The Experts below are selected from a list of 96 Experts worldwide ranked by ideXlab platform
Christine M Szymanski - One of the best experts on this subject based on the ideXlab platform.
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Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages
MDPI AG, 2016Co-Authors: David J. Simpson, Jessica C. Sacher, Christine M SzymanskiAbstract:Recently, a large number of new technologies have been developed that exploit the unique properties of Bacteriophage Receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, Receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other Bacteriophages
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a Receptor binding protein of campylobacter jejuni Bacteriophage nctc 12673 recognizes flagellin glycosylated with acetamidino modified pseudaminic acid
Molecular Microbiology, 2015Co-Authors: Muhammad Afzal Javed, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Lieke B Van Alphen, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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A Receptor‐binding protein of Campylobacter jejuni Bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino‐modified pseudaminic acid
Molecular Microbiology, 2014Co-Authors: Muhammad Afzal Javed, Lieke B. Van Alphen, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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Phage Receptor binding protein-based magnetic enrichment method as an aid for real time PCR detection of foodborne bacteria.
The Analyst, 2013Co-Authors: Somayyeh Poshtiban, Muhammad Afzal Javed, Christine M Szymanski, Denis Arutyunov, Amit Kumar Singh, Graham S. Banting, Stephane EvoyAbstract:We present a novel phage Receptor binding protein-based magnetic separation and pre-enrichment method as an alternative to the immunomagnetic separation methods by replacing antibodies with Bacteriophage Receptor binding proteins (RBPs). We couple the proposed RBP-based magnetic separation with real time PCR for rapid, sensitive and specific detection of Campylobacter jejuni cells in artificially contaminated skim milk, milk with 2% fat and chicken broth. Recovery rates, assessed by real time PCR, were greater than 80% for the samples spiked with as low as 100 cfu mL−1 of C. jejuni cells. The specificity of capture was confirmed using Salmonella Typhimurium as a negative control where no bacteria were captured on the RBP-derivatized magnetic beads. The combination of RBP-based magnetic separation and real time PCR improved PCR sensitivity and allowed the detection of C. jejuni cells in milk and chicken broth samples without a time consuming pre-enrichment step through culturing. The total sample preparation and analysis time in the proposed RBP-based enrichment method coupled with real time PCR was less than 3 h.
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Bacteriophage Receptor binding protein based assays for the simultaneous detection of Campylobacter jejuni and Campylobacter coli.
PloS one, 2013Co-Authors: Muhammad Afzal Javed, Somayyeh Poshtiban, Denis Arutyunov, Stephane Evoy, Christine M SzymanskiAbstract:Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barre syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their Receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host Receptor binding protein, Gp047. In the current study, we localized the Receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.
Muhammad Afzal Javed - One of the best experts on this subject based on the ideXlab platform.
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a Receptor binding protein of campylobacter jejuni Bacteriophage nctc 12673 recognizes flagellin glycosylated with acetamidino modified pseudaminic acid
Molecular Microbiology, 2015Co-Authors: Muhammad Afzal Javed, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Lieke B Van Alphen, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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A Receptor‐binding protein of Campylobacter jejuni Bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino‐modified pseudaminic acid
Molecular Microbiology, 2014Co-Authors: Muhammad Afzal Javed, Lieke B. Van Alphen, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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Phage Receptor binding protein-based magnetic enrichment method as an aid for real time PCR detection of foodborne bacteria.
The Analyst, 2013Co-Authors: Somayyeh Poshtiban, Muhammad Afzal Javed, Christine M Szymanski, Denis Arutyunov, Amit Kumar Singh, Graham S. Banting, Stephane EvoyAbstract:We present a novel phage Receptor binding protein-based magnetic separation and pre-enrichment method as an alternative to the immunomagnetic separation methods by replacing antibodies with Bacteriophage Receptor binding proteins (RBPs). We couple the proposed RBP-based magnetic separation with real time PCR for rapid, sensitive and specific detection of Campylobacter jejuni cells in artificially contaminated skim milk, milk with 2% fat and chicken broth. Recovery rates, assessed by real time PCR, were greater than 80% for the samples spiked with as low as 100 cfu mL−1 of C. jejuni cells. The specificity of capture was confirmed using Salmonella Typhimurium as a negative control where no bacteria were captured on the RBP-derivatized magnetic beads. The combination of RBP-based magnetic separation and real time PCR improved PCR sensitivity and allowed the detection of C. jejuni cells in milk and chicken broth samples without a time consuming pre-enrichment step through culturing. The total sample preparation and analysis time in the proposed RBP-based enrichment method coupled with real time PCR was less than 3 h.
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Bacteriophage Receptor binding protein based assays for the simultaneous detection of Campylobacter jejuni and Campylobacter coli.
PloS one, 2013Co-Authors: Muhammad Afzal Javed, Somayyeh Poshtiban, Denis Arutyunov, Stephane Evoy, Christine M SzymanskiAbstract:Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barre syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their Receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host Receptor binding protein, Gp047. In the current study, we localized the Receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.
John F Kelly - One of the best experts on this subject based on the ideXlab platform.
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a Receptor binding protein of campylobacter jejuni Bacteriophage nctc 12673 recognizes flagellin glycosylated with acetamidino modified pseudaminic acid
Molecular Microbiology, 2015Co-Authors: Muhammad Afzal Javed, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Lieke B Van Alphen, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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A Receptor‐binding protein of Campylobacter jejuni Bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino‐modified pseudaminic acid
Molecular Microbiology, 2014Co-Authors: Muhammad Afzal Javed, Lieke B. Van Alphen, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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Genome and proteome of Campylobacter jejuni Bacteriophage NCTC 12673
Applied and environmental microbiology, 2011Co-Authors: Andrew M. Kropinski, Wen Ding, Denis Arutyunov, Stephane Evoy, Amit Kumar Singh, Mary Foss, Anna M. Cunningham, Andrey R. Pavlov, Matthew J. Henry, John F KellyAbstract:Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni Bacteriophage NCTC 12673 and the exploitation of its Receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the Receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage Receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms.
Wen Ding - One of the best experts on this subject based on the ideXlab platform.
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a Receptor binding protein of campylobacter jejuni Bacteriophage nctc 12673 recognizes flagellin glycosylated with acetamidino modified pseudaminic acid
Molecular Microbiology, 2015Co-Authors: Muhammad Afzal Javed, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Lieke B Van Alphen, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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A Receptor‐binding protein of Campylobacter jejuni Bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino‐modified pseudaminic acid
Molecular Microbiology, 2014Co-Authors: Muhammad Afzal Javed, Lieke B. Van Alphen, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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Genome and proteome of Campylobacter jejuni Bacteriophage NCTC 12673
Applied and environmental microbiology, 2011Co-Authors: Andrew M. Kropinski, Wen Ding, Denis Arutyunov, Stephane Evoy, Amit Kumar Singh, Mary Foss, Anna M. Cunningham, Andrey R. Pavlov, Matthew J. Henry, John F KellyAbstract:Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni Bacteriophage NCTC 12673 and the exploitation of its Receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the Receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage Receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms.
Jessica C. Sacher - One of the best experts on this subject based on the ideXlab platform.
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Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages
MDPI AG, 2016Co-Authors: David J. Simpson, Jessica C. Sacher, Christine M SzymanskiAbstract:Recently, a large number of new technologies have been developed that exploit the unique properties of Bacteriophage Receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, Receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other Bacteriophages
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a Receptor binding protein of campylobacter jejuni Bacteriophage nctc 12673 recognizes flagellin glycosylated with acetamidino modified pseudaminic acid
Molecular Microbiology, 2015Co-Authors: Muhammad Afzal Javed, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Lieke B Van Alphen, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
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A Receptor‐binding protein of Campylobacter jejuni Bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino‐modified pseudaminic acid
Molecular Microbiology, 2014Co-Authors: Muhammad Afzal Javed, Lieke B. Van Alphen, Cheryl E. Nargang, Jessica C. Sacher, John F Kelly, Richard D. Cummings, Wen Ding, David F Smith, Christine M SzymanskiAbstract:Bacteriophage Receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the Receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.
