This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antib... more This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antibodies (MAbs) produced to be specific for Porphyromonas gingivalis whole cells which we had previously shown to bind to epitopes recognized by sera from periodontitis patients. Preliminary data had suggested that the arginine-specific proteases of P. gingivalis (ArgI, ArgIA, and ArgIB) contained the antigenic determinants of four of these antibodies (MAbs 1A1, 2B/H9, 7D5, and 3B1). The location of the binding sites was examined with purified P. gingivalis enzymes and recombinant regions of the ArgI polyprotein expressed by subclones of the prpR1 gene in Escherichia coli XL-1 Blue cells. All four antibodies were reactive with protein determinants within the beta subunit, a hemagglutinin and/or adhesin component, of the ArgI dimer. MAb 1A1 strongly inhibited the agglutination of human erythrocytes by P. gingivalis W50 culture supernatant, suggesting that the binding site for this antibody c...
ABSTRACTProteases ofPorphyromonas gingivalisare considered to be important virulence determinants... more ABSTRACTProteases ofPorphyromonas gingivalisare considered to be important virulence determinants of this periodontal bacterium. Several biochemical isoforms of arginine-specific proteases are derived fromrgpAandrgpB. HRgpA is a heterodimer composed of the catalytic α chain noncovalently associated with a β adhesin chain derived from the C terminus of the initial full-length translation product. The catalytic α chain is also present as a monomer (RgpA) either free in solution or associated with membranes.rgpBlacks the coding region for the adhesin domain present inrgpAand yields only monomeric forms (RgpB) which again may be soluble or membrane associated. In this study, the catalytic chains of this unusual group of enzymes are shown to be differentially modified by the posttranslational addition of carbohydrate. A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did not react with the corresponding recombinant RgpA α chain expressed inEscherichia colibut was immunoreactiv...
Porphyromonas gingivalis produces outer-membrane vesicles (OMVs) rich in virulence factors includ... more Porphyromonas gingivalis produces outer-membrane vesicles (OMVs) rich in virulence factors including cysteine-proteases and A-LPS, one of the two lipopolysaccharides (LPS) produced by this organism. Previous studies had suggested that A-LPS together with PG0027, an outer-membrane (OM) protein, may be involved in OMV-formation. Their roles in this process were examined using parent W50 and mutant ΔPG0027 strains. Inactivation of PG0027 caused a reduction in yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and ΔPG0027 strains were analysed by matrix-assisted laser-desorption-ionisation-time of flight mass spectrometry (MALDI-TOF- MS). Lipid A (W50 cells) contained bis-P-pentaacyl-, mono-P-pentaacyl-, mono-P-tetraacyl-, non-P-pentaacyl- and non-P-tetraacyl- species whereas lipid A (ΔPG0027 cells) contained only phosphorylated-species: non-phosphorylated-species were absent. MALDI-TOF/TOF-tandem-MS of mono-P-pentaacyl-(m/z 1688)-lipid A and mono-P-tetraacyl-(m/z 1448)- li...
Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe unable to synthesise haem (... more Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe unable to synthesise haem (Fe (II)-protoporphyrin IX) or hemin (Fe(III)-protoporphyrin IX-Cl) which are important growth/virulence-factors, and must therefore derive them from the host P. gingivalis expresses several proteinaceous hemin -binding-sites which are important in the binding/ transport of haem/hemin from the host. P. gingivalis also synthesises several virulence factors, namely cysteine-proteases Arg- and Lys-gingipains and two lipopolysaccharides (LPS), O-LPS and A-LPS. The gingipains are required for the production of the black pigment, μ-oxo-bishaem ([Fe(III)PPIX]2 O), which is derived from hemoglobin and deposited on the bacterial cell-surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of μ-oxo-bishaem on the cell-surface. A P. gingivalis mutant defective in the biosynthesis of Arg-gingipains, namely rgpA/rgp...
For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to human... more For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host–pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and gender...
ABSTRACT One hundred years ago King Oscar II frequented the meeting place for the Fifth European ... more ABSTRACT One hundred years ago King Oscar II frequented the meeting place for the Fifth European Oral Microbiology Workshop at Särö, Gothenburg, Sweden. This was convened from 9th to 11th May 1996, in the splendid royal summer house by the sea. The workshop was organised by Professor Gunnar Dahlén, Dr Panos Papanou, Dr Maude Wikström and colleagues from the Department of Oral Microbiology at Gothenburg University. Eighty-two delegates from 11 European countries and Australia attended the meeting. The workshop was organised into seven sessions over 2 days.
Additional file 1. Supplementary Table 1. Peptides generated from mass spectroscopy analysis of t... more Additional file 1. Supplementary Table 1. Peptides generated from mass spectroscopy analysis of the honey mushroom aflatoxin-degrading enzyme.
Background Aflatoxins are carcinogenic compounds produced by certain species of Aspergillus fungi... more Background Aflatoxins are carcinogenic compounds produced by certain species of Aspergillus fungi. The consumption of crops contaminated with this toxin cause serious detrimental health effects, including death, in both livestock and humans. As a consequence, both the detection and quantification of this toxin in food/feed items is tightly regulated with crops exceeding the allowed limits eliminated from food chains. Globally, this toxin causes massive agricultural and economic losses each year. Results In this paper we investigate the feasibility of using an aflatoxin-degrading enzyme strategy to reduce/eliminate aflatoxin loads in developing maize kernels. We used an endoplasmic reticulum (ER) targeted sub-cellular compartmentalization stabilizing strategy to accumulate an aflatoxin-degrading enzyme isolated from the edible Honey mushroom Armillariella tabescens and expressed it in embryo tissue in developing maize kernels. Three transgenic maize lines that were determined to be e...
The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is an important insect pest in c... more The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is an important insect pest in cotton that feeds on reproductive fruit, contributing to yield loss. Economically damaging infestations of L. lineolaris have doubled in Virginia since 2013. Escalation of L. lineolaris abundance may increase Fusarium hardlock disease observed in this region, compounding economic losses. Research has linked Fusarium hardlock with fungal species Fusarium verticillioides and F. proliferatum. Field and greenhouse experiments were performed to investigate (i) Fusarium hardlock occurrence in field plots managed and unmanaged for L. lineolaris, (ii) severity of F. verticillioides infection of cotton bolls with and without the presence of L. lineolaris feeding in a greenhouse setting, and (iii) Fusarium species composition and prevalence within field-collected L. lineolaris and cotton lint with and without insect feeding injury and hardlock symptoms present. Nearly twice the amount of hardlock (...
Previous studies of the serum immunoglobulin G antibody response of periodontal patients have dem... more Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels. In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated. ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer composed of the catalytically active alpha component alone. The gene encoding ArgI (prpR1 encoding protease polyprotein ArgI) was cloned from Sau3AI digests of P. gingivalis W50 DNA into pUC18. Sequence analysis demonstrated that the alpha and beta components are contiguous on the initial translation product and are flanked by large N- and C-terminal extensions. prpR1 is 97.5% identical to the rgp-1 gene from P. gingivalis H66. prpR1 expression in Escherichia coli demonstrated the presence o...
The prpR1 of Porphyromonas gingivalis codes for three distinct enzymes with specificity for argin... more The prpR1 of Porphyromonas gingivalis codes for three distinct enzymes with specificity for arginyl peptide bonds termed RI, RIA, and RIB. These three isoforms comprise the majority of the extracellular, arginine-specific protease activity in P. gingivalis W50. RI is a heterodimer in which the catalytic α chain is noncovalently associated with a second chain involved in adherence phenomena. RIA and RIB are both monomeric species. RIA represents the free α chain, and RIB is a highly posttranslationally modified form of the α chain which is exclusively vesicle or membrane associated and migrates as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In previous studies, insertional inactivation of the prpR1 demonstrated that arginine-specific protease activity can also arise from a closely related second gene, prR2 . In the present work, the prR2 was insertionally inactivated in P. gingivalis W50 in order to establish the contribution of this locus to the argi...
Brown marmorated stink bug (Halyomorpha halys Stål) is an invasive agricultural pest that causes ... more Brown marmorated stink bug (Halyomorpha halys Stål) is an invasive agricultural pest that causes severe damage to many crops. To determine potential associations between H. halys feeding damage, Fusarium infection, and mycotoxin contamination in field corn, a field survey was conducted in eight counties in Virginia. Results indicated an association between H. halys feeding damage and fumonisin contamination. Subsequent field experiments in Delaware, Maryland, and Virginia examined the ability of H. halys to increase Fusarium verticillioides (Sacc.) Nirenberg infection and fumonisin concentrations in corn. At the milk stage, H. halys (0 or 4 adults) and Fusarium (with or without F. verticillioides inoculum) treatments were applied to bagged ears in a two by two factorial randomized complete block design with 12 replicates. H. halys treatments increased levels of feeding damage (P < 0.0001) and Fusarium infection (P = 0.0380). Interaction between H. halys and Fusarium treatments in...
Objectives: The production of outer membrane vesicles (OMV) in gram-negative bacteria is consider... more Objectives: The production of outer membrane vesicles (OMV) in gram-negative bacteria is considered to be an important mechanism of virulence factor delivery, inter-species communication and pathogenic interactions with the host. In recent investigations, we established that the periodontal organism, Porphyromonas gingivalis, has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we showed a critical role for lipopolysaccharide in directing this sorting mechanism. In the current work we aimed to describe the machinery required for OMV biogenesis in this bacterium. Methods: The protein composition of P. gingivalis OMV was examined by proteomic analysis to determine whether OMV specific proteins (not detectable in the bacterial outer membrane) were present as these may be involved in OMV formation. Targetted gene mutagenesis b...
Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPS), O-LPS and A-LPS. The structur... more Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPS), O-LPS and A-LPS. The structure of the core oligosaccharide (OS) of O-LPS and the attachment-site of O-polysaccharide (PS) repeating unit [ →3)-α-d-Galp-(1→6)-α-d-Glcp-(1→4)-α-l-Rhap-(1→3)-β-d-GalNAcp-(1→] to the core, have been elucidated using mutant strains ΔPG1051(WaaL, O-antigen ligase) and ΔPG1142 (Wzy, O-antigen polymerase) respectively. The core OS occurs as an "uncapped" glycoform devoid of O-PS and a "capped" glycoform which contains the attachment-site of O-PS via β-d-GalNAc at position O-3 of the terminal α-(1→3)-linked mannose (Man) residue. In this study, the attachment site of A-PS to core-OS was determined based on structural analysis of SR-type-LPS [O-LPS and A-LPS] isolated from P. gingivalis mutant strain ΔPG1142 by extraction with aqueous hot-phenol to minimize the destruction of A-LPS. Application of one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) in com...
Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal ... more Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N- and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in the processing of KGP. A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) an...
Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the a... more Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either N-acylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5alpha. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant pro...
This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antib... more This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antibodies (MAbs) produced to be specific for Porphyromonas gingivalis whole cells which we had previously shown to bind to epitopes recognized by sera from periodontitis patients. Preliminary data had suggested that the arginine-specific proteases of P. gingivalis (ArgI, ArgIA, and ArgIB) contained the antigenic determinants of four of these antibodies (MAbs 1A1, 2B/H9, 7D5, and 3B1). The location of the binding sites was examined with purified P. gingivalis enzymes and recombinant regions of the ArgI polyprotein expressed by subclones of the prpR1 gene in Escherichia coli XL-1 Blue cells. All four antibodies were reactive with protein determinants within the beta subunit, a hemagglutinin and/or adhesin component, of the ArgI dimer. MAb 1A1 strongly inhibited the agglutination of human erythrocytes by P. gingivalis W50 culture supernatant, suggesting that the binding site for this antibody c...
ABSTRACTProteases ofPorphyromonas gingivalisare considered to be important virulence determinants... more ABSTRACTProteases ofPorphyromonas gingivalisare considered to be important virulence determinants of this periodontal bacterium. Several biochemical isoforms of arginine-specific proteases are derived fromrgpAandrgpB. HRgpA is a heterodimer composed of the catalytic α chain noncovalently associated with a β adhesin chain derived from the C terminus of the initial full-length translation product. The catalytic α chain is also present as a monomer (RgpA) either free in solution or associated with membranes.rgpBlacks the coding region for the adhesin domain present inrgpAand yields only monomeric forms (RgpB) which again may be soluble or membrane associated. In this study, the catalytic chains of this unusual group of enzymes are shown to be differentially modified by the posttranslational addition of carbohydrate. A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did not react with the corresponding recombinant RgpA α chain expressed inEscherichia colibut was immunoreactiv...
Porphyromonas gingivalis produces outer-membrane vesicles (OMVs) rich in virulence factors includ... more Porphyromonas gingivalis produces outer-membrane vesicles (OMVs) rich in virulence factors including cysteine-proteases and A-LPS, one of the two lipopolysaccharides (LPS) produced by this organism. Previous studies had suggested that A-LPS together with PG0027, an outer-membrane (OM) protein, may be involved in OMV-formation. Their roles in this process were examined using parent W50 and mutant ΔPG0027 strains. Inactivation of PG0027 caused a reduction in yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and ΔPG0027 strains were analysed by matrix-assisted laser-desorption-ionisation-time of flight mass spectrometry (MALDI-TOF- MS). Lipid A (W50 cells) contained bis-P-pentaacyl-, mono-P-pentaacyl-, mono-P-tetraacyl-, non-P-pentaacyl- and non-P-tetraacyl- species whereas lipid A (ΔPG0027 cells) contained only phosphorylated-species: non-phosphorylated-species were absent. MALDI-TOF/TOF-tandem-MS of mono-P-pentaacyl-(m/z 1688)-lipid A and mono-P-tetraacyl-(m/z 1448)- li...
Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe unable to synthesise haem (... more Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe unable to synthesise haem (Fe (II)-protoporphyrin IX) or hemin (Fe(III)-protoporphyrin IX-Cl) which are important growth/virulence-factors, and must therefore derive them from the host P. gingivalis expresses several proteinaceous hemin -binding-sites which are important in the binding/ transport of haem/hemin from the host. P. gingivalis also synthesises several virulence factors, namely cysteine-proteases Arg- and Lys-gingipains and two lipopolysaccharides (LPS), O-LPS and A-LPS. The gingipains are required for the production of the black pigment, μ-oxo-bishaem ([Fe(III)PPIX]2 O), which is derived from hemoglobin and deposited on the bacterial cell-surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of μ-oxo-bishaem on the cell-surface. A P. gingivalis mutant defective in the biosynthesis of Arg-gingipains, namely rgpA/rgp...
For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to human... more For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host–pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and gender...
ABSTRACT One hundred years ago King Oscar II frequented the meeting place for the Fifth European ... more ABSTRACT One hundred years ago King Oscar II frequented the meeting place for the Fifth European Oral Microbiology Workshop at Särö, Gothenburg, Sweden. This was convened from 9th to 11th May 1996, in the splendid royal summer house by the sea. The workshop was organised by Professor Gunnar Dahlén, Dr Panos Papanou, Dr Maude Wikström and colleagues from the Department of Oral Microbiology at Gothenburg University. Eighty-two delegates from 11 European countries and Australia attended the meeting. The workshop was organised into seven sessions over 2 days.
Additional file 1. Supplementary Table 1. Peptides generated from mass spectroscopy analysis of t... more Additional file 1. Supplementary Table 1. Peptides generated from mass spectroscopy analysis of the honey mushroom aflatoxin-degrading enzyme.
Background Aflatoxins are carcinogenic compounds produced by certain species of Aspergillus fungi... more Background Aflatoxins are carcinogenic compounds produced by certain species of Aspergillus fungi. The consumption of crops contaminated with this toxin cause serious detrimental health effects, including death, in both livestock and humans. As a consequence, both the detection and quantification of this toxin in food/feed items is tightly regulated with crops exceeding the allowed limits eliminated from food chains. Globally, this toxin causes massive agricultural and economic losses each year. Results In this paper we investigate the feasibility of using an aflatoxin-degrading enzyme strategy to reduce/eliminate aflatoxin loads in developing maize kernels. We used an endoplasmic reticulum (ER) targeted sub-cellular compartmentalization stabilizing strategy to accumulate an aflatoxin-degrading enzyme isolated from the edible Honey mushroom Armillariella tabescens and expressed it in embryo tissue in developing maize kernels. Three transgenic maize lines that were determined to be e...
The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is an important insect pest in c... more The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is an important insect pest in cotton that feeds on reproductive fruit, contributing to yield loss. Economically damaging infestations of L. lineolaris have doubled in Virginia since 2013. Escalation of L. lineolaris abundance may increase Fusarium hardlock disease observed in this region, compounding economic losses. Research has linked Fusarium hardlock with fungal species Fusarium verticillioides and F. proliferatum. Field and greenhouse experiments were performed to investigate (i) Fusarium hardlock occurrence in field plots managed and unmanaged for L. lineolaris, (ii) severity of F. verticillioides infection of cotton bolls with and without the presence of L. lineolaris feeding in a greenhouse setting, and (iii) Fusarium species composition and prevalence within field-collected L. lineolaris and cotton lint with and without insect feeding injury and hardlock symptoms present. Nearly twice the amount of hardlock (...
Previous studies of the serum immunoglobulin G antibody response of periodontal patients have dem... more Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels. In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated. ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer composed of the catalytically active alpha component alone. The gene encoding ArgI (prpR1 encoding protease polyprotein ArgI) was cloned from Sau3AI digests of P. gingivalis W50 DNA into pUC18. Sequence analysis demonstrated that the alpha and beta components are contiguous on the initial translation product and are flanked by large N- and C-terminal extensions. prpR1 is 97.5% identical to the rgp-1 gene from P. gingivalis H66. prpR1 expression in Escherichia coli demonstrated the presence o...
The prpR1 of Porphyromonas gingivalis codes for three distinct enzymes with specificity for argin... more The prpR1 of Porphyromonas gingivalis codes for three distinct enzymes with specificity for arginyl peptide bonds termed RI, RIA, and RIB. These three isoforms comprise the majority of the extracellular, arginine-specific protease activity in P. gingivalis W50. RI is a heterodimer in which the catalytic α chain is noncovalently associated with a second chain involved in adherence phenomena. RIA and RIB are both monomeric species. RIA represents the free α chain, and RIB is a highly posttranslationally modified form of the α chain which is exclusively vesicle or membrane associated and migrates as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In previous studies, insertional inactivation of the prpR1 demonstrated that arginine-specific protease activity can also arise from a closely related second gene, prR2 . In the present work, the prR2 was insertionally inactivated in P. gingivalis W50 in order to establish the contribution of this locus to the argi...
Brown marmorated stink bug (Halyomorpha halys Stål) is an invasive agricultural pest that causes ... more Brown marmorated stink bug (Halyomorpha halys Stål) is an invasive agricultural pest that causes severe damage to many crops. To determine potential associations between H. halys feeding damage, Fusarium infection, and mycotoxin contamination in field corn, a field survey was conducted in eight counties in Virginia. Results indicated an association between H. halys feeding damage and fumonisin contamination. Subsequent field experiments in Delaware, Maryland, and Virginia examined the ability of H. halys to increase Fusarium verticillioides (Sacc.) Nirenberg infection and fumonisin concentrations in corn. At the milk stage, H. halys (0 or 4 adults) and Fusarium (with or without F. verticillioides inoculum) treatments were applied to bagged ears in a two by two factorial randomized complete block design with 12 replicates. H. halys treatments increased levels of feeding damage (P < 0.0001) and Fusarium infection (P = 0.0380). Interaction between H. halys and Fusarium treatments in...
Objectives: The production of outer membrane vesicles (OMV) in gram-negative bacteria is consider... more Objectives: The production of outer membrane vesicles (OMV) in gram-negative bacteria is considered to be an important mechanism of virulence factor delivery, inter-species communication and pathogenic interactions with the host. In recent investigations, we established that the periodontal organism, Porphyromonas gingivalis, has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we showed a critical role for lipopolysaccharide in directing this sorting mechanism. In the current work we aimed to describe the machinery required for OMV biogenesis in this bacterium. Methods: The protein composition of P. gingivalis OMV was examined by proteomic analysis to determine whether OMV specific proteins (not detectable in the bacterial outer membrane) were present as these may be involved in OMV formation. Targetted gene mutagenesis b...
Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPS), O-LPS and A-LPS. The structur... more Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPS), O-LPS and A-LPS. The structure of the core oligosaccharide (OS) of O-LPS and the attachment-site of O-polysaccharide (PS) repeating unit [ →3)-α-d-Galp-(1→6)-α-d-Glcp-(1→4)-α-l-Rhap-(1→3)-β-d-GalNAcp-(1→] to the core, have been elucidated using mutant strains ΔPG1051(WaaL, O-antigen ligase) and ΔPG1142 (Wzy, O-antigen polymerase) respectively. The core OS occurs as an "uncapped" glycoform devoid of O-PS and a "capped" glycoform which contains the attachment-site of O-PS via β-d-GalNAc at position O-3 of the terminal α-(1→3)-linked mannose (Man) residue. In this study, the attachment site of A-PS to core-OS was determined based on structural analysis of SR-type-LPS [O-LPS and A-LPS] isolated from P. gingivalis mutant strain ΔPG1142 by extraction with aqueous hot-phenol to minimize the destruction of A-LPS. Application of one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) in com...
Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal ... more Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N- and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in the processing of KGP. A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) an...
Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the a... more Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either N-acylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5alpha. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant pro...
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