A copper site is required for iron transport by the periplasmic proteins P19 and FetP.

Campylobacter jejuni is a leading cause of food-borne gastrointestinal disease in humans and uropathogenic Escherichia coli is a leading cause of urinary tract infections. Both human pathogens harbour a homologous iron uptake system (termed cjFetM-P19 in C. jejuni and ecFetM-FetP in E. coli). Although these systems are important for growth under iron limitation, the mechanisms by which these systems function during iron transport remain undefined. The copper ions bound to P19 and FetP, the homologous periplasmic proteins, are coordinated in an uncommon penta-dentate manner involving a Met-Glu-His3 motif and exhibit positional plasticity. Here we demonstrate the function of the Met and Glu residues in modulating copper binding and controlling copper positioning through site-directed variants, binding assays, and crystal structures. Growth of C. jejuni strains with these p19 variants is impaired under iron limited conditions as compared to the wild-type strain. Additionally, an acidic residue-rich secondary site is required for binding iron and function in vivo. Finally, western blot analyses demonstrate direct and specific interactions between periplasmic P19 and FetP with the large periplasmic domain of their respective inner membrane transporters cjFetM and ecFetM.

Crystal structure of the usher chaperone YadV reveals a monomer with the proline lock in closed conformation suggestive of an intermediate state.

Cell surface pili assembled by the chaperone-usher (CU) pathway play a crucial role in the adhesion of uropathogenic Escherichia coli. YadV is the chaperone component of the CU pathway of Yad pili. Here, we report the crystal structure of YadV from E. coli. In contrast to major usher chaperones, YadV is a monomer in solution as well as in the crystallographic symmetry, and the monomeric form is a preferred state for interacting with pilus subunits. Moreover, we observed a closed conformation for the proline lock, a crucial structural element for chaperone-pilus subunit interaction. MD simulation shows that the closed state of the proline lock is not energetically stable. Thus, the structure of monomeric YadV with its closed proline lock may serve as an intermediate state to provide suitable access to pilus subunits.

Physical Extraction and Fast Protein Liquid Chromatography for Purifying Flagella Filament From Uropathogenic Escherichia coli for Immune Assay.

Flagella are expressed on the surface of a wide range of bacteria, conferring motility and contributing to virulence and innate immune stimulation. Host-pathogen interaction studies of the roles of flagella in infection, including due to uropathogenic Escherichia coli (UPEC), have used various methods to purify and examine the biology of the major flagella subunit protein, FliC. These studies have offered insight into the ways in which flagella proteins interact with host cells. However, previous methods used to extract and purify FliC, such as mechanical shearing, ultracentrifugation, heterologous expression in laboratory E. coli strains, and precipitation-inducing chemical treatments have various limitations; as a result, there are few observations based on highly purified, non-denatured FliC in the literature. This is especially relevant to host-pathogen interaction studies such as immune assays that are designed to parallel, as closely as possible, naturally-occurring interactions between host cells and flagella. In this study, we sought to establish a new, carefully optimized method to extract and purify non-denatured, native FliC from the reference UPEC strain CFT073 to be suitable for immune assays. To achieve purification of FliC to homogeneity, we used a mutant CFT073 strain containing deletions in four major chaperone-usher fimbriae operons (type 1, F1C and two P fimbrial gene clusters; CFT073Δ4). A sequential flagella extraction method based on mechanical shearing, ultracentrifugation, size exclusion chromatography, protein concentration and endotoxin removal was applied to CFT073Δ4. Protein purity and integrity was assessed using SDS-PAGE, Western blots with anti-flagellin antisera, and native-PAGE. We also generated a fliC-deficient strain, CFT073Δ4ΔfliC, to enable the concurrent preparation of a suitable carrier control to be applied in downstream assays. Innate immune stimulation was examined by exposing J774A.1 macrophages to 0.05-1 µg of purified FliC for 5 h; the supernatants were analyzed for cytokines known to be induced by flagella, including TNF-α, IL-6, and IL-12; the results were assessed in the context of prior literature. Macrophage responses to purified FliC encompassed significant levels of several cytokines consistent with prior literature reports. The purification method described here establishes a new approach to examine highly purified FliC in the context of host-pathogen interaction model systems.

CTX-M-15-producing uropathogenic Escherichia coli isolates at Rio de Janeiro, Brazil: Molecular epidemiology and MALDI-TOF MS.

Because of the high prevalence of CTX-M-15-producing Escherichia coli isolates causing urinary tract infections in Rio de Janeiro, we have investigated bla-CTX-M-15 gene presence, as well as CTX-M-15 production, in 32 E. coli isolates recovered from the urine of outpatients assisted at a public hospital located in the west zone of Rio. Molecular epidemiology was assessed by PFGE and phylo-typing methods. The work highlights the good performance of MALDI-TOF MS as an alternative tool to detect extended-spectrum beta-lactamases among CTX-M-15-producing E. coli isolates.

TRPV1 and the MCP-1/CCR2 Axis Modulate Post-UTI Chronic Pain.

The etiology of chronic pelvic pain syndromes remains unknown. In a murine urinary tract infection (UTI) model, lipopolysaccharide of uropathogenic E. coli and its receptor TLR4 are required for post-UTI chronic pain development. However, downstream mechanisms of post-UTI chronic pelvic pain remain unclear. Because the TRPV1 and MCP-1/CCR2 pathways are implicated in chronic neuropathic pain, we explored their role in post-UTI chronic pain. Mice were infected with the E. coli strain SΦ874, known to produce chronic allodynia, and treated with the TRPV1 antagonist capsazepine. Mice treated with capsazepine at the time of SΦ874 infection failed to develop chronic allodynia, whereas capsazepine treatment of mice at two weeks following SΦ874 infection did not reduce chronic allodynia. TRPV1-deficient mice did not develop chronic allodynia either. Similar results were found using novelty-suppressed feeding (NSF) to assess depressive behavior associated with neuropathic pain. Imaging of reporter mice also revealed induction of MCP-1 and CCR2 expression in sacral dorsal root ganglia following SΦ874 infection. Treatment with a CCR2 receptor antagonist at two weeks post-infection reduced chronic allodynia. Taken together, these results suggest that TRPV1 has a role in the establishment of post-UTI chronic pain, and CCR2 has a role in maintenance of post-UTI chronic pain.

Phytochemicals As Uropathognic Escherichia Coli FimH Antagonist: In Vitro And In Silico Approach.

BACKGROUND: Urinary tract infection (UTI) is caused by uropathogenic Escherichia coli (UPEC). The UPEC initiate pathogenesis by expressing type 1 pili, which attach to membrane receptors on the uroepithelial cells. Inhibition of attachment can provide a valuable target for prophylaxis in symptom-free milieu. METHODS: The antibacterial efficacy of alcoholic, hydroalcoholic and aqueous extracts of four plants namely Achyranthes aspera, Andrographis paniculata, Artemissia vulgaris and Glycyrrhiza glabra was evaluated against seven isolated bacterial strains and procured E. coli (UTI89/UPEC) strain. Screening of isolated strains was based on morphological characteristics and biofilm forming ability followed by physiological and biochemical analysis. RESULTS: The hydroalcoholic extracts of G. glabra at 50 µg/ml showed an impending antioxidant (DPPH) effect of 95.65% compared to ascorbic acid. The MIC values of all the plant extracts against selected bacterial strains ranged between 125 to 1000 µg/ml. In silico molecular docking performed to make out the antiadhesive role of 115 documented phytochemicals from selected plants identified quercetin-3-glucoside, ethyl caffeate, liquiritoside, liquiritin and isoliquiritigenin as potential phytochemicals. Molecular dynamics simulation performed by PTRAJ module of Amber11 package to monitor the stability of hydrogen bond showed that quercetin-3-glucoside and ethyl caffeate are potential phytochemicals as antiadhesive forming H-bonds with the FimH protein ligand. CONCLUSIONS: Aforesaid phytochemicals demonstrate effective antibacterial activity through the anti-adhesion mechanism.

Unraveling the essential role of CysK in CDI toxin activation.

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI(+) bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT(EC536) deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT(EC536) binary complex and the neutralized ternary complex of CysK/CdiA-CT/CdiI(EC536) CdiA-CT(EC536) inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT(EC536) exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT(EC536) thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT(EC536), and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.

Characterization of a highly potent antimicrobial peptide microcin N from uropathogenic Escherichia coli.

Microcin N is a low-molecular weight, highly active antimicrobial peptide produced by uropathogenic Escherichia coli In this study, the native peptide was expressed and purified from pGOB18 plasmid carrying E. coli in low yield. The pure peptide was characterized using mass spectrometry, N-terminal sequencing by Edman degradation as well as trypsin digestion. We found that the peptide is 74-residue long, cationic (+2 total charge), highly hydrophobic and consists of glycine as the first N-terminal residue. The minimum inhibitory concentration of the peptide against Salmonella enteritidis was found to be 150 nM. Evaluation of the solution conformation of the peptide using circular dichroism spectroscopy showed that the peptide is well folded in 40% trifluoroethanol with helical structure whereas the folded structure is lost in aqueous solution. To increase the yield of this potent peptide, we overexpressed GST-tagged microcin N using E. coli BL21. Recombinant GST-tagged microcin N was successfully expressed in E. coli BL21; however, the cleaved mature microcin N did not show activity against the indicator strain (S. enterica) most likely due to the extreme hydrophobic nature of the peptide. Efforts to produce active microcin N in large scale are discussed as this peptide has huge potential to be the next generation antimicrobial agent.

Siderophore biosynthesis coordinately modulated the virulence-associated interactive metabolome of uropathogenic Escherichia coli and human urine.

Uropathogenic Escherichia coli (UPEC) growth in women's bladders during urinary tract infection (UTI) incurs substantial chemical exchange, termed the "interactive metabolome", which primarily accounts for the metabolic costs (utilized metabolome) and metabolic donations (excreted metabolome) between UPEC and human urine. Here, we attempted to identify the individualized interactive metabolome between UPEC and human urine. We were able to distinguish UPEC from non-UPEC by employing a combination of metabolomics and genetics. Our results revealed that the interactive metabolome between UPEC and human urine was markedly different from that between non-UPEC and human urine, and that UPEC triggered much stronger perturbations in the interactive metabolome in human urine. Furthermore, siderophore biosynthesis coordinately modulated the individualized interactive metabolome, which we found to be a critical component of UPEC virulence. The individualized virulence-associated interactive metabolome contained 31 different metabolites and 17 central metabolic pathways that were annotated to host these different metabolites, including energetic metabolism, amino acid metabolism, and gut microbe metabolism. Changes in the activities of these pathways mechanistically pinpointed the virulent capability of siderophore biosynthesis. Together, our findings provide novel insights into UPEC virulence, and we propose that siderophores are potential targets for further discovery of drugs to treat UPEC-induced UTI.

Molecular and Structural Characterization of a Novel Escherichia coli Interleukin Receptor Mimic Protein.

UNLABELLED: Urinary tract infection (UTI) is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA). We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R), interleukin-4 receptor (IL-4R), and interleukin-10 receptor (IL-10R) binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection. IMPORTANCE: Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infection (UTI), a disease of major significance to human health. Globally, the incidence of UPEC-mediated UTI is strongly associated with increasing antibiotic resistance, making this extremely common infection a major public health concern. In this report, we describe the regulatory, structural, functional, and immunogenic properties of a candidate UPEC vaccine antigen, IrmA. We demonstrate that IrmA is a small UPEC protein that forms a unique domain-swapped dimer with structural mimicry to several human cytokine receptors. We also show that IrmA binds to IL-2, IL-4, and IL-10, is strongly immunogenic in urosepsis patients, and is coexpressed with factors associated with biofilm formation. Overall, this work suggests a potential novel contribution for IrmA in UPEC infection.

Nitazoxanide Inhibits Pilus Biogenesis by Interfering with Folding of the Usher Protein in the Outer Membrane.

Many bacterial pathogens assemble surface fibers termed pili or fimbriae that facilitate attachment to host cells and colonization of host tissues. The chaperone/usher (CU) pathway is a conserved secretion system that is responsible for the assembly of virulence-associated pili by many different Gram-negative bacteria. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and an integral outer membrane (OM) assembly and secretion platform termed the usher. Nitazoxanide (NTZ), an antiparasitic drug, was previously shown to inhibit the function of aggregative adherence fimbriae and type 1 pili assembled by the CU pathway in enteroaggregativeEscherichia coli, an important causative agent of diarrhea. We show here that NTZ also inhibits the function of type 1 and P pili from uropathogenicE. coli(UPEC). UPEC is the primary causative agent of urinary tract infections, and type 1 and P pili mediate colonization of the bladder and kidneys, respectively. By analysis of the different stages of the CU pilus biogenesis pathway, we show that treatment of bacteria with NTZ causes a reduction in the number of usher molecules in the OM, resulting in a loss of pilus assembly on the bacterial surface. In addition, we determine that NTZ specifically prevents proper folding of the usher ß-barrel domain in the OM. Our findings demonstrate that NTZ is a pilicide with a novel mechanism of action and activity against diverse CU pathways. This suggests that further development of the NTZ scaffold may lead to new antivirulence agents that target the usher to prevent pilus assembly.

Hybrid Structure of the Type 1 Pilus of Uropathogenic Escherichia coli.

Type 1 pili are filamentous protein assemblies on the surface of Gram-negative bacteria that mediate adhesion to host cells during the infection process. The molecular structure of type 1 pili remains elusive on the atomic scale owing to their insolubility and noncrystallinity. Herein we describe an approach for hybrid-structure determination that is based on data from solution-state NMR spectroscopy on the soluble subunit and solid-state NMR spectroscopy and STEM data on the assembled pilus. Our approach is based on iterative modeling driven by structural information extracted from different sources and provides a general tool to access pseudo atomic structures of protein assemblies with complex subunit folds. By using this methodology, we determined the local conformation of the FimA pilus subunit in the context of the assembled type 1 pilus, determined the exact helical pilus architecture, and elucidated the intermolecular interfaces contributing to pilus assembly and stability with atomic detail.

Crystal structure analysis of c4763, a uropathogenic Escherichia coli-specific protein.

Urinary-tract infections (UTIs), which are some of the most common infectious diseases in humans, can cause sepsis and death without proper treatment. Therefore, it is necessary to understand their pathogenicity for proper diagnosis and therapeutics. Uropathogenic Escherichia coli, the major causative agents of UTIs, contain several genes that are absent in nonpathogenic strains and are therefore considered to be relevant to UTI pathogenicity. c4763 is one of the uropathogenic E. coli-specific proteins, but its function is unknown. To investigate the function of c4763 and its possible role in UTI pathogenicity, its crystal structure was determined at a resolution of 1.45 Šby a multiple-wavelength anomalous diffraction method. c4763 is a homodimer with 129 residues in one subunit that contains a GGCT-like domain with five α-helices and seven ß-strands. c4763 shows structural similarity to the C-terminal domain of allophanate hydrolase from Kluyveromyces lactis, which is involved in the degradation of urea. These results suggest that c4763 might be involved in the utilization of urea, which is necessary for bacterial survival in the urinary tract. Further biochemical and physiological investigation will elucidate its functional relevance in UTIs.

Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB.

Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone-usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.

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.

The C-glycosyltransferase IroB from pathogenic Escherichia coli: identification of residues required for efficient catalysis.

Escherichia coli C-glycosyltransferase IroB catalyzes the formation of a CC bond between enterobactin and the glucose moiety of UDP-glucose, resulting in the production of mono-, di- and tri-glucosylated enterobactin (MGE, DGE, TGE). To identify catalytic residues, we generated a homology model of IroB from aligned structures of two similar C-glycosyltransferases as templates. Superposition of our homology model onto the structure of a TDP-bound orthologue revealed residue W264 as a possible stabilizer of UDP-glucose. D304 in our model was located near the predicted site of the glucose moiety of UDP-glucose. A loop containing possible catalytic residues (H65, H66, E67) was found at the predicted enterobactin-binding site. We generated IroB variants at positions 65-67, 264, and 304 and investigated variant protein conformations and enzymatic activities. Variants were found to have Tm values similar to wild-type IroB. Fluorescence emission spectra of H65A/H66A, E67A, and D304N were superimposable with wild-type IroB. However, the emission spectrum of W264L was blue-shifted, suggesting solvent exposure of W264. While H65A/H66A retained activity (92% conversion of enterobactin, with MGE as a major product), all other IroB variants were impaired in their abilities to glucosylate enterobactin: E67A catalyzed partial (29%) conversion of enterobactin to MGE; W264L converted 55% of enterobactin to MGE; D304N was completely inactive. Activity-impaired variants were found to bind enterobactin with affinities within 2.5-fold of wild-type IroB. Given our outcomes, we propose that IroB W264 and D304 are required for binding and orienting UDP-glucose, while E67, possibly supported by H65/H66, participates in enterobactin/MGE/DGE deprotonation.

Implementation of Fourier transform infrared spectroscopy for the rapid typing of uropathogenic Escherichia coli.

In this paper, we demonstrate that Fourier transform infrared (FT-IR) spectroscopy is able to discriminate rapidly between uropathogenic Escherichia coli (UPEC) of key lineages with only relatively simple sample preparation. A total of 95 bacteria from six different epidemiologically important multilocus sequence types (ST10, ST69, ST95, ST73, ST127 and ST131) were used in this project and principal component-discriminant function analysis (PC-DFA) of these samples produced clear separate clustering of isolates, based on the ST. Analysis of data using partial least squares-discriminant analysis (PLS-DA), incorporating cross-validation, indicated a high prediction accuracy of 91.19% for ST131. These results suggest that FT-IR spectroscopy could be a useful method for the rapid identification of members of important UPEC STs.

Crystal structure of c5321: a protective antigen present in uropathogenic Escherichia coli strains displaying an SLR fold.

BACKGROUND: Increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties. RESULTS: We report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats. CONCLUSIONS: The structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen.

Assessment of immune responses of the flagellin (FliC) fused to FimH adhesin of Uropathogenic Escherichia coli.

Urinary tract infection (UTI) caused by Uropathogenic Escherichia coli (UPEC) is one of the most common infectious diseases in the world. Despite extensive efforts, a vaccine that protects humans against UTI is currently missing. In this study, the immunogenicity of flagellin (FliC) of UPEC strain in different vaccine combinations with FimH antigen of UPEC and conventional adjuvant Montanide ISA 206 was assessed. Finally, efficacy of the immune responses was evaluated for protection of the bladder and kidney of challenged immunized mice. Mice immunized with the fusion FimH·FliC induced significantly higher anti-FliC humoral (IgG1) and cellular (Th1 and Th2) immune responses than with FliC alone or FliC admixed with FimH. The Montanide enhanced the immune responses of FliC antigen and directed the anti-FliC responses preferentially toward Th1. The FliC vaccine combinations reduced bladder infection as compared to control mice. The fusion FimH·FliC and FliC admixed with FimH and Montanide combinations gave the best results in protection of kidney infection, compared to the control mice. The results of this study propose new promising vaccine combinations based on the FliC antigen and Montanide against UTI caused by UPEC.

Second order rate constants of donor-strand exchange reveal individual amino acid residues important in determining the subunit specificity of pilus biogenesis.

P pili are hair-like adhesive structures that are assembled on the outer membrane (OM) of uropathogenic Escherichia coli by the chaperone-usher pathway. In this pathway, chaperone-subunit complexes are formed in the periplasm and targeted to an OM assembly platform, the usher. Pilus subunits display a large groove caused by a missing ß-strand which, in the chaperone-subunit complex, is provided by the chaperone. At the usher, pilus subunits are assembled in a mechanism termed "donor-strand exchange (DSE)" whereby the ß-strand provided by the chaperone is exchanged by the incoming subunit's N-terminal extension (Nte). This occurs in a zip-in-zip-out fashion, starting with a defined residue, P5, in the Nte inserting into a defined site in the groove, the P5 pocket. Here, electrospray ionization-mass spectrometry (ESI-MS) has been used to measure DSE rates in vitro. Second order rate constants between the chaperone-subunit complex and a range of Nte peptides substituted at different residues confirmed the importance of the P5 residue of the Nte in determining the rate of DSE. In addition, residues either side of the P5 residue (P5 + 1 and P5 - 1), the side-chains of which are directed away from the subunit groove, also modulate the rates of DSE, most likely by aiding the docking of the Nte into the P5 pocket on the accepting subunit prior to DSE. The ESI-MS approach developed is applicable to the measurement of rates of DSE in pilus biogenesis in general and demonstrates the scope of ESI-MS in determining biomolecular processes in molecular detail.