Phage-displayed Random Library Research Articles

Pro-Fibrotic Effects of CCL18 on Human Lung Fibroblasts Are Mediated via CCR6.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown origin, with a median patient survival time of ~3 years after diagnosis without anti-fibrotic therapy. It is characterized by progressive fibrosis indicated by increased collagen deposition and high numbers of fibroblasts in the lung. It has been demonstrated that CCL18 induces collagen and αSMA synthesis in fibroblasts. We aimed to identify the CCL18 receptor responsible for its pro-fibrotic activities. We used a random phage display library to screen for potential CCL18-binding peptides, demonstrated its expression in human lungs and fibroblast lines by PCR and immunostaining and verified its function in cell lines. We identified CCR6 (CD196) as a CCL18 receptor and found its expression in fibrotic lung tissue and lung fibroblast lines derived from fibrotic lungs, but it was almost absent in control lines and tissue. CCL18 induced receptor internalization in a CCR6-overexpressing cell line. CCR6 blockade in primary human lung fibroblasts reduced CCL18-induced FGF2 release as well as collagen-1 and αSMA expression. Knockdown of CCR6 in a mouse fibroblast cell line abolished the induction of collagen and α-smooth muscle actin expression. Our data indicate that CCL18 triggers pro-fibrotic processes via CCR6, highlighting its role in fibrogenesis.

Development of highly specific peptibody against Porcine epidemic diarrhea virus

Abstract Porcine epidemic diarrhea virus (PEDV) is an etiological agent of porcine epidemic diarrhea (PED), causing dehydration, vomiting, and acute watery diarrhea. Because it has a high mortality rate in neonatal piglets, PEDV is one of the critical pathogens affecting the swine industry. Since PEDV has shown rapid spreading in herds of swine, early diagnosis of this virus is very important to control the disease. Serological diagnosis test is a simple, rapid method for detecting PEDV infected pigs, and the development of PEDV-specific antibodies is essential for an accurate serological diagnostic test. Because conventional methods of development of monoclonal antibodies are time and hand-consuming work, recombinant antibodies are considered as an alternative strategy for the development of target-specific monoclonal antibodies. Peptibody is one of the promising recombinant antibodies that target-specific peptide is fused with the Fc domain of immunoglobulin G. The peptibody which have a specificity of peptides and immune function of Fc region as advantages have developed for therapeutic and diagnostic purpose. Here, we generated a PEDV-specific peptibody (PEDV-PB) that is fused with PEDV-specific peptide and mouse IgG2b Fc region. PEDV-specific peptide sequences were discovered by random phage display library and PEDV-PB was produced from Chinese hamster ovary (CHO) cells. PEDV-PB showed highly specific binding in several serological tests for PEDV, such as western blotting, immunofluorescence assay, and enzyme-linked immunosorbent assay. Our results suggest that PEDV-PB is very useful for the development of a rapid detection kit for PEDV.

Biotechnological and Immunological Platforms Based on PGL-I Carbohydrate-Like Peptide of Mycobacterium leprae for Antibodies Detection Among Leprosy Clinical Forms.

Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.

Identification of Plasmodium knowlesi Merozoite Surface Protein-119 (PkMSP-119 ) novel binding peptides from a phage display library.

Plasmodium knowlesi, the fifth human malaria parasite, has caused mortality in humans. We aimed to identify P.knowlesi novel binding peptides through a random linear dodecapeptide phage display targeting the 19-kDa fragment of Merozoite Surface Protein-1 protein. rPkMSP-119 protein was heterologously expressed using Expresso® Solubility and Expression Screening System and competent E.cloni® 10G cells according to protocol. Three rounds of biopanning were performed on purified rPkMSP-119 to identify binding peptides towards rPkMSP-119 using Ph.D.™-12 random phage display library. Binding sites of the identified peptides to PkMSP-119 were in silico predicted using the CABS-dock web server. Four phage peptide variants that bound to PkMSP-119 were identified after three rounds of biopanning, namely Pkd1, Pkd2, Pkd3 and Pkd4. The sequences of both Pkd1 and Pkd2 consist of a large number of histidine residues. Pkd1 showed positive binding signal with 6.1× vs. BSA control. Docking results showed that Pkd1 and Pkd2 were ideal binding peptides for PkMSP-119 . We identified two novel binding peptides of PkMSP-119 , Pkd1 (HFPFHHHKLRAH) and Pkd2 (HPMHMLHKRQHG), through phage display. They provide a valuable starting point for the development of novel therapeutics.

Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (Kd,app) was calculated to be 810nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5μg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

The Relationship between B-cell Epitope and Mimotope Sequences.

B-cell epitope is a group of residues which is on the surface of an antigen. It invokes humoral responses. Locating B-cell epitope is important for effective vaccine design, and the development of diagnostic reagents. Mimotope-based B-cell epitope prediction method is a kind of conformational B-cell epitope prediction, and the core idea of the method is mapping the mimotope sequences which are obtained from a random phage display library. However, current mimotope-based B-cell epitope prediction methods cannot maintain a high degree of satisfaction in the circumstances of employing only mimotope sequences. In this study, we did a multi-perspective analysis on parameters for conformational B-cell epitopes and characteristics between epitope and mimotope on a benchmark datasets which contains 67 mimotope sets, corresponding to 40 unique complex structures. In these 67 cases, there are 25 antigen-antibody complexes and 42 protein-protein interactions. We analyzed the two parts separately. The results showed the mimotope sequences do have some epitope features, but there are also some epitope properties that mimotope sequences do not contain. In addition, the numbers of epitope segments with different lengths were obviously different between the antigen-antibody complexes and the protein-protein interactions. This study reflects how similar do mimotope sequence and genuine epitopes have; and evaluates existing mimotope-based B-cell epitope prediction methods from a novel viewpoint.

Identification of Immunotopes against <i>Mycobacterium leprae</i> as Immune Targets Using PhDTm- 12mer Phage Display Peptide Library

Purpose: To determine the surface epitopes of Mycobacterium leprae ( M. leprae ) and evaluate their efficacy in the production of anti- M. leprae antibodies in an animal model. Methods: Blood samples were obtained from 34 patients suffering from lepromatous leprosy. Antibodies were obtained from the samples, semi-purified and used to coat the wells of ELISA microplate, and M13 random-peptides library was added to the wells. After four rounds of panning, three clones were isolated and their peptide mimotopes were sequenced. Western blot was used to evaluate the interaction of the isolated mimotopes. Results: Three selective clones were tested by direct enzyme-linked immunosorbent assay (ELISA) and western blot. anti-leprae antibodies in various dilutions and were found to be serological active. Sequencing of the isolated peptides showed identities between the two clones that were able to successfully induce anti-Leprae humoral response in mice. Conclusion: The findings indicate that the isolated peptides can potentially be used for early diagnosis. However, further research is required to improve their potency as new vaccines against leprosy. Keywords: Bacteriophage, Vaccine, Leprosy, Mycobacterium leprae , Random Peptide Phage Display Library

Identification and evaluation of a novel peptide binding to the cell surface of Staphylococcus aureus

Identification of short peptides that serve as specific ligands to biological materials such as microbial cell surfaces has major implications in better understanding the molecular recognition of cell surfaces. In this study we screened a commercially available random phage-display library against Staphylococcus aureus cells and identified peptides specifically binding to the bacteria. A synthetic peptide (SA5-1) representing the consensus sequence (VPHNPGLISLQG) of the bacteria-binding peptide was evaluated for its binding potential against S. aureus. Dot-blot, immunoblot assay and ELISA results revealed the SA5-1 peptide to be highly specific to S. aureus. The SA5-1 peptide binding was optimal between pH 6.0 and 8.0. Nanogold Transmission Electron Microscopy demonstrated that the SA5-1 binds to the outer membrane surface of S. aureus. Diagnostic potential of the SA5-1 peptide was evaluated in human platelet samples spiked with S. aureus and specific detection of the bacteria by biotinylated-SA5-1 and streptavidin-conjugated fluorescent quantum dots. Fluorometry results indicated that the peptide was able to detect ∼100 organisms per ml in a spiked biological sample providing a proof-of-concept towards potential of this peptide as a S. aureus diagnostic tool that can be of use in different detection platforms.

Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread

BackgroundA33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus.ResultsBy screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture.ConclusionsThese results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may interfere with the function of A33 in vivo. This information will also be useful for designing improved assays to evaluate the potency of monoclonal and polyclonal products that target A33 or A33-modulated EV dissemination.

Selection of Triptolide Ligands from a Random Phage Display Library and Primary Verification of Their Combination

Triptolide is an extract from the Chinese herb Tripterygium wilfordii Hook f.We screened out a peptide from a C7 phage display library and presumed it was a potential peptide ligand for triptolide.The specificity of the selected clone for triptolide was confirmed by ELISA and immunoprecipitation.After DNA sequencing,a BLAST search was carried out and 77 matching sequences was retrieved.The most promising candidate is human steroidogenic factor-1(hSF-1),a member of the nuclear receptor family that controls the synthesis of steroid hormones by regulating steroidogenic enzyme genes.We purified the ligand binding domain(LBD) of hSF-1 and then used it for further experiments.Fluorescence spectrum experiments showed that triptolide could cause fluorescence quenching of hSF-1-LBD,isothermal titration calorimetric(ITC) measurements showed a enthalpy-driven interaction between triptolide and hSF-1-LBD,and a dose-dependent interaction between them was observed by surface plasmon resonance(SPR).All these results confirmed the specific interaction between hSF-1 and triptolide.

MimoPro: a more efficient Web-based tool for epitope prediction using phage display libraries

BackgroundA B-cell epitope is a group of residues on the surface of an antigen which stimulates humoral responses. Locating these epitopes on antigens is important for the purpose of effective vaccine design. In recent years, mapping affinity-selected peptides screened from a random phage display library to the native epitope has become popular in epitope prediction. These peptides, also known as mimotopes, share the similar structure and function with the corresponding native epitopes. Great effort has been made in using this similarity between such mimotopes and native epitopes in prediction, which has resulted in better outcomes than statistics-based methods can. However, it cannot maintain a high degree of satisfaction in various circumstances.ResultsIn this study, we propose a new method that maps a group of mimotopes back to a source antigen so as to locate the interacting epitope on the antigen. The core of this method is a searching algorithm that is incorporated with both dynamic programming (DP) and branch and bound (BB) optimization and operated on a series of overlapping patches on the surface of a protein. These patches are then transformed to a number of graphs using an adaptable distance threshold (ADT) regulated by an appropriate compactness factor (CF), a novel parameter proposed in this study. Compared with both Pep-3D-Search and PepSurf, two leading graph-based search tools, on average from the results of 18 test cases, MimoPro, the Web-based implementation of our proposed method, performed better in sensitivity, precision, and Matthews correlation coefficient (MCC) than both did in epitope prediction. In addition, MimoPro is significantly faster than both Pep-3D-Search and PepSurf in processing.ConclusionsOur search algorithm designed for processing well constructed graphs using an ADT regulated by CF is more sensitive and significantly faster than other graph-based approaches in epitope prediction. MimoPro is a viable alternative to both PepSurf and Pep-3D-Search for epitope prediction in the same kind, and freely accessible through the MimoPro server located at http://informatics.nenu.edu.cn/MimoPro.

A peptide with similarity to baculovirus ODV-E66 binds the gut epithelium of Heliothis virescens and impedes infection with Autographa californica multiple nucleopolyhedrovirus

Baculoviruses infect their lepidopteran hosts via the midgut epithelium through binding of occlusion-derived virus (ODV) and fusion between the virus envelope and microvillar membranes. To identify genes and sequences that are involved in this process, a random phage display library was screened for peptides that bound to brush border membrane vesicles (BBMV) derived from the midgut epithelium of Heliothis virescens. Seventeen peptides that bound to BBMV were recovered. Two of these, HV1 and HV2, had sequence similarity to the ODV envelope protein ODV-E66 that is found in five species of alphabaculoviruses. Chemically synthesized versions of HV1 and HV2, and two peptides (AcE66A and AcE66B) derived from similar sequences of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ODV-E66, bound to unfixed cryosections of whole midgut tissues. AcE66A, but not HV1, bound to H. virescens gut BBMV proteins on a far-Western blot. Competition assays with HV1 and purified AcMNPV ODV resulted in decreased mortality of H. virescens larvae at a dose of 1 LD(50), and a significant increase in survival time at higher virus concentrations. These results suggest a role for ODV-E66 in baculovirus infection of lepidopteran larval midgut epithelium.

Mapping of binding epitopes of a human decay-accelerating factor monoclonal antibody capable of enhancing rituximab-mediated complement-dependent cytotoxicity

Complement-dependent cytotoxicity (CDC) is thought to be one of the most important mechanisms of action of therapeutic monoclonal antibodies (mAbs). The decay-accelerating factor (DAF) overexpressed in certain tumors limits the CDC effect of the therapeutic anticancer antibodies. The use of DAF blocking antibodies targeted specifically at cancer cells in combination with immunotherapeutic mAbs of cancer may improve the therapeutic effect in cancer patients. In this study, the lysis of Raji cells mediated by CDC was determined after blocking DAF function by anti-DAF polyclonal antibody and 3 mAbs (DG3, DG9, DA11) prepared in our laboratory, respectively, in the presence of the anti-CD20 chimeric mAb rituximab. The binding domains of the three anti-DAF mAbs were identified using yeast surface display technique, and the mimic epitopes of mAb DG3 were screened from a random phage-display nonapeptide library. The results showed that blocking DAF function by anti-DAF polyclonal antibody enhanced complement-mediated killing of Raji cells. Among the 3 mAbs against DAF, only DG3 was found to be able to remarkably enhance the CDC effect of the therapeutic mAb rituximab. DG3 bound to the third short consensus repeat (SCR) of DAF. Binding of DG3 to immobilized DAF was inhibited by mimic epitope peptides screened from the peptide library. Our results suggest that a higher level of DAF expressed by certain tumor cells is significant to abolish the CDC effect of therapeutic anticancer antibodies, and mAbs binding to SCR3 can enhance the complement-mediated killing of Raji cells. It is of significance to identify the DAF epitopes required in inhibiting CDC not only for better understanding of the relationship between the structure and function of DAF, but also for designing and developing anti-DAF mAbs capable of enhancing CDC.

Identification of Their Epitope Reveals the Structural Basis for the Mechanism of Action of the Immunosuppressive Antibodies Basiliximab and Daclizumab

Interleukin-2 (IL-2) and its receptor (IL-2R) play a major role in cellular immunity. The monoclonal antibodies basiliximab and daclizumab directed against the IL-2R subunit CD25 are widely used to prevent graft or host rejection after allogeneic tissue transplantation. Although these antibodies have been used for this purpose for many years, their common epitope within the CD25 protein is unknown. We screened a random phage display library to isolate peptides specifically binding to basiliximab. A striking amino acid sequence motif was enriched. This motif is homologous to the peptide ERIYHFV comprising amino acid positions 116 to 122 within the extracellular domain of CD25, suggesting that this is the basiliximab epitope. Basiliximab and daclizumab binding of selected phage was specific, as no binding was observed to isotype antibody controls. Phage binding could be inhibited by the cognate peptide. In cells expressing mutant CD25, binding of basiliximab was abolished when two or more amino acids of the suspected epitope were changed. In contrast, basiliximab binding remained unaffected in cells expressing CD25 versions with mutations outside this epitope. We therefore conclude that the (116)ERIYHFV(122) string within CD25 is the epitope recognized by basiliximab and daclizumab. This epitope overlaps with the interaction site of CD25 and IL-2, thus revealing the structural basis for the inhibition of IL-2R binding by this class of immunosuppressive antibodies.

Inhibitory effects of a specific phage-displayed peptide on high peritoneal metastasis of gastric cancer

Peritoneal dissemination in gastric cancer is the most frequent cause of the noncurative resection and recurrence after curative resection. We, therefore, evaluated the feasibility of a peptide, which was obtained by screening a random phage display library, in the treatment of peritoneal metastases of gastric cancer. In this study, a novel cell line, GC9811-P, with a high potential peritoneal metastasis of gastric cancer derived from its parental cell line, GC9811, was established. Using a phage display library, we isolated a specific peptide that selectively bound to GC9811-P cells rather than its parental GC9811cells. The isolated phage-displaying peptide, SMSIASPYIALE (named peptide PIII), was obtained after four rounds of selection, showing a tendency to preferentially bind to GC9811-P cells compared with a panel of other gastric cancer cell lines, and preferentially accumulate in peritoneal metastasis tumor tissue in comparison with control organs, peritoneum, liver, pancreas, spleen, lung, and kidney. Further study showed that synthetic peptide PIII could significantly inhibit adhesive and invasional ability of GC9811-P cells and could effectively block the corresponding phage binding to the GC9811-P cells, whereas, exposure of the cells to various concentrations of peptide PIII showed no obvious cell growth inhibition. Furthermore, a highly reproducible animal experimental model of gastric cancer with peritoneal dissemination was established in nude mice by injecting a suspension of the cell line into the gastric wall of nude mice. Animals intraperitoneally treated with peptide PIII in this model or another animal model of gastric cancer with peritoneal dissemination established using MKN45 cells showed suppressed tumor metastasis to peritoneum and significantly prolonged survival. In conclusion, the selected peptide PIII was a biologically active peptide and could effectively inhibit peritoneal dissemination of gastric cancer.

One-step affinity purification of recombinant urokinase-type plasminogen activator receptor using a synthetic peptide developed by combinatorial chemistry

Several lines of evidence have pointed to a role of urokinase-type plasminogen activator receptor (uPAR) as a modulator of certain biochemical processes that are active during tumor invasion and metastasis. Consequently, the structure and function of this receptor have been studied extensively, using recombinantly produced uPAR that has been purified by either affinity chromatography using its cognate ligand, the urokinase-type plasminogen activator (uPA), or a monoclonal anti-uPAR antibody (R2), or by hydroxyapatite. Here, we present a new method for the efficient one-step affinity purification of recombinant uPAR exploiting a high-affinity synthetic peptide antagonist (AE152). The corresponding parent peptide was originally identified in a random phage-display library and subsequently subjected to affinity maturation by combinatorial chemistry. This study compares the affinity purification of a soluble, recombinant uPAR using the monoclonal antibody R2 or the peptide AE152 immobilized on Sepharose. The two affinity ligands perform equally well in purifying uPAR from Drosophila melanogaster Schneider 2 cell culture medium and yield products of comparable purity, activity, and stability as judged by SDS–PAGE, size exclusion chromatography and surface plasmon resonance analysis. The general availability of peptide synthesis renders the present AE152-based affinity purification of uPAR more accessible than the traditional protein-based affinity purification strategies. In this way, large amounts of recombinant uPAR can conveniently be purified for further structural and functional studies.

Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies

Interference with fat hydrolysis results in the reduced use of ingested lipids. Inhibition of pancreatic lipase reduces the efficiency of fat absorption in the small intestine and thereby initiates modest long-term reduction in body weight. In an attempt to select peptides with affinity for the surface of pancreatic lipase and potential inhibitory activity, a random, cyclic heptapeptide phage-displayed library was used. Five independent selections, differing in elution step, were performed. In three selection protocols, a sequential elution strategy was applied in anticipation of improving the selection of high-affinity clones. Four heptapeptides with the highest affinity, seemingly for pancreatic lipase, were selected, synthesized, and characterized for their capacity to inhibit enzyme function. Although no clear consensus among the sequenced peptides was found, one of the selected peptides inhibited pancreatic lipase with an apparent inhibition constant of 16 muM.

Efficient isolation of peptide ligands for the endothelial cell protein C receptor (EPCR) using candidate receptor phage display biopanning

Phage display biopanning has been used for a number of applications including ligand generation for targeted drug delivery, targeting gene therapy vectors and identification of protein–protein interaction sites. In this study, a random phage display library was used to isolate peptide ligands to the endothelial protein C receptor (EPCR), identifying 74 different peptide sequences and several motifs. Binding to EPCR was characterized by a solid phase binding assay, demonstrating that 95% of isolated peptides were specific for EPCR. Several homologies with potential relevance to EPCR biology were identified, the most notable being leukolysin (MT-MMP6) and cerastocytin.

Use of Phage Display To Identify Potential Pseudomonas aeruginosa Gene Products Relevant to Early Cystic Fibrosis Airway Infections

Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis. Treatment of established infections is difficult, even with microbiologically active agents. Thus, prevention of infection is an important goal of management. Isolates from cystic fibrosis patients appear to originate from the environment but adapt to the milieu of the airway of the cystic fibrosis patient and evolve toward a common phenotype. Identification of the antigens expressed early in infection may lead to novel targets for vaccine development. Immunogenic peptides were identified in a J404 random nonapeptide phage display library with serum from cystic fibrosis patients obtained within the first year of P. aeruginosa infection. One hundred sixty-five reactive clones were verified by plaque lift assays, and their inserts were sequenced. The sequenced nonapeptides were compared with the published sequence of strain PAO1, identifying homologies to 76 genes encoding outer membrane and secreted proteins. The majority of these were proteins involved in small-molecule transport, membrane structural proteins, and secreted factors. An in silico analysis was performed that suggested that the occurrence of multiple matches to predominantly outer membrane and secreted proteins was not attributable to random chance. Finally, gene expression array data from early isolates of P. aeruginosa from cystic fibrosis patients was compared with the results from phage display analysis. Eleven outer membrane and secreted proteins were common between the two data sets. These included genes involved in iron acquisition, antibiotic efflux, fimbrial biogenesis, and pyocin synthesis. These results demonstrate the feasibility and validity of this novel approach and suggest potential targets for future development.

Using capillary electrophoresis to study methylation effect on RNA-peptide interaction.

Methylation of RNA and proteins is one of a broad spectrum of post-transcriptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA(Phe) anticodon stem and loop domain (ASL(Phe)) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm(32)), 2'-O-methylguanine (Gm(34)) and 5-methylcytidine (m(5)C(40))) (ASL(Phe)-Cm(32),Gm(34),m(5)C(40)) and a 15-amino-acid peptide (named t(F)2: Ser(1)-Ile-Ser-Pro-Trp(5)-Gly-Phe-Ser-Gly-Leu(10)-Leu- Arg-Trp-Ser-Tyr(15)) selected from a random phage display library (RPL). A peptide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL(Phe) shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASL(Phe) was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49-57)-NH(2) (named Tat1: Arg(49)-Lys-Lys-Arg(52)-Arg-Gln-Arg-Arg- Arg(57)-NH(2)). In the presence of Tat(49-57)-NH(2) a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg(52)-->Arg(Me)(2), crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.