Outer Membrane Usher Protein Research Articles

Structural basis for adhesin secretion by the outer-membrane usher in type 1 pili

Gram-negative bacteria produce chaperone-usher pathway pili, which are extracellular protein fibers tipped with an adhesive protein that binds to a receptor with stereochemical specificity to determine host and tissue tropism. The outer-membrane usher protein, together with a periplasmic chaperone, assembles thousands of pilin subunits into a highly ordered pilus fiber. The tip adhesin in complex with its cognate chaperone activates the usher to allow extrusion across the outer membrane. The structural requirements to translocate the adhesin through the usher pore from the periplasm to the extracellular space remains incompletely understood. Here, we present a cryoelectron microscopy structure of a quaternary tip complex in the type 1 pilus system from Escherichia coli, which consists of the usher FimD, chaperone FimC, adhesin FimH, and the tip adapter FimF. In this structure, the usher FimD is caught in the act of secreting its cognate adhesin FimH. Comparison with previous structures depicting the adhesin either first entering or having completely exited the usher pore reveals remarkable structural plasticity of the two-domain adhesin during translocation. Moreover, a piliation assay demonstrated that the structural plasticity, enabled by a flexible linker between the two domains, is a prerequisite for adhesin translocation through the usher pore and thus pilus biogenesis. Overall, this study provides molecular details of adhesin translocation across the outer membrane and elucidates a unique conformational state adopted by the adhesin during stepwise secretion through the usher pore. This study elucidates fundamental aspects of FimH and usher dynamics critical in urinary tract infections and is leading to antibiotic-sparing therapeutics.

Bioinformatics and immunoinformatics assisted multiepitope vaccine construct against Burkholderia anthina

Burkholderia anthina is a pathogenic bacterial species belonging to the Burkholderiaceae family and it is mainly considered the etiological agent of chronic obstructive pulmonary diseases associated with cystic fibrosis, due to being intrinsic antibiotic resistant making it difficult to treat pulmonary infections. Hence increased rate of antibiotic-resistant bacterial species vaccine development is the priority to tackle this problem. In research work, we designed a multi-epitope-based vaccine construct against B. anthina using reverse vaccinology immunoinformatics and biophysical approaches. Based on the subtractive proteomic screening of core proteins we identified 3 probable antigenic proteins and good vaccine targets namely, type VI secretion system tube protein hcpBurkholderia, fimbria/pilus periplasmic chaperone and fimbrial biogenesis outer membrane usher protein. The selected 3 proteins were used for B and B cells B-derived T-cell epitopes prediction. In epitopes prediction, different epitopes were predicted with various lengths and percentile scores and subjected to further immunoinformatics analysis. In immunoinformatics screening a total number of 06, IDDGNANAL, KTVKPDPRY, SEVESGSAP, YGGDLTVEV, SVSHDTNGR, and GSKADGYQR epitopes were considered good vaccine target candidates and shortlisted for vaccine construct designing. The vaccine construct was designed by joining selected epitopes with the help of a GPGPG linker and additionally linked with cholera toxin b subunit adjuvant to increase the efficacy of the vaccine construct the sequence of the said adjuvant were retrieved from protein data bank through its (PDB ID: 5ELD). The designed vaccine construct was evaluated for its physiochemical properties analysis in which we reported that the vaccine construct comprises 216 amino acids with a molecular weight of 22.37499 kilo Dalton, 15.55 instability index (II) is computed, and this classifies that the vaccine construct is properly stable. VaxiJen v2.0 web server predicted that the vaccine construct is probable antigenic in nature with 0.6320 predicted value. Furthermore AllerTOP v. 2.0 tool predicted that the designed vaccine construct is non allergic in nature. Molecular docking analysis was done for analysis of the binding affinity of the vaccine construct with TLR-2 (PDB ID: 6NIG), the docking results predicted 799.2 kcal/mol binding energy score that represents the vaccine construct has a good binding ability with TLR-2. Moreover, molecular dynamic simulation analysis results revealed that the vaccine construct and immune cell receptor has proper binding stability over various environmental condition, i.e. change in pressure range, temperature, and motion. After each analysis, we observed that the vaccine construct is safe stable, and probably antigenic and could generate an immune response against the target pathogen but in the future, experimental analysis is still needed to verify in silico base results.

Assessment of the growth inhibition and anti-biofilm activity of aptamer (PmA2G02) against Proteus mirabilis 1429T

Proteus mirabilis is known to cause Catheter-associated urinary tract infections (CAUTIs), which exhibit virulence factors linked to forming biofilms. Aptamers have recently been explored as potential anti-biofilm agents. This study demonstrates the anti-biofilm activity of aptamer (PmA2G02) targeting P. mirabilis 1429T, a pathogenic bacteria known to cause Catheter-associated urinary tract infections (CAUTIs). The studied aptamer inhibited biofilm formation, swarming motility, and cell viability at a concentration of 3 μM. The study also showed that the PmA2G02 had a binding affinity towards fimbrial outer membrane usher protein (PMI1466), flagellin protein (PMI1619), and regulator of swarming behavior (rsbA), which are responsible for adhesion, motility, and quorum sensing, respectively. Crystal violet assay, SEM, and confocal imaging confirmed the effectiveness of the PmA2G02 as an anti-biofilm agent. Moreover, as verified by qPCR, the expression levels of fimD, fliC2, and rsbA were significantly reduced compared to the untreated group. This study suggests that aptamer may be a potential alternative to traditional antibiotics for the treatment of CAUTIs caused by P. mirabilis. These findings shed light on the mechanisms by which the aptamer inhibits biofilm formation.

Computational study to investigate Proteus mirabilis proteomes for multi-epitope vaccine construct design

Proteus mirabilis is a gram-negative bacterium particularly known for its unique swarming ability. The swarming gives the bacteria ability to enhance adherence to the catheter surface and epithelium cells of the urethra to cause catheter associated urinary tract infections. P. mirabilis has evolved resistant to antibiotics. Additionally, there is an approved vaccine against P. mirabilis, thus demanding for identification of new vaccine targets. This gram-negative bacterium consists of 19,502 core proteins, out of which 19,063 are redundant proteins and remaining 439 are non-redundant proteins. The non-redundant proteins have 21 proteins present on the cell surface out of which 11 proteins are virulent. Antigenicity analysis predicted only 2 proteins as antigenic (fimbrial biogenesis outer membrane usher protein and ligand-gated channel protein). Four and seven B-cells epitopes were predicted from the former and later proteins, respectively. The predicted B-cells epitopes were used for T- cells epitopes prediction. The predicted epitopes were linked to each other through GPGPG linkers and joined with cholera toxin beta subunit adjuvant. A multi-epitopes vaccine construct consisting of 226 residues was docked with MHC-I, MHC-II and TLR-4. The best docked complex in each case has binding energy of −714.6, −744.6 and −829.5 kcal/mol, respectively. Moreover, the docking results were validated through molecular dynamics simulation and binding free energies estimation. The net energy of −137.2 kcal/mol was calculated for vaccine-MHC-I complex, −133.39 kcal/mol for vaccine-MHC-II and −158.68 kcal/mol for vaccine-TLR-4 complex. The designed vaccine construct could provoke immune responses against targeted pathogen and may be used in experimental testing. Communicated by Ramaswamy H. Sarma

Identification and construction of a multi-epitopes vaccine design against Klebsiella aerogenes: molecular modeling study

A rapid rise in antibiotic resistance by bacterial pathogens is due to these pathogens adaptation to the changing environmental conditions. Antibiotic resistance infections can be reduced by a number of ways such as development of safe and effective vaccine. Klebsiella aerogene is a gram-negative, rod-shaped bacterium resistant to a variety of antibiotics and no commercial vaccine is available against the pathogen. Identifying antigens that can be easily evaluated experimentally would be crucial to successfully vaccine development. Reverse vaccinology (RV) was used to identify vaccine candidates based on complete pathogen proteomic information. The fully sequenced proteomes include 44,115 total proteins of which 43,316 are redundant and 799 are non-redundant. Subcellular localization showed that only 1 protein in extracellular matrix, 7 were found in outer-membrane proteins, and 27 in the periplasm space. A total of 3 proteins were found virulent. Next in the B-cell-derived T-cell epitopes mapping phase, the 3 proteins (Fe2+− enterobactin, ABC transporter substrate-binding protein, and fimbriae biogenesis outer membrane usher protein) were tested positive for antigenicity, toxicity, and solubility. GPGPG linkers were used to prepare a vaccine construct composed of 7 epitopes and an adjuvant of toxin B subunit (CTBS). Molecular docking of vaccine construct with major histocompatibility-I (MHC-I), major histocompatibility-II (MHC-II), and Toll-like receptor 4 (TLR4) revealed vaccine robust interactions and stable binding pose to the receptors. By using molecular dynamics simulations, the vaccine-receptors complexes unveiled stable dynamics and uniform root mean square deviation (rmsd). Further, binding energies of complex were computed that again depicted strong intermolecular bindings and formation of stable conformation.

An In-Silico Investigation to Design a Multi-Epitopes Vaccine against Multi-Drug Resistant Hafnia alvei.

Antimicrobial resistance has become a significant health issue because of the misuse of antibiotics in our daily lives, resulting in high rates of morbidity and mortality. Hafnia alvei is a rod-shaped, Gram-negative and facultative anaerobic bacteria. The medical community has emphasized H. alvei’s possible association with gastroenteritis. As of now, there is no licensed vaccine for H. alvei, and as such, computer aided vaccine design approaches could be an ideal approach to highlight the potential vaccine epitopes against this bacteria. By using bacterial pan-genome analysis (BPGA), we were able to study the entire proteomes of H. alvei with the aim of developing a vaccine. Based on the analysis, 20,370 proteins were identified as core proteins, which were further used in identifying potential vaccine targets based on several vaccine candidacy parameters. The prioritized vaccine targets against the bacteria are; type 1 fimbrial protein, flagellar hook length control protein (FliK), flagellar hook associated protein (FlgK), curli production assembly/transport protein (CsgF), fimbria/pilus outer membrane usher protein, fimbria/pilus outer membrane usher protein, molecular chaperone, flagellar filament capping protein (FliD), TonB-dependent hemoglobin /transferrin/lactoferrin family receptor, Porin (OmpA), flagellar basal body rod protein (FlgF) and flagellar hook-basal body complex protein (FliE). During the epitope prediction phase, different antigenic, immunogenic, non-Allergenic, and non-Toxic epitopes were predicted for the above-mentioned proteins. The selected epitopes were combined to generate a multi-epitope vaccine construct and a cholera toxin B subunit (adjuvant) was added to enhance the vaccine’s antigenicity. Downward analyses of vaccines were performed using a vaccine three-dimensional model. Docking studies have confirmed that the vaccine strongly binds with MHC-I, MHC-II, and TLR-4 immune cell receptors. Additionally, molecular dynamics simulations confirmed that the vaccine epitopes were exposed to nature and to the host immune system and interpreted strong intermolecular binding between the vaccine and receptors. Based on the results of the study, the model vaccine construct seems to have the capacity to produce protective immune responses in the host, making it an attractive candidate for further in vitro and in vivo studies.

Molecular events confirming antimutagenicity to abscisic acid derived from a floral honey establishing its functional relevance

Natural dietary products of health promoting and disease preventive functional relevance are gaining significant prominence. Current investigation was aimed to decipher the underlying molecular mechanism responsible for the antimutagenic action contributing to functional relevance of floral honey (‘Pongammia pinnata’, Karanj honey) derived abscisic acid (ABA) against ethyl methanesulfonate (EMS) induced mutagenesis. Differential expression of proteins under different treatment conditions was ascertained by 2D gel electrophoresis. Selectively up-regulated characterized using MALDI-TOF MS/MS were identified as polyribonucleotide nucleotidyl transferse (PNPase), LPS-assembly lipoprotein (LptE), Outer membrane Usher protein (HtrE), ATP-dependent DNA helicase (RecG), and Phosphomethyl pyrimidine synthase (ThiC). Antimutagenicity exerted by ABA against EMS was ∼78% in wild type E. coli MG1655 strain however, in E. coli MG1655 ΔthiC, ΔpnpA, ΔrecG, and ΔhtrE this activity was found to be ∼60, 10, 9 and 10%, respectively. Proteomic analysis and antimutagenicity studies using E. coli single gene knockout strains thus indicated about the possible role of thiC, htrE, lptE, recG and pnp in observed antimutagenicity. Cyclic voltametry as well as competition kinetics through pulse radiolysis confirmed lack of antioxidant capacity in abscisic acid apparently ruling out the possibility of scavenging of electrophilic intermediates generated by ethyl methanesulfonate. It is proposed that ABA is exerting antimutagenicity through its involvement at the cellular level leading to physiological adaptation, strengthening of cell wall proteins and up-regulation of the repair proteins. This study provides a novel dimension to the functional role of abscisic acid from its nutraceutical perspective.

Reverse Vaccinology and Immunoinformatic Assisted Designing of a Multi-Epitopes Based Vaccine Against Nosocomial Burkholderia cepacia.

Burkholderia cepacia is a Gram-negative nosocomial pathogen and is considered as a troublesome bacterium due to its resistance to many common antibiotics. There is no licensed vaccine available to prevent the pathogen infections, thus making the condition more alarming and warrant the search for novel therapeutic and prophylactic approaches. In order to identify protective antigens from pathogen proteome, substantial efforts are put forth to prioritized potential vaccine targets and antigens that can be easily evaluated experimentally. In this vaccine design investigation, it was found that B. cepacia completely sequenced proteomes available in NCBI genome database has a total of 28,966 core proteins. Out of total, 25,282 proteins were found redundant while 3,684 were non-redundant. Subcellular localization revealed that 18 proteins were extracellular, 31 were part of the outer membrane, 75 proteins were localized in the periplasm, and 23 were virulent proteins. Five proteins namely flagellar hook protein (FlgE), fimbria biogenesis outer membrane usher protein, Type IV pilus secretin (PilQ), cytochrome c4, flagellar hook basal body complex protein (FliE) were tested for positive for antigenic, non-toxic, and soluble epitopes during predication of B-cell derived T-cell epitopes. A vaccine peptide of 14 epitopes (joined together via GPGPG linkers) and cholera toxin B subunit (CTBS) adjuvant (joined to epitopes peptide via EAAAK linker) was constructed. Binding interaction of the modeled vaccine with MHC-I, MHC-II, and Toll-like receptor 4 (TLR-4) immune receptors was studied using molecular docking studies and further analyzed in molecular dynamics simulations that affirms strong intermolecular binding and stable dynamics. The maximum root mean square deviation (RMSD) score of complexes in the simulation time touches to 2 Å. Additionally, complexes binding free energies were determined that concluded robust interaction energies dominated by van der Waals. The total energy of each complex is < −190 kcal/mol. In summary, the designed vaccine showed promising protective immunity against B. cepacia and needs to be examined in experiments.

Immunoinformatics approach for the determination of antigenic epitope candidates against Burkholderia cenocepacia

Cystic fibrosis (CF) is a genetic disorder that affects mainly the lungs but also the liver, intestine, and kidneys. It causes an increase in dehydrated mucus which results in the proliferation of numerous pathobionts. Lung inflammation in CF patients is commonly observed due to the opportunistic infection of the Burkholderia cepacia complex (Bcc) that includes one of the deadly pathogens named B. cenocepacia. Although a low incidence rate is present, B. cenocepacia infection is still a major problem for the CF community. In the study, it was aimed to uncover T and B cell epitopes of novel antigenic proteins of B. cenocepacia that can be potential targets for vaccine development studies. The whole proteome of B. cenocepacia ATCC BAA-245 was first analyzed to eliminate the non-redundant proteins which were then subjected to Vaxign analysis for the determination of vaccine candidates. After subcellular localization analysis, the shortlisted proteins were directed to the antigenicity, virulence, and allergenicity analyses, resulting in nine antigenic, virulent, and non-allergenic candidates. The epitope prediction was then carried out for these candidates and seven proteins containing four TonB-dependent receptors, ornibactin receptor, phospholipase C, and putative outer membrane usher protein were found to contain common T and B cell epitopes. Besides, the epitopes of the three candidates had the best exposed topology that can be used to construct multi-epitope vaccines against Bcc.

CupA1/cupA5 gene overexpressed at subinhibitory concentration of carbapenem in biofilm forming Pseudomonas aeruginosa: Transcriptomic study from India

Background: Pseudomonas aeruginosa, an opportunistic pathogen associated with hospital infections is a potent biofilm forming bacteria. A new class of genes termed the chaperone/usher (cup) encoding fimbrial subunit, chaperone and outer membrane usher protein have been identified which facilitates biofilm formation in Pseudomonas aeruginosa independent of the Type IV pili. Since antibiotics at low concentrations can change biological response in bacteria; therefore in the present study the response of cupA1 and cupA5 genes was studied at a sub-inhibitory concentration of carbapenem to check if it influences biofilm formation. Methods and materials: Four clinical isolates of Pseudomonas aeruginosa harboring blaOXA-48&blaNDM associated with hospital infection were selected for the study. All the isolates had MIC above breakpoint against imipenem and meropenem. The isolates were cultured overnight in fresh Luria Bertani broth with and without 2 μg/mL carbapenem (imipenem, meropenem) stress respectively. Total RNA was isolated from the overnight cultures grown to log phase using RNeasy kit (Qiagen, India); cDNA was prepared using the Quanti Tect Reverse Transcription kit (Qiagen, India) and was then subjected to quantitative real-time PCR to estimate the relative expression of cupA1 and cupA5 genes in the study isolates using the ΔΔCt method. Results: The transcriptional analysis of cupA1 and cupA5 genes in the isolates revealed that the expression levels of the genes in all of the four isolates under normal condition i.e. without any antibiotic pressure were lower than the expression level in control strain Pseudomonas aeruginosa ATCC 278533 (RQ=1). Whereas, overexpression of the genes was observed in all the isolates when these were treated with 2 μg/mL imipenem and meropenem respectively. The expression of cupA5 gene increased multiple folds under meropenem stress in all the isolates. Conclusion: The results of the present study imply that carbapenem at a concentration below the lethal dose increases the expression of cupA1 and cupA5 genes which facilitates biofilm formation. Thus from the present study, it can be concluded that imipenem and meropenem at sub-inhibitory concentration favours the biofilm forming ability in clinical strains of P. aeruginosa establishing infection and thus complicating antibiotic therapies which may further lead to treatment failure.

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 β-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.

Combating tigecycline resistant Acinetobacter baumannii: A leap forward towards multi-epitope based vaccine discovery

Global emergence of Tigecycline resistant Acinetobacter baumannii (TRAB) is on the horizon and poses a very serious threat to human health. There is a pressing demand for suitable therapeutics against this pathogen, particularly a vaccine to protect against TRAB infections. We present a comprehensive investigation of the complete proteome of a TRAB AB031 strain to predict promiscuous antigenic, non-allergenic, virulent B-cell derived T-cell epitopes and formulate a multi-epitope vaccine against the pathogen. We identified epitopes from three proteins: outer membrane protein assembly factor (BamA), fimbrial biogenesis outer membrane usher protein (FimD) and type IV secretion protein (Rhs) that are appropriate for vaccine design. These proteins constitute the core proteome of the pathogen, are essential, localized at the pathogen surface, non-homologous to humans, mice and to the beneficial probiotic bacteria that reside the human gut. Moreover, these proteins are ideal candidates for experimental investigation as they have favorable physicochemical properties and have strong cellular interacting networks. The predicted epitopes: FPLNDKPGD (BamA), FVHAEEAAA (FimD) and YVVAGTAAA (Rhs) have exo-membrane topology for efficient recognition of the host immune system and high affinity for the most prevalent allele in human populations, the DRB*0101. These epitopes were linked and attached to an adjuvant to enhance its antigenicity. The multi-epitope vaccine-construct was docked with the TLR4 receptor to assess its affinity for the protein and thus its presentation to the host immune system. Docking results were validated through molecular dynamics simulations and binding free energies were calculated using the molecular mechanics/generalized Born (MM-GBSA) method. In conclusion, we expect the designed chimeric vaccine is highly likely to be effective against infections caused by TRAB.

Immuno-informatics driven proteome-wide investigation revealed novel peptide-based vaccine targets against emerging multiple drug resistant Providencia stuartii

The bacterium Providencia stuartii, is associated with urinary tract infections and is the most common cause of purple urine bag syndrome. The increasing multi-drug resistance pattern shown by the pathogen and lack of licensed vaccines make treatment of infections caused by P. stuartii challenging. As vaccinology data against the pathogen is scarce, an in silico proteome based Reverse Vaccinology (RV) protocol, in combination with subtractive proteomics is introduced in this work to screen potential vaccine candidates against P. stuartii. The analysis identified three potential vaccine candidates for designing broad-spectrum and strain-specific peptide vaccines: FimD4, FimD6, and FimD8. These proteins are essential for pathogen survival, localized in the outer membrane, virulent, and antigenic in nature. Immunoproteomic tools mapped surface exposed and non-allergenic 9mer B-cell derived T-cell antigenic epitopes for the proteins. The epitopes also show stable and rich interactions with the most predominant HLA allele (DRB1*0101) in the human population. Metabolic pathway annotation of the proteins indicated that fimbrial biogenesis outer membrane usher protein (FimD6) is the most suitable candidate for vaccine design, due to its involvement in several significant pathways. These pathways include: the bacterial secretion system, two-component system, β-lactam resistance, and cationic antimicrobial peptide pathways. The predicted epitopes may provide a basis for designing a peptide-based vaccine against P. stuartii.

The Structure of the CS1 Pilus of Enterotoxigenic Escherichia coli Reveals Structural Polymorphism

Enterotoxigenic Escherichia coli (ETEC) is a bacterial pathogen that causes diarrhea in children and travelers in developing countries. ETEC adheres to host epithelial cells in the small intestine via a variety of different pili. The CS1 pilus is a prototype for a family of related pili, including the CFA/I pili, present on ETEC and other Gram-negative bacterial pathogens. These pili are assembled by an outer membrane usher protein that catalyzes subunit polymerization via donor strand complementation, in which the N terminus of each incoming pilin subunit fits into a hydrophobic groove in the terminal subunit, completing a β-sheet in the Ig fold. Here we determined a crystal structure of the CS1 major pilin subunit, CooA, to a 1.6-Å resolution. CooA is a globular protein with an Ig fold and is similar in structure to the CFA/I major pilin CfaB. We determined three distinct negative-stain electron microscopic reconstructions of the CS1 pilus and generated pseudoatomic-resolution pilus structures using the CooA crystal structure. CS1 pili adopt multiple structural states with differences in subunit orientations and packing. We propose that the structural perturbations are accommodated by flexibility in the N-terminal donor strand of CooA and by plasticity in interactions between exposed flexible loops on adjacent subunits. Our results suggest that CS1 and other pili of this class are extensible filaments that can be stretched in response to mechanical stress encountered during colonization.

Cloning, purification, crystallization and preliminary X-ray diffraction studies of Escherichia coli PapD-like protein (EcpD).

Many Gram-negative bacteria are characterized by hair-like proteinaceous appendages on their surface known as fimbriae. In uropathogenic strains of Escherichia coli, fimbriae mediate attachment by binding to receptors on the host cell, often contributing to virulence and disease. E. coli PapD-like protein (EcpD) is a periplasmic chaperone that plays an important role in the proper folding and guiding of Yad fimbrial proteins to the outer membrane usher protein in a process known as pilus biogenesis. EcpD is essential for pilus biogenesis in uropathogenic E. coli and plays an important role in virulence. In the present study, EcpD was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.67 Å resolution and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 100.3, b = 127.6, c = 45.9 Å. There was a single molecule in the asymmetric unit and the corresponding Matthews coefficient was calculated to be 3.02 Å(3) Da(-1), with 59% solvent content. Initial phases were determined by molecular replacement.

Use of Enterobacterial Repetitive Intergenic Consensus PCR in Detecting Target(s) of Hapalindole-T, From a Cyanobacterium, in &lt;i&gt;Escherichia Coli&lt;/i&gt;: &lt;i&gt;In Silico&lt;/i&gt; Validation

Problem statement: Identification of newer biomolecule as well as targets are a rising concern because of increasing drug resistance in bacteria. We have isolated a broad spectrum antibiotic biomolecule Hapalindole-T from a cyanobacterium, Fisherella sp. growing on local Azadirachta indica tree bark. A model bacterium E. coli was screened spontaneously for Hap-T resistance. These resistant strains of E. coli were used to identify Hap-T target(s). Approach: These strains were subjected to Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR analysis and compared with the sensitive one. We have used different bioinformatic tools like Clustalw, NJ Plot and Docking server. The Swiss-model server was used for homology modelling. Predicted 3D structure was refined by energy minimization and quality was assessed by Procheck. The model protein hailed from fimbrial biogenesis outer membrane usher protein (ADK89122.1). The interaction between the predicted structure of Model-1 protein and Hap-T biomolecule was analysed in silico using Autodock and Mopac parameters. Results: An additional band of DNA fragment (~500 base pairs) was found on agarose gel run after amplified genome of resistant strain. The results indicated that certain residues (Tyr-28), (Phe-54), (Leu-36) and (Val-52) were highly conserved and present in active sites. Conclusion/Recommendations: Thus understanding of microbial adhesion can act as an alternative approach in development of broad spectrum antibiotics.

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 ( capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone–subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A C). Caf1A C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone–subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone–usher systems.

Snapshots of usher-mediated protein secretion and ordered pilus assembly.

Type 1 pilus biogenesis was used as a paradigm to investigate ordered macromolecular assembly at the outer cell membrane. The ability of Gram-negative bacteria to secrete proteins across their outer membrane and to assemble adhesive macromolecular structures on their surface is a defining event in pathogenesis. We elucidated genetic, biochemical, and biophysical requirements for assembly of functional type 1 pili. We discovered that the minor pilus protein FimG plays a critical role in nucleating the formation of the adhesive tip fibrillum. Genetic methods were used to trap pilus subunits during their translocation through the outer membrane usher protein, providing data on the structural interactions that occur between subunit components during type 1 pilus formation. Electron microscopic and biochemical analyses of these stepwise assembly intermediates demonstrated that translocation of pilus subunits occurs linearly through the usher's central channel, with formation of the pilus helix occurring extracellularly. Specialized pilin subunits play unique roles both in this multimerization and in the final ultrastructure of the adhesive pilus.

Specific high affinity binding of human interleukin 1β by Caf1A usher protein of Yersinia pestis

Understanding the interaction of Yersinia pestis with the key components of the immune system is important for elucidation of the pathogenesis of bubonic plague, one of the most severe and acute bacterial diseases. Here we report the specific, high affinity binding ( K d = 1.40 × 10 −10 M ± 0.14 × 10 −10) of radiolabelled human interleukin 1β (hIL-1β) to E. coli cells carrying the capsular fl operon of Y. pestis. Caf1A outer membrane usher protein was isolated to greater than 98% purity. Competition studies with purified Caf1, together with immunoblotting studies, identified Caf1A as the hIL-1β receptor. Competition between Caf1 subunit and hIL-1β for the same or an overlapping binding site on CaflA was demonstrated. Relevance of these results to the pathogenesis of Y. pestis and other Gram negative bacterial pathogens with homologous outer membrane usher proteins is discussed.