis and that are disrupted by the bacteria. Comparison of macrophages infected with wild-type or ∆tolC F. tularensis revealed that the bacteria interfere with TLR2-MYD88-p38 signaling at early times post infection to delay apoptosis, dampen innate host responses, and preserve the intracellular replicative niche. Experiments using the mouse pneumonic tularemia model confirmed the in vivo relevance of these findings, revealing contributions of TLR2 and MYD88 signaling to the protective host response to F. tularensis, which is modulated by the bacteria to promote virulence. IMPORTANCE Francisella tularensis is a Gram-negative intracellular bacterial pathogen and the causative agent of the zoonotic disease tularemia. F. tularensis, like other intracellular pathogens, modulates host-programmed cell death pathways to ensure its replication and survival. We previously identified the outer membrane channel protein TolC as required for the ability of F. tularensis to delay host cell death. However, the mechanism by which F. tularensis delays cell death pathways during intracellular replication is unclear despite being critical to pathogenesis. In the present study, we address this gap in knowledge by taking advantage of ∆tolC mutants of F. tularensis to uncover signaling pathways governing host apoptotic responses to F. tularensis and which are modulated by the bacteria during infection to promote virulence. These findings reveal mechanisms by which intracellular pathogens subvert host responses and enhance our understanding of the pathogenesis of tularemia.

Since its discovery in the United States in 2017, the Asian longhorned tick (Haemaphysalis longicornis) has been detected in most eastern states between Rhode Island and Georgia. Long Island, east of New York City, a recognized high-risk area for tick-borne diseases, is geographically close to New Jersey and New York sites where H. longicornis was originally found. However, extensive tick surveys conducted in 2018 did not identify H. longicornis on Long Island. In stark contrast, our 2022 tick survey suggests that H. longicornis has rapidly invaded and expanded in multiple surveying sites on Long Island (12 out of 17 sites). Overall, the relative abundance of H. longicornis was similar to that of lone star ticks, Amblyomma americanum, a previously recog-nized tick species abundantly present on Long Island. Interestingly, our survey suggests that H. longicornis has expanded within the Appalachian forest ecological zone of Long Island's north shore compared to the Pine Barrens located on the south shore of Long Island. The rapid invasion and expansion of H. longicornis into an insular environment are different from the historical invasion and expansion of two native tick species, Ixodes scapularis (blacklegged tick or deer tick) and A. americanum, in Long Island. The implications of H. longicornis transmitting or introducing tick-borne pathogens of public health importance remain unknown.

In times of crisis, including the current COVID-19 pandemic, the supply chain of filtering facepiece respirators, such as N95 respirators, are disrupted. To combat shortages of N95 respirators, many institutions were forced to decontaminate and reuse respirators. While several reports have evaluated the impact on filtration as a measurement of preservation of respirator function after decontamination, the equally important fact of maintaining proper fit to the users' face has been understudied. In the current study, we demonstrate the complete inactivation of SARS-CoV-2 and preservation of fit test performance of N95 respirators following treatment with dry heat. We apply scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS), X-ray diffraction (XRD) measurements, Raman spectroscopy, and contact angle measurements to analyze filter material changes as a consequence of different decontamination treatments. We further compared the integrity of the respirator after autoclaving versus dry heat treatment via quantitative fit testing and found that autoclaving, but not dry heat, causes the fit of the respirator onto the users face to fail, thereby rendering the decontaminated respirator unusable. Our findings highlight the importance to account for both efficacy of disinfection and mask fit when reprocessing respirators to for clinical redeployment.

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.

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I (MP25)1 Sep 2021MP25-06 STRUCTURES OF P PILUS ASSEMBLY INTERMEDIATES REVEAL NOVEL INTERACTIONS AND DRUG TARGETS AT THE USHER PLATFORM IN UROPATHOGENIC ESCHERICHIA COLI Glenn T Werneburg, Minge Du, Zuanning Yuan, Nadine Henderson, Hemil Chauhan, Amanda Kovach, Gongpu Zhao, Jessica Johl, Huilin Li, and David Thanassi Glenn T WerneburgGlenn T Werneburg More articles by this author , Minge DuMinge Du More articles by this author , Zuanning YuanZuanning Yuan More articles by this author , Nadine HendersonNadine Henderson More articles by this author , Hemil ChauhanHemil Chauhan More articles by this author , Amanda KovachAmanda Kovach More articles by this author , Gongpu ZhaoGongpu Zhao More articles by this author , Jessica JohlJessica Johl More articles by this author , Huilin LiHuilin Li More articles by this author , and David ThanassiDavid Thanassi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002022.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Uropathogenic Escherichia coli (UPEC) generate adhesive surface structures termed pili to initiate urinary tract infection. UPEC adherence to the kidney urothelium is mediated by P pili, facilitating pyelonephritis. P pili are assembled through the chaperone-usher (CU) pathway. Here, a dedicated chaperone, PapD, promotes periplasmic pilus subunit folding and an integral outer membrane usher protein, PapC, provides the assembly platform and secretion channel for the growing pilus fiber. The structural basis for P pilus biogenesis has remained elusive due to the variable-length P pilus tip fiber, creating difficulties in the isolation of homogenous complexes suitable for structural analysis. Given their central roles in initiating and sustaining kidney infection, there is great interest in the molecular mechanisms of P pilus assembly and function. METHODS: Using a series of molecular genetics approaches, a method to circumvent the variability in P pilus length was devised. PapCDKFG and PapCDKG protein complexes were thus expressed and purified. Bacterial adherence function was confirmed using hemagglutination assays. The structures of the purified complexes were solved using cryo-electron microscopy. RESULTS: Structures were solved for the three-subunit PapCDKFG complex and for two, two-subunit PapCDKG complexes, at 7.6, 3.8 and 7.2 Å resolution, respectively (Figure panels a, b, c, respectively). A novel usher binding site at the PapD chaperone apex was identified, and a series of point mutations at this site disrupted pilus assembly. CONCLUSIONS: We present the first structures of the activated PapC usher captured during the pilus assembly process. The P pilus structures elucidate processive steps in pilus biogenesis, reveal differences between P and type 1 pili, and capture novel conformational dynamics of the usher assembly machine. Further, we identify a new important interaction site at the PapD chaperone and demonstrate its functional significance. The results open new avenues for novel therapeutics designed against P pili and other virulence factors. The molecules may serve as alternatives to traditional antibiotics in the treatment of urinary tract infections and other infectious diseases, without contributing to resistance or disrupting gut flora. Source of Funding: This study was supported by US National Institutes of Health grants F30 AI 112252 (to G.T.W.), R01 GM062987 (to D.G.T.) and R35 GM131754 (to H.L.), and by the Van Andel Institute (to H.L.) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e455-e455 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Glenn T Werneburg More articles by this author Minge Du More articles by this author Zuanning Yuan More articles by this author Nadine Henderson More articles by this author Hemil Chauhan More articles by this author Amanda Kovach More articles by this author Gongpu Zhao More articles by this author Jessica Johl More articles by this author Huilin Li More articles by this author David Thanassi More articles by this author Expand All Advertisement Loading ...

Yersinia pseudotuberculosisis a foodborne pathogen that subverts immune function by translocation ofYersiniaouter protein (Yop) effectors into host cells. As adaptive γδ T cells protect the intestinal mucosa from pathogen invasion, we assessed whetherY.pseudotuberculosissubverts these cells in mice and humans. Tracking Yop translocation revealed that the preferential delivery of Yop effectors directly into murine Vγ4 and human Vδ2+T cells inhibited anti-microbial IFNγ production. Subversion was mediated by the adhesin YadA, injectisome component YopB, and translocated YopJ effector. A broad anti-pathogen gene signature and STAT4 phosphorylation levels were inhibited by translocated YopJ. Thus,Y.pseudotuberculosisattachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of immune subversion in mice and humans. This study uncovered a conservedY.pseudotuberculosispathway that subverts adaptive γδ T cell function to promote pathogenicity.

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder I (MP46)1 Apr 2020MP46-18 ANTIBIOTIC-RESISTANT UROPATHOGENS FORM BIOFILMS PREDOMINANTLY ON DISTAL AND LUMINAL ASPECTS OF CATHETERS AND ARE INHIBITED BY A NOVEL CETYLPYRIDINIUM CHLORIDE-BASED FORMULATION Glenn Werneburg*, Nadine Henderson, Raymond Rackley, Anh Nguyen, Daniel Shoskes, Amanda Le Sueur, Anthony Corcoran, Aaron Katz, Jason Kim, Annie Rohan, and David Thanassi Glenn Werneburg*Glenn Werneburg* More articles by this author , Nadine HendersonNadine Henderson More articles by this author , Raymond RackleyRaymond Rackley More articles by this author , Anh NguyenAnh Nguyen More articles by this author , Daniel ShoskesDaniel Shoskes More articles by this author , Amanda Le SueurAmanda Le Sueur More articles by this author , Anthony CorcoranAnthony Corcoran More articles by this author , Aaron KatzAaron Katz More articles by this author , Jason KimJason Kim More articles by this author , Annie RohanAnnie Rohan More articles by this author , and David ThanassiDavid Thanassi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000901.018AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urinary catheter biofilms promote bacterial resistance to antibiotics and host cell defenses. Biofilm formation is critical for the pathogenesis of catheter-associated UTI. Here we sought to determine the bacterial composition and antibiotic resistance patterns of bacterial biofilms, as well as their locations on catheters over time. Further, we aimed to test a novel antiseptic formulation, containing cetylpyridinium chloride (CPC) as the active product ingredient combined with pro-adherence factors, for growth inhibition of the biofilm bacterial isolates in liquid medium. METHODS: Urinary catheters were collected following removal from patients, were sectioned and stained for biofilms using crystal violet, and spectrophotometry was used for quantitation. Viability was confirmed via growth in liquid culture, and next generation sequencing via the MicroGenDX platform was employed. Five isolates were cultured, normalized, and subcultured in the presence of the 1:10 or 1:100 antiseptic formulation, or a normal saline control, and growth was measured with spectrophotometry. RESULTS: Thirty-three urinary catheters were included in the study. Biofilm formation increased as a function of time up to 5 weeks. Biofilms predominated on the luminal, balloon, and distal (p=0.034 vs. proximal end) portions of catheters. Uropathogenic bacteria were detected in 10 of 10 samples analyzed using next-generation sequencing (Figure 1a). Genes conferring bacterial resistance to multiple antibiotics were detected in isolates. Relative to the normal saline control, the CPC formulation consistently inhibited growth of the bacterial isolates in a dose-dependent manner (Figure 1b). CONCLUSIONS: Biofilms were composed of uropathogenic bacteria, which were often resistant to common antibiotics. A novel antiseptic formulation inhibited growth of biofilm isolates in a dose-response manner. Biofilm reduction techniques including routine antiseptic irrigation warrant further investigation, as they are well-positioned to target the luminal-predominant biofilms identified in our study and thus may reduce the risk of CAUTI. Source of Funding: NIH T32GM008444 and F30AI112252 (GTW), R01GM062987 (DGT), AACN-Sigma Theta Tau Critical Care Award (AJR) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e680-e681 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Glenn Werneburg* More articles by this author Nadine Henderson More articles by this author Raymond Rackley More articles by this author Anh Nguyen More articles by this author Daniel Shoskes More articles by this author Amanda Le Sueur More articles by this author Anthony Corcoran More articles by this author Aaron Katz More articles by this author Jason Kim More articles by this author Annie Rohan More articles by this author David Thanassi More articles by this author Expand All Advertisement PDF downloadLoading ...

Periodontitis is a progressive inflammatory disease that affects roughly half of American adults. Colonization of the oral cavity by the Gram-negative bacterial pathogen Porphyromonas gingivalis is a key event in the initiation and development of periodontal disease.

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of tularemia. Previous studies with the attenuated live vaccine strain (LVS) identified a role for the outer membrane protein TolC in modulation of host cell responses during infection and virulence in the mouse model of tularemia.

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder II (MP77)1 Apr 2019MP77-10 PROGRESSION AND FORMATION OF BACTERIAL BIOFILMS ON INDWELLING URINARY CATHETERS OVER TIME Glenn T. Werneburg*, Anh Nguyen, Nadine Henderson, Amanda Le Sueur, Anthony Corcoran, Aaron Katz, Jason Kim, Annie Rohan, and David G. Thanassi Glenn T. Werneburg*Glenn T. Werneburg* More articles by this author , Anh NguyenAnh Nguyen More articles by this author , Nadine HendersonNadine Henderson More articles by this author , Amanda Le SueurAmanda Le Sueur More articles by this author , Anthony CorcoranAnthony Corcoran More articles by this author , Aaron KatzAaron Katz More articles by this author , Jason KimJason Kim More articles by this author , Annie RohanAnnie Rohan More articles by this author , and David G. ThanassiDavid G. Thanassi More articles by this author View All Author Informationhttps://doi.org/10.1097/01.JU.0000557322.77175.6fAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVES: Urinary catheter biofilm formation is common and consists of adherent bacteria, metabolic products, and host components. Through biofilms, bacteria can resist both antibiotics and host defense mechanisms. Biofilm formation on urinary catheters is a critical step in the pathogenesis of catheter-associated UTI. We sought to determine the catheter locations of onset and progression of biofilm formation. METHODS: Urinary catheters of 0-5 weeks indwelling time were collected. Catheters were then analyzed using spectrophotometry. Specifically, catheters were sectioned and stained with crystal violet, and excess dye was removed. Adherent crystal violet, which represented adherent biofilm, was then dissolved, and the concentration was spectrophotometrically quantitated relative to controls. RESULTS: 33 urinary catheters were collected: 10 in week 1, 6 in week 2, 3 in week 3, 8 in week 4, and 6 in week 5. Biofilms progressed throughout all aspects of the catheter over the 5 weeks indwelling time. The distal end of the catheter exhibited significantly greater staining relative to the proximal side (Figure). The balloon of the catheter consistently showed intense staining, indicative of significant biofilm adherence. CONCLUSIONS: Biofilms progressed as indwelling time increased, and were detected following as little as 1 day indwelling time. The locations of greatest biofilm predominance were the distal portion of the urinary catheter as well as the balloon. We expect that these findings will inform the design and production of novel urinary catheters, which may benefit from focusing antiseptic strategies on the distal and balloon portions of the catheter. Importantly, our findings suggest that biofilms may proliferate through a multimodal etiology, and that sterile insertion technique may prevent early proximal biofilm formation, but may not reduce distal growth. Ongoing studies will determine the bacterial species responsible for biofilm formation, and whether biofilm proliferation is affected by patient sex. Source of Funding: GTW was supported by award T32GM008444 and Award F30AI112252 from the NIH. Shaker Heights, OH; Stony Brook, NY; Mineola, NY; Stony Brook, NY© 2019 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 201Issue Supplement 4April 2019Page: e1136-e1136 Advertisement Copyright & Permissions© 2019 by American Urological Association Education and Research, Inc.MetricsAuthor Information Glenn T. Werneburg* More articles by this author Anh Nguyen More articles by this author Nadine Henderson More articles by this author Amanda Le Sueur More articles by this author Anthony Corcoran More articles by this author Aaron Katz More articles by this author Jason Kim More articles by this author Annie Rohan More articles by this author David G. Thanassi More articles by this author Expand All Advertisement PDF downloadLoading ...

The chaperone-usher (CU) pathway is a conserved secretion system dedicated to the assembly of a superfamily of virulence-associated surface structures by a wide range of Gram-negative bacteria. Pilus biogenesis by the CU pathway requires two specialized assembly components: a dedicated periplasmic chaperone and an integral outer membrane assembly and secretion platform termed the usher. The CU pathway assembles a variety of surface fibers, ranging from thin, flexible filaments to rigid, rod-like organelles. Pili typically act as adhesins and function as virulence factors that mediate contact with host cells and colonization of host tissues. Pilus-mediated adhesion is critical for early stages of infection, allowing bacteria to establish a foothold within the host. Pili are also involved in modulation of host cell signaling pathways, bacterial invasion into host cells, and biofilm formation. Pili are critical for initiating and sustaining infection and thus represent attractive targets for the development of antivirulence therapeutics. Such therapeutics offer a promising alternative to broad-spectrum antibiotics and provide a means to combat antibiotic resistance and treat infection while preserving the beneficial microbiota. A number of strategies have been taken to develop antipilus therapeutics, including vaccines against pilus proteins, competitive inhibitors of pilus-mediated adhesion, and small molecules that disrupt pilus biogenesis. Here we provide an overview of the function and assembly of CU pili and describe current efforts aimed at interfering with these critical virulence structures.

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 ?-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.

We report that the nanometer-scale architecture of polymer chains plays a crucial role in its protein resistant property over surface chemistry. Protein-repellent (noncharged), few nanometer thick polymer layers were designed with homopolymer chains physisorbed on solids. We evaluated the antifouling property of the hydrophilic or hydrophobic adsorbed homopolymer chains against bovine serum albumin in water. Molecular dynamics simulations along with sum frequency generation spectroscopy data revealed the self-organized nanoarchitecture of the adsorbed chains composed of inner nematic-like ordered segments and outer brush-like segments across homopolymer systems with different interactions among a polymer, substrate, and interfacial water. We propose that this structure acts as a dual barrier against protein adsorption.

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder II1 Apr 2018MP23-01 THE UROPATHOGENIC ESCHERICHIA COLI TYPE 1 PILUS USHER EMPLOYS A 2-STEP VERIFICATION PROCESS DURING INITIATION OF PILUS ASSEMBLY Glenn T. Werneburg, Hemil Chauhan, Nadine S. Henderson, and David G. Thanassi Glenn T. WerneburgGlenn T. Werneburg More articles by this author , Hemil ChauhanHemil Chauhan More articles by this author , Nadine S. HendersonNadine S. Henderson More articles by this author , and David G. ThanassiDavid G. Thanassi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.733AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Uropathogenic strains of Escherichia coli use the chaperone/usher (CU) pathway to construct adhesive pili. Pili facilitate binding to bladder and kidney epithelial cells, thereby promoting urinary tract colonization and infection. The outer membrane usher catalyzes the exchange of chaperone-subunit for subunit-subunit interactions, and promotes ordered polymerization and secretion of subunits into the mature pilus fiber. An essential and well-regulated step of pilus assembly is the activation of the usher, wherein it undergoes a transformation from a plug-gated membrane pore to an engaged assembly machine. We sought to gain a mechanistic understanding of how the usher is activated and controls the pilus assembly. METHODS We monitored pilus subunit interactions with different usher domains using genetic, biochemical, and biophysical approaches including Forster Resonance Energy Transfer (FRET). RESULTS Using a FRET-based affinity approach, we identified particular regions of the activating pilus subunit (adhesin) that are essential for discrete steps of the usher activation process. Specifically, we demonstrated that the C-terminal domain (pilin domain) of the activating subunit is necessary for subunit recruitment to the usher platform (Panel C, Kd=194 nM), and that the N-terminal domain (lectin domain) of the activating subunit is necessary for subsequent usher plug expulsion (Panel E, act1/2=4 nM) and priming for pilus assembly (Panels F-G, Kd=4 nM). Further, using a mutagenesis approach, we demonstrated that the addition of the lectin domain is sufficient to confer usher-activating potential to a non-activating pilus subunit. CONCLUSIONS Our results suggest that the usher employs a ″2-step verification″ process in which it recognizes and verifies both the N and C domains of the initiating adhesin subunit in discrete steps of the activation process. This process may ensure that every assembled pilus has an adhesin at its tip, in a location to facilitate adhesion and UTI. These discoveries open avenues for drug design to target these newly-elucidated mechanistic steps, thus potentially preventing or treating UTI, without selecting for resistant bacterial strains. Our FRET-based approaches are poised for the screening of molecules to disrupt this process. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e281 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Glenn T. Werneburg More articles by this author Hemil Chauhan More articles by this author Nadine S. Henderson More articles by this author David G. Thanassi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...