RESUMO
A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular "velcro" enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.
Assuntos
Anticorpos Antibacterianos , Anticorpos Monoclonais , Bordetella pertussis/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Peroxidase do Rábano Silvestre/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Citometria de Fluxo , Hibridomas/imunologia , Sensibilidade e EspecificidadeRESUMO
Pilin is the major structural protein that forms type IV pili of various pathogenic bacteria, including Pseudomonas aeruginosa. Pilin is involved in attachment of the bacterium to host cells during infection, in the initiation of immune response, and serves as a receptor for a variety of bacteriophage. We have used (15)N nuclear magnetic resonance relaxation measurements to probe the backbone dynamics of an N-terminally truncated monomeric pilin from P. aeruginosa strain K122-4. (15)N-T(1), -T(2), and [(1)H]-(15)N nuclear Overhauser enhancement measurements were carried out at three magnetic field strengths. The measurements were interpreted using the Lipari-Szabo model-free analysis, which reveals the amplitude of spatial restriction for backbone N-NH bond vectors with respect to nano- to picosecond time-scale motions. Regions of well-defined secondary structure exhibited consistently low-amplitude spatial fluctuations, while the terminal and loop regions showed larger amplitude motions in the subnano- to picosecond time-scale. Interestingly, the C-terminal disulfide loop region that contains the receptor binding domain was found to be relatively rigid on the pico- to nanosecond time-scale but exhibited motion in the micro- to millisecond time-scale. It is notable that this disulfide loop displays a conserved antigenic epitope and mediates binding to the asialo-GM(1) cell surface receptor. The present study suggests that a rigid backbone scaffold mediates attachment to the host cell receptor, and also maintains the conformation of the conserved antigenic epitope for antibody recognition. In addition, slower millisecond time-scale motions are likely to be crucial for conferring a range of specificity for these interactions. Characterization of pilin dynamics will aid in developing a detailed understanding of infection, and will facilitate the design of more efficient anti-adhesin synthetic vaccines and therapeutics against pathogenic bacteria containing type IV pili.
Assuntos
Antígenos de Bactérias/química , Proteínas de Membrana/química , Pseudomonas aeruginosa/química , Anisotropia , Proteínas de Fímbrias , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Gangliosídeo G(M1)/metabolismo , Proteínas de Membrana/metabolismo , Modelos Químicos , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , TermodinâmicaRESUMO
Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal alpha-helix. Engineering of a truncated pilin from Pseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an alpha-helix at the N terminus that lies across a 4-stranded antiparallel beta-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Proteínas de Bactérias/uso terapêutico , Proteínas de Membrana/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/uso terapêutico , Proteínas de Bactérias/genética , Vacinas Bacterianas , Ligação Competitiva , Método Duplo-Cego , Proteínas de Fímbrias , Proteínas de Membrana/genética , Proteínas de Membrana/uso terapêutico , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/imunologia , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Taxa de SobrevidaRESUMO
A novel bacterial protein secretion system was used to produce vaccine candidates against Pseudomonas aeruginosa. The surface protein (RsaA) of Caulobacter crescentus was adapted to produce recombinant vaccine proteins based on the pilus tip epitope ('adhesintope') of P. aeruginosa. Two versions of the adhesintope, with (PCK) or without (PE) the cysteine residues that flank the epitope were investigated, fused to the C-terminus or inserted into full-length RsaA. When expressed in caulobacter the fusion proteins were secreted as aggregates. Full length RsaA-containing adhesintopes assembled on the cell surface as an S-layer; these were recovered by low pH extraction. Vaccine candidates were evaluated in a mouse challenge model. PCK-containing proteins produced at least 1000-fold higher antibody titers against the adhesintope, compared to the PE version, exceeding titers achievable with any other vaccine preparation method. Immunoglobulin isotyping indicated a balanced IgG1/IgG2 response, though when challenged with P. aeruginosa, the PE- and PCK-containing proteins did not afford mice a significant level of protection. Overall, we describe a new system for vaccine production that shows promise as a fast, economical way to construct, evaluate and produce vaccine proteins.
Assuntos
Vacinas Bacterianas/isolamento & purificação , Glicoproteínas de Membrana , Pseudomonas aeruginosa/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Vacinas Bacterianas/genética , Vacinas Bacterianas/farmacologia , Sequência de Bases , Caulobacter/genética , Primers do DNA/genética , Expressão Gênica , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos A , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus. The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions. These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology. More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P. aeruginosa.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Fímbrias Bacterianas/imunologia , Proteínas de Membrana/imunologia , Pseudomonas aeruginosa/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/química , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos de Bactérias/química , Vacinas Bacterianas/química , Sítios de Ligação , Reações Cruzadas , Desenho de Fármacos , Proteínas de Fímbrias , Fímbrias Bacterianas/química , Isomerismo , Proteínas de Membrana/química , Dados de Sequência Molecular , Movimento (Física) , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
The backbone dynamics of a 15N-labeled recombinant PAK pilin peptide spanning residues 128-144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys128-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Se r-Lys144) were probed by measurements of 15N NMR relaxation. This PAK(128-144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The 15N longitudinal (T1) and transverse (T2) relaxation rates and the steady-state heteronuclear [1H]-15N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 degrees C) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the 'model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153-162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II beta-turns spanning residues Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.
Assuntos
Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Pseudomonas aeruginosa/química , Proteínas de Fímbrias , Proteínas de Membrana/imunologia , Pili Sexual , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , TemperaturaRESUMO
Fibers of pilin monomers (pili) form the dominant adhesin of Pseudomonas aeruginosa, and they play an important role in infections by this opportunistic bacterial pathogen. Blocking adhesion is therefore a target for vaccine development. The receptor-binding site is located in a C-terminal disulphide-bonded loop of each pilin monomer, but functional binding sites are displayed only at the tip of the pilus. A factor complicating vaccination is that different bacterial strains produce distinct, and sometimes highly divergent, pilin variants. It is surprising that all strains still appear to bind a common receptor, asialo-GM1. Here, we present the 1.63 A crystal structure of pilin from P. aeruginosa strain PAK. The structure shows that the proposed receptor-binding site is formed by two beta-turns that create a surface dominated by main-chain atoms. Receptor specificity could therefore be maintained, whilst allowing side-chain variation, if the main-chain conformation is conserved. The location of the binding site relative to the proposed packing of the pilus fiber raises new issues and suggests that the current fiber model may have to be reconsidered. Finally, the structure of the C-terminal disulphide-bonded loop will provide the template for the structure-based design of a consensus sequence vaccine.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/classificação , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Vacinas Bacterianas/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dissulfetos/metabolismo , Proteínas de Fímbrias , Gangliosídeo G(M1)/metabolismo , Glicosilação , Proteínas de Membrana/classificação , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Vacinas Sintéticas/químicaAssuntos
Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Infecções Bacterianas/prevenção & controle , Glicoproteínas de Membrana , Streptococcus mutans/patogenicidade , Adesinas Bacterianas/efeitos dos fármacos , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Ensaios Clínicos como Assunto , Resistência Microbiana a Medicamentos , Humanos , Lectinas/efeitos dos fármacos , Lectinas/genética , Lectinas/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Dente/microbiologiaRESUMO
Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. Research has shown that the C-terminal region of the pilin monomer contains the epithelial cell binding domain, which is semiconserved in seven different strains of this bacterium. Antibodies to this region of the pilin molecule are also able to block and prevent the infection process. As there is a degree of sequence and structural homology in the C-terminal region and all strains examined have been shown to bind to the same cell surface receptor, we reasoned that it should be possible to produce a synthetic peptide consensus sequence which would provide cross-reactive antiserum from a single peptide immunogen inhibiting the adherence of the known strains of P. aeruginosa. In this article we examine the cross-reactivity of five rabbit polyclonal antisera. One has been raised against the cell-surface receptor binding domain of native PAK strain pilin (residues 128-144) while the others have been raised to analogues of this region. Analysis of the cross-reactivity of these antisera, using competitive ELISA assay, has shown that it is possible to manipulate the amino acid sequence of a peptide immunogen to generate antiserum, which exhibits enhanced cross-reactivity to various strains of P. aeruginosa. Furthermore, when this peptide is conjugated to tetanus toxoid and used to vaccinate mice it provided cross-reactive protection against heterologous challenge with PAO strain bacteria. The results of these experiments are analyzed, and the applicability of our hypothesis and the implications of this approach to the design of a strain-independent consensus vaccine for immunization against Pseudomonas aeruginosa are discussed.
Assuntos
Vacinas Bacterianas/química , Desenho de Fármacos , Peptídeos/química , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/química , Antígenos de Bactérias/química , Camundongos , Dados de Sequência Molecular , CoelhosRESUMO
The two-stranded alpha-helical coiled-coil is now recognized as one of nature's favorite ways of creating a dimerization motif. Based on the knowledge of protein folding studies and de novo design model systems, a novel heterodimeric coiled-coil protein was synthesized. The heterodimeric E/K coiled-coil was constructed with two distinct peptides (E and K) that will spontaneously associate into a full helical coiled-coil structure in solution. Equilibrium CD, NMR and real time biosensor kinetics experiments showed that the E/K coiled-coil is both structurally (deltaG(unfold)=11.3 kcal/mol) and kinetically (Kd approximately 1 nM) stable in solution at neutral pH. The engineered coiled-coil had been applied as a dimerization and capture domain for biosensor based applications and used in an expression/detection/affinity chromatography system. Specific test examples demonstrated the usefulness of the E/K heterodimeric system in these applications. The universality of coiled-coil as a dimerization motif in nature and our ability to design and synthesize these proteins suggest a wide variety of applications.
Assuntos
Técnicas Biossensoriais , Cromatografia de Afinidade/instrumentação , Sequência de Aminoácidos , Dados de Sequência Molecular , Conformação Proteica , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surface receptors. Previously, it has been shown that the pilus adhesin of P. aeruginosa PAK binds to the ganglioside asialo-GM1. In particular, it was found that the carbohydrate sequence beta-D-GalNAc(1-->4)beta-D-Gal is the minimal carbohydrate receptor sequence of asialo-GM1. To study the binding specificity of P. aeruginosa, O-modified and N-modified sugar analogs, where each hydroxyl group was substituted either by O-methyl or O-propyl and the acetamido group was changed to a propionamido group, were synthesized. The sugar analogs were evaluated as inhibitors in a competitive solid phase binding assay. The results demonstrate that the pili of P. aeruginosa PAK accepts a variety of sugar analogs possessing the sequence beta-D-GalNAc(1-->4)beta-D-Gal. Most sugar analogs bind with a similar order of magnitude (50% inhibitory concentration (IC50) = 60-130 microM) except for the 2-O-propyl derivative 7 (IC50 = 8 +/- 4 microM) compared with an IC50 of 79 +/- 18 microM for the native compound. The significant increase in binding affinity of 2-O-propyl derivative 7 suggests that improved inhibitors of adhesion may be prepared by introducing a hydrophobic side chain at the 2-position of galactose.
Assuntos
Aderência Bacteriana , Metabolismo dos Carboidratos , Fímbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Receptores Imunológicos/metabolismo , Sítios de Ligação , Ligação Competitiva , Sequência de Carboidratos , Epitélio/metabolismo , Gangliosídeo G(M1)/metabolismoRESUMO
The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has recently been the target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. We have successfully cloned and bacterially expressed a 15N-labeled PAK pilin peptide spanning residues 128-144 of the intact PAK pilin protein, PAK 128-144(Hs145), and have determined the solution secondary structure of this peptide using heteronuclear multidimensional NMR spectroscopy. The oxidized recombinant peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around the Ile138-Pro139 peptide bond. The pattern of NOEs, temperature coefficients, and coupling constants observed for the trans isomer demonstrate the presence of a type I beta-turn and a type II beta-turn spanning Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively. This is in agreement with the NMR solution structure of the trans isomer of a synthetic PAK 128-144 peptide which showed a type I and a type II beta-turn in these same regions of the sequence [McInnes, C., Sönnichsen, F. D., Kay, C. M., Hodges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hodges, R. S., and Sykes, B. D. (1995) Biochemistry 34, 16255-16268]. The pattern of NOEs, temperature coefficients, and coupling constants observed for the cis isomer also demonstrate a type II beta-turn spanning Pro139-Lys-Gly-Cys142, but suggest a second beta-turn spanning Asp132-Gln-Asp-Glu135. Thus, the cis isomer may also possess a double-turn motif (like the trans isomer), but with different spacing between the turns and a different placement of the first turn in the sequence. The discovery of a double-turn motif in the trans (and cis) recombinant PAK pilin peptide is an extremely important result since the double turn has been implicated as a structural requirement for the recognition of both receptor and antibody. These results pave the way for future isotope-edited NMR studies of the labeled recombinant PAK pilin peptide bound to antibody and receptor, studies integral to the design of an effective synthetic peptide vaccine.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Proteínas da Membrana Bacteriana Externa/biossíntese , Vacinas Bacterianas , Sítios de Ligação , Clonagem Molecular , Escherichia coli , Proteínas de Fímbrias , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Pili Sexual , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Soluções , Termodinâmica , Vacinas SintéticasRESUMO
The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems.
Assuntos
Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Fragmentos de Peptídeos/química , Pseudomonas aeruginosa/química , Receptores de Superfície Celular/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas , Dissacarídeos/química , Dissacarídeos/metabolismo , Epitopos/química , Proteínas de Fímbrias , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência , Vacinas SintéticasAssuntos
Engenharia de Proteínas , Proteínas Recombinantes/isolamento & purificação , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Dimerização , Proteínas de Fímbrias , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Estrutura Secundária de Proteína , Pseudomonas aeruginosa , Proteínas Recombinantes/químicaRESUMO
Using the techniques of genetic engineering and the principles of protein de novo design, we have developed a unique affinity matrix protein tag system as a rapid, convenient and sensitive method to detect, purify and characterize newly expressed recombinant peptides or proteins from cell extracts. The method utilizes two de novo-designed linear peptide sequences that can selectively dimerize to form the stable protein motif, the two-stranded alpha-helical coiled-coil. In this method, a recombinant bacterial expression vector pRLDE has been engineered so that one of the dimerization strands (E-coil) is expressed as a C-terminal fusion tag on newly expressed peptides or proteins, while the other (K-coil) is either biotin-labeled for detection in a Western blot-type format or immobilized on an insoluble silica support for selective dimerization affinity chromatography. Recombinantly expressed peptides from Escherichia coli containing the dimerization tag have been produced, detected and purified using this method. The recombinant peptides were easily and clearly identified using the biotin-labeled coil, while the single-step affinity purification results indicated the purity of the affinity purified expressed peptides to be > 95%, as assessed by reversed-phase chromatography. The stability of the dimerization domain also allows for the purified peptide to be left attached to the matrix, thus creating a new peptide-bound column that can be used to study peptide-protein or peptide-ligand interactions. Therefore this system offers a new alternative to existing peptide or protein fusion tags and demonstrates the utility of a de novo-designed system.
Assuntos
Cromatografia de Afinidade/métodos , Vetores Genéticos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Dimerização , Escherichia coli/genética , Proteínas de Fímbrias , Dados de Sequência Molecular , Plasmídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The surface plasmon resonance (SPR) technique was used to study the formation kinetics of a de novo designed coiled-coil (E/K coil). The E/K coil is made up of two distinct peptides (E and K) each with five heptad (g-a-b-c-d-e-f) repeats. The E peptide's heptad sequence is E-V-S-A-L-E-K, and the K peptide's heptad sequence is K-V-S-A-L-K-E. A linker C-nL-G-G-G (nL = norleucine) is present at the C-terminus of the E peptide and at the N-terminus of the K peptide for the SPR studies. Heterodimer formation involves both electrostatic and hydrophobic interactions at the dimer interface. Under conditions that favor the heterodimer formation, the CD signal ([theta]222) varied as a function of peptide concentration. The estimated dissociation constant (Kd) was 2.45 +/- 0.71 nM. Denaturation studies with guanidine-HCI (GdnHC11/2 = 3.9 M) suggested a value of 3.53 +/- 0.48 nM. For the SPR investigation, the peptides were biotinylated and linked to streptavidin in order to increase their effective molecular weight and consequently enhance the signal intensity. Biotinylation in itself did not impede coiled-coil formation based on CD measurements. The biosensor study revealed a slow dissociation rate constant for the heterodimer (kd approximately 2 x 10(-4) s-1) and a moderately fast association rate constant [ka approximately (4.27-4.53) x 10(5) M-1 s-1). This gives a calculated Kd of 0.47-0.50 nM, which agrees reasonably well with the equilibrium CD studies. Therefore, based on the SPR data, the preference for heterodimer formation is due to a combination of moderately fast association and slow dissociation rates.
Assuntos
Oligopeptídeos/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Dicroísmo Circular , Dimerização , Eletroquímica , Cinética , Análise dos Mínimos Quadrados , Oligopeptídeos/síntese química , Software , Análise Espectral/métodos , Relação Estrutura-Atividade , UltracentrifugaçãoRESUMO
Pseudomonas aeruginosa and Candida albicans were reported to adhere to the glycosphingolipid asialo-GM1 by means of pili and fimbriae, respectively. These diverse adhesins have been previously reported to have an immunologically conserved antigenic epitope and the role of this cross-reactive epitope in adherence to asialo-GM1 was investigated in this study. Both the unbiotinylated PAK pilus and fimbrial adhesins inhibited biotinylated pili from P. aeruginosa PAK and biotinylated C. albicans fimbriae binding to asialo-GM1 and receptors present on human buccal epithelial cells (BECs), which suggested that the same receptor sites were recognized by the two adhesins. Monoclonal antibodies PK99H and Fm16 raised against the P. aeruginosa PAK pili and C. albicans fimbriae, respectively, recognized a conserved epitope present on the two adhesins. Both Fm16 and PK99H blocked fimbriae binding to asialo-GM1 and BEC receptors and also inhibited P. aeruginosa and C. albicans whole cell binding to BECs. These data suggested that the conserved epitope confers receptor-binding properties to the adhesins, demonstrated that (i) asialo-GM1-like receptors present on epithelial cell surfaces are utilized by the pilus and fimbrial adhesins and (ii) the binding to these glycoreceptors is mediated by a conserved epitope that has receptor-binding properties.
Assuntos
Adesinas Bacterianas/metabolismo , Moléculas de Adesão Celular , Proteínas Fúngicas/metabolismo , Mucosa Bucal/metabolismo , Mucosa Bucal/microbiologia , Receptores de Superfície Celular/metabolismo , Adesinas Bacterianas/imunologia , Anticorpos Bloqueadores , Anticorpos Monoclonais , Aderência Bacteriana , Candida albicans/imunologia , Candida albicans/metabolismo , Adesão Celular , Bochecha , Epitélio/metabolismo , Epitélio/microbiologia , Epitopos/metabolismo , Proteínas Fúngicas/imunologia , Gangliosídeo G(M1)/metabolismo , Humanos , Técnicas In Vitro , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismoRESUMO
Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt-P. aeruginosa) and C. albicans whole cells (Bt-C. albicans). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.
Assuntos
Adesinas Bacterianas/metabolismo , Candida albicans/metabolismo , Moléculas de Adesão Celular , Proteínas Fúngicas/metabolismo , Lectinas , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Anticorpos , Sítios de Ligação , Sequência de Carboidratos , Células Cultivadas , Sequência Conservada , Dissacarídeos/metabolismo , Gangliosídeo G(M1)/metabolismo , Humanos , Dados de Sequência MolecularRESUMO
The pili of Pseudomonas aeruginosa mediate bacterial binding to human epithelial cell surfaces. We have previously shown that a 17-residue synthetic peptide, KCTSDQDEQFIPKGCSK, corresponding to the C-terminal sequence of the PAK pilin protein (residues 128-144) contains the adherence binding domain. Another pilin strain, KB7, has been cloned and sequenced [Paranchych et al. (1990) in Pseudomonas Biotransformations, Pathogenesis and Evolving Biotechnology, pp 343-351, American Society for Microbiology, Washington, DC]. The C-terminal 17-residue sequence of the KB7 pilin is SCATTVDAKFRPNGCTD, which is semiconserved as compared to the PAK sequence. In this study, the interactions between the A549 human lung carcinoma cells and the two P. aeruginosa pilin strains were elucidated using a single alanine replacement analysis on the C-terminal 17-residue synthetic peptide of the pilins. The ability of these peptide analogs to inhibit the binding of the biotinylated PAK pili to A549 cells was assessed. Six PAK amino acid side chains (Ser131, Gln136, Ile138, Pro139, Gly141, and Lys144) and nine KB7 side chains (Ala130, Thr131, Thr132, Val133, Asp134, Ala135, Lys136, Arg138, and Pro139) were found to be important in mediating the pilus adhesin binding to A549 cells. In addition, a flexible peptide analog with both cysteine residues replaced by alanine failed to inhibit the binding of PAK pili to A549 cells. This suggests that the interactions between the pilin ligand and the A549 cell surface receptors are dependent on the conformation mediated by the disulfide bridge (Cys129 and Cys142). The residues considered to contribute to bacterial adherence are referred to as the "adhesintope". Four PAK and three KB7 side chains were located in a structurally more rigid region of the disulfide-bridged peptide as revealed by two-dimensional NMR studies [McInnes et al. (1993) Biochemistry 32, 13432-13440]. The structural aspects of the pilin-receptor interactions related to the mapped adhesintope sequences are discussed. The dissimilarities between the PAK and KB7 adhesintopes may suggest that compensatory mutations could occur among different pilin strains so as to allow the pilin adhesins to interact with the same receptor.