The occurrence of carbapenem- and colistin-resistance among Gram-negative bacteria is increasing ... more The occurrence of carbapenem- and colistin-resistance among Gram-negative bacteria is increasing worldwide. The aim of this study was to understand the distribution of carbapenem- and colistin-resistance in two areas in Tamil Nadu, India. The clinical isolates (n=89) used in this study were collected from two diagnostic centres in Tamil Nadu, India. The bacterial isolates were screened for meropenem- and colistin-resistance. Further, resistance genes blaNDM-1, blaOXA-48-like, blaIMP, blaVIM, blaKPC, mcr-1 and mcr-2 and integrons were studied. The synergistic effect of meropenem in combination with colistin was assessed. A total of 89 bacterial isolates were studied which included Escherichia coli (n=43), Klebsiella pneumoniae (n=18), Pseudomonas aeruginosa (n=10), Enterobacter cloacae (n=6), Acinetobacter baumannii (n=5), Klebsiella oxytoca (n=4), Proteus mirabilis (n=2) and Salmonella paratyphi (n=1). MIC testing showed that 58/89 (65 %) and 29/89 (32 %) isolates were resistant to ...
Previous studies have shown that VimA, an acetyltransferase, can modulate gingipain biogenesis in... more Previous studies have shown that VimA, an acetyltransferase, can modulate gingipain biogenesis in Porphyromonas gingivalis. Inactivation of the vimA gene resulted in isogenic mutants that showed a late onset of gingipain activity that only occurred during the stationary growth phase. To further elucidate the role and contribution of the gingipains in this VimA-dependent process, isogenic mutants defective in the gingipain genes in the vimA-deficient genetic background were evaluated. In contrast to the wild-type strain, RgpB and Kgp gingipain activities were absent in exponential phase in the ∆rgpA::tetQ-vimA::ermF mutant. However, these activities increased to 31% and 53%, respectively, of that of the wild-type during stationary phase. In the ∆rgpA::cat-∆kgp::tetQ-vimA::ermF mutant, the RgpB protein was observed in the extracellular fraction but no activity was present even at the stationary growth phase. There was no gingipain activity observed in the ∆rgpB::cat-∆kgp::tetQ-vimA::e...
Three fowl adenovirus 4 (FAV4) isolates from chicken and one from quail, all from Tamil Nadu, Ind... more Three fowl adenovirus 4 (FAV4) isolates from chicken and one from quail, all from Tamil Nadu, India were analyzed. The L1 loop variable region of hexon gene of these isolates was amplified by PCR and sequenced. The nucleotide sequences (442 bp) and deduced amino acid sequences of the four isolates were compared with those of other isolates of FAV4. The nucleotide sequences of the four isolates had a 98% homology with other Indian isolates and a 96% homology with Belgian and Russian isolates. The amino acid sequences of the four Indian isolates had a more than 98% homology with other Indian isolates and a more than 92% homology with Belgian and Russian isolates. Hence, the variable of L1 loop region of hexon gene was found to be highly homologous in all the FAV4 isolates tested both at nucleotide and amino acid level.
Previous studies have shown that the Porphyromonas gingivalis vimA gene plays a significant role ... more Previous studies have shown that the Porphyromonas gingivalis vimA gene plays a significant role in gingipain biogenesis. In contrast to the wild-type strain, a 90% reduction was seen in both Arg-x- (Rgp) and Lys-x- (Kgp) specific gingipain activities in the vimA-defective mutant (FLL92) during the exponential growth phase. These activities, however, increased to approximately 60% of that of the wild-type strain during stationary phase. The Rgp is encoded by two genes rgpA and rgpB. The source of the activation is unclear since RgpA and RgpB can contribute to the Arg-x-specific gingipain activities and only RgpB was membrane associated in the FLL92. Objective: To identify the source of the late onset gingipain activity in the P. gingivalis vimA-defective mutant. Methods: Allelic exchange mutagenesis was used to inactivate the rgpA, and rgpB genes in P. gingivalis FLL92. Whole cell and extracellular protein fractions were assayed for gingipain activity using N-p-benzoyl-DL-arginine p...
Introduction: The VimA protein of Porphyromonas gingivalis plays a significant role in several ce... more Introduction: The VimA protein of Porphyromonas gingivalis plays a significant role in several cellular processes including: gingipain maturation, fimbrial synthesis, lipid polysaccharide polymerization and anchorage of outer-membrane proteins. In silico analysis predicts the VimA protein is part of the FemXAB non-ribosomal peptidyltransferase family which is involved in peptidoglycan synthesis and the DUF482 Super-family which is involved in protein sorting. Objective: To evaluate the role of the VimA in peptidoglycan biogenesis and protein sorting. Methods: The peptidoglycan of the vimA mutant and wild-type W83 was isolated and purified from cells grown to log and stationary growth phase. Peptidoglycan thickness and elasticity was measured using atomic force microscopy. The chemical composition of the peptidoglycan as well as the O-Acetylation profile was examined using HPLC-based organic acid analysis. Mass Spectrometry was used to identify aberrantly expressed extracellular prot...
Introduction: Gingipains are major virulence factors produced by Porphyromonas gingivalis, an imp... more Introduction: Gingipains are major virulence factors produced by Porphyromonas gingivalis, an important etiological agent of adult periodontitis. Several genes are involved in the activation/maturation of these gingipains. Previous studies have shown that inactivation of vimF, a putative glycosyltransferase gene, alters glycosylation and activation of the gingipains in Porphyromonas gingivalis W83. Objective: To determine the cellular localization of the VimF protein. Methods: The vimF gene was cloned with a 6x His tag at the C terminal end using pTrcHis-2 expression vector and expressed in E.coli. The recombinant protein, rVimF, was purified using nickel column and an anti-VimF antibody was raised in rabbits. This antibody was used to localize VimF in Porphyromonas gingivalis using Immunoelectron Microscopy (IEM) and western blot analysis. Results: Bioinformatic analysis suggests the VimF protein to be a secreted glycosyltransferase 1 with a GTB type fold. IEM using anti-rVimF anti...
PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis,... more PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P. gingivalis FLL350 showed significant reduction in gingipain activity compared to the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P. gingivalis, its genetic properties were further characterized. A 5'-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off in vitro transcription assay using the purified rPG0162 and RNAP core enzyme from E.coli with the PG0162 promoter as template. Since an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P. gingivalis FLL391 was created. Compared to the wild-type strain, transcriptome analysis of P. gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than 2 fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P. gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P. gingivalis. This article is protected by copyright. All rights reserved.
Introduction: Transcriptome analysis in P. gingivalis demonstrated that oxidative stress can modu... more Introduction: Transcriptome analysis in P. gingivalis demonstrated that oxidative stress can modulate several functional classes of genes depending on the severity and duration of the exposure. There was a 4.0 up-regulation of the hypothetical protein, PG0686, in P. gingivalis in the presence of 0.25 mM hydrogen peroxide for 15 minutes. In silicoanalysis of PG0686 protein identified 6 domains, including hemerythrin, DUF-1858 (domain of unknown function), and a possible sensory box domain. Objective: To study the relative significance of PG0686 in oxidative stress resistance in P. gingivalis. Methods: DNA microarray analysis and Real-time RT-PCR were used to determine gene induction under oxidative stress. Upstream and downstream flanking regions of the 1.5 kb gene were fused with the ermF antibiotic resistance cassette, and used to create a PG0686 deletion mutant, designated FLL361, by allelic exchange mutagenesis. The PG0686 open reading frame was cloned into the pEXP5-NT vector, a...
Introduction: The bcp-recA-vimA-vimF-vimE-aroG operon in Porphyromonas gingivalis is known to be ... more Introduction: The bcp-recA-vimA-vimF-vimE-aroG operon in Porphyromonas gingivalis is known to be important in the regulation of virulence via the posttranslational modification of the gingipains. The aroG gene is further characterized to evaluate its relative significance in this process. In other bacteria this gene encodes for a chorimate mutase which is involved in the synthesis of aromatic amino acids and is important for survival. Objective: We hypothesize that aroG is important in virulence modulation in P. gingivalis. Methods: Bioinformatics analysis and protein modeling were performed to evaluate the structural characteristics of AroG. Overlapping extension PCR was used to fuse flanking upstream and downstream DNA of the aroG gene with the ermF cassette. Allelic exchange mutagenesis was used to inactivate the aroG gene in P. gingivalis W83. Isogenic mutant colonies were confirmed by colony PCR and DNA sequencing. The mutant strain was characterized for their virulence potenti...
Porphyromonas gingivalis, the major etiologic agent in adult periodontitis produces large amounts... more Porphyromonas gingivalis, the major etiologic agent in adult periodontitis produces large amounts of proteases that are important for its survival and pathogenesis. Maturation/activation of gingipains, a major protease, in P. gingivalis involves networks of poorly understood processes. We have previously shown that genes of the vim locus including vimF, a putative glycosyltransferase (GTase) are involved in glycosylation/maturation of gingipains. Objective: To further characterize the putative glycosyltansferase activity of VimF by testing its ability to transfer glycans to P. gingivalis proteins including the gingipains. Methods: The purified rVimF was used in a calorimetric assay to estimate glycosyltransferase activity in the presence of UDP sugars including UDP-galactose, UDP-glucose, UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine as the donor substrate and glucose, galactose, lactose, N-acetyleglucosamine, N-acetylegalactosamine and mannose as acceptor substrate. Results...
Introduction: To survive in the inflammatory environment of the periodontal pocket would suggest ... more Introduction: To survive in the inflammatory environment of the periodontal pocket would suggest Porphyromonas gingivalis can adapt to and protect itself from the deleterious effects of oxidative stress. Transcriptome analysis identified a 2.7 fold up-regulation of PG2212 in P. gingivalis W83 exposed to hydrogen peroxide. Bioinformatics analysis showed PG2212 to be a putative regulatory protein with a Cys2-His2 zinc-finger DNA binding domain. Objective: To evaluate the regulatory transcription properties of PG2212 and assess its role in oxidative stress resistance in P. gingivalis. Method: The PG2212 gene in P. gingivalis was inactive by allelic exchange mutagenesis using the ermF cassette. DNA-microarray and Real-time quantitative PCR were used to evaluate differentially gene expression in the PG2212-defective mutant. EMSA was used to study protein binding to the promoter region of the PG2212 regulated genes. Result: Compared to the parent strain, the PG2212-defective isogenic muta...
The occurrence of carbapenem- and colistin-resistance among Gram-negative bacteria is increasing ... more The occurrence of carbapenem- and colistin-resistance among Gram-negative bacteria is increasing worldwide. The aim of this study was to understand the distribution of carbapenem- and colistin-resistance in two areas in Tamil Nadu, India. The clinical isolates (n=89) used in this study were collected from two diagnostic centres in Tamil Nadu, India. The bacterial isolates were screened for meropenem- and colistin-resistance. Further, resistance genes blaNDM-1, blaOXA-48-like, blaIMP, blaVIM, blaKPC, mcr-1 and mcr-2 and integrons were studied. The synergistic effect of meropenem in combination with colistin was assessed. A total of 89 bacterial isolates were studied which included Escherichia coli (n=43), Klebsiella pneumoniae (n=18), Pseudomonas aeruginosa (n=10), Enterobacter cloacae (n=6), Acinetobacter baumannii (n=5), Klebsiella oxytoca (n=4), Proteus mirabilis (n=2) and Salmonella paratyphi (n=1). MIC testing showed that 58/89 (65 %) and 29/89 (32 %) isolates were resistant to ...
Previous studies have shown that VimA, an acetyltransferase, can modulate gingipain biogenesis in... more Previous studies have shown that VimA, an acetyltransferase, can modulate gingipain biogenesis in Porphyromonas gingivalis. Inactivation of the vimA gene resulted in isogenic mutants that showed a late onset of gingipain activity that only occurred during the stationary growth phase. To further elucidate the role and contribution of the gingipains in this VimA-dependent process, isogenic mutants defective in the gingipain genes in the vimA-deficient genetic background were evaluated. In contrast to the wild-type strain, RgpB and Kgp gingipain activities were absent in exponential phase in the ∆rgpA::tetQ-vimA::ermF mutant. However, these activities increased to 31% and 53%, respectively, of that of the wild-type during stationary phase. In the ∆rgpA::cat-∆kgp::tetQ-vimA::ermF mutant, the RgpB protein was observed in the extracellular fraction but no activity was present even at the stationary growth phase. There was no gingipain activity observed in the ∆rgpB::cat-∆kgp::tetQ-vimA::e...
Three fowl adenovirus 4 (FAV4) isolates from chicken and one from quail, all from Tamil Nadu, Ind... more Three fowl adenovirus 4 (FAV4) isolates from chicken and one from quail, all from Tamil Nadu, India were analyzed. The L1 loop variable region of hexon gene of these isolates was amplified by PCR and sequenced. The nucleotide sequences (442 bp) and deduced amino acid sequences of the four isolates were compared with those of other isolates of FAV4. The nucleotide sequences of the four isolates had a 98% homology with other Indian isolates and a 96% homology with Belgian and Russian isolates. The amino acid sequences of the four Indian isolates had a more than 98% homology with other Indian isolates and a more than 92% homology with Belgian and Russian isolates. Hence, the variable of L1 loop region of hexon gene was found to be highly homologous in all the FAV4 isolates tested both at nucleotide and amino acid level.
Previous studies have shown that the Porphyromonas gingivalis vimA gene plays a significant role ... more Previous studies have shown that the Porphyromonas gingivalis vimA gene plays a significant role in gingipain biogenesis. In contrast to the wild-type strain, a 90% reduction was seen in both Arg-x- (Rgp) and Lys-x- (Kgp) specific gingipain activities in the vimA-defective mutant (FLL92) during the exponential growth phase. These activities, however, increased to approximately 60% of that of the wild-type strain during stationary phase. The Rgp is encoded by two genes rgpA and rgpB. The source of the activation is unclear since RgpA and RgpB can contribute to the Arg-x-specific gingipain activities and only RgpB was membrane associated in the FLL92. Objective: To identify the source of the late onset gingipain activity in the P. gingivalis vimA-defective mutant. Methods: Allelic exchange mutagenesis was used to inactivate the rgpA, and rgpB genes in P. gingivalis FLL92. Whole cell and extracellular protein fractions were assayed for gingipain activity using N-p-benzoyl-DL-arginine p...
Introduction: The VimA protein of Porphyromonas gingivalis plays a significant role in several ce... more Introduction: The VimA protein of Porphyromonas gingivalis plays a significant role in several cellular processes including: gingipain maturation, fimbrial synthesis, lipid polysaccharide polymerization and anchorage of outer-membrane proteins. In silico analysis predicts the VimA protein is part of the FemXAB non-ribosomal peptidyltransferase family which is involved in peptidoglycan synthesis and the DUF482 Super-family which is involved in protein sorting. Objective: To evaluate the role of the VimA in peptidoglycan biogenesis and protein sorting. Methods: The peptidoglycan of the vimA mutant and wild-type W83 was isolated and purified from cells grown to log and stationary growth phase. Peptidoglycan thickness and elasticity was measured using atomic force microscopy. The chemical composition of the peptidoglycan as well as the O-Acetylation profile was examined using HPLC-based organic acid analysis. Mass Spectrometry was used to identify aberrantly expressed extracellular prot...
Introduction: Gingipains are major virulence factors produced by Porphyromonas gingivalis, an imp... more Introduction: Gingipains are major virulence factors produced by Porphyromonas gingivalis, an important etiological agent of adult periodontitis. Several genes are involved in the activation/maturation of these gingipains. Previous studies have shown that inactivation of vimF, a putative glycosyltransferase gene, alters glycosylation and activation of the gingipains in Porphyromonas gingivalis W83. Objective: To determine the cellular localization of the VimF protein. Methods: The vimF gene was cloned with a 6x His tag at the C terminal end using pTrcHis-2 expression vector and expressed in E.coli. The recombinant protein, rVimF, was purified using nickel column and an anti-VimF antibody was raised in rabbits. This antibody was used to localize VimF in Porphyromonas gingivalis using Immunoelectron Microscopy (IEM) and western blot analysis. Results: Bioinformatic analysis suggests the VimF protein to be a secreted glycosyltransferase 1 with a GTB type fold. IEM using anti-rVimF anti...
PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis,... more PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P. gingivalis FLL350 showed significant reduction in gingipain activity compared to the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P. gingivalis, its genetic properties were further characterized. A 5'-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off in vitro transcription assay using the purified rPG0162 and RNAP core enzyme from E.coli with the PG0162 promoter as template. Since an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P. gingivalis FLL391 was created. Compared to the wild-type strain, transcriptome analysis of P. gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than 2 fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P. gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P. gingivalis. This article is protected by copyright. All rights reserved.
Introduction: Transcriptome analysis in P. gingivalis demonstrated that oxidative stress can modu... more Introduction: Transcriptome analysis in P. gingivalis demonstrated that oxidative stress can modulate several functional classes of genes depending on the severity and duration of the exposure. There was a 4.0 up-regulation of the hypothetical protein, PG0686, in P. gingivalis in the presence of 0.25 mM hydrogen peroxide for 15 minutes. In silicoanalysis of PG0686 protein identified 6 domains, including hemerythrin, DUF-1858 (domain of unknown function), and a possible sensory box domain. Objective: To study the relative significance of PG0686 in oxidative stress resistance in P. gingivalis. Methods: DNA microarray analysis and Real-time RT-PCR were used to determine gene induction under oxidative stress. Upstream and downstream flanking regions of the 1.5 kb gene were fused with the ermF antibiotic resistance cassette, and used to create a PG0686 deletion mutant, designated FLL361, by allelic exchange mutagenesis. The PG0686 open reading frame was cloned into the pEXP5-NT vector, a...
Introduction: The bcp-recA-vimA-vimF-vimE-aroG operon in Porphyromonas gingivalis is known to be ... more Introduction: The bcp-recA-vimA-vimF-vimE-aroG operon in Porphyromonas gingivalis is known to be important in the regulation of virulence via the posttranslational modification of the gingipains. The aroG gene is further characterized to evaluate its relative significance in this process. In other bacteria this gene encodes for a chorimate mutase which is involved in the synthesis of aromatic amino acids and is important for survival. Objective: We hypothesize that aroG is important in virulence modulation in P. gingivalis. Methods: Bioinformatics analysis and protein modeling were performed to evaluate the structural characteristics of AroG. Overlapping extension PCR was used to fuse flanking upstream and downstream DNA of the aroG gene with the ermF cassette. Allelic exchange mutagenesis was used to inactivate the aroG gene in P. gingivalis W83. Isogenic mutant colonies were confirmed by colony PCR and DNA sequencing. The mutant strain was characterized for their virulence potenti...
Porphyromonas gingivalis, the major etiologic agent in adult periodontitis produces large amounts... more Porphyromonas gingivalis, the major etiologic agent in adult periodontitis produces large amounts of proteases that are important for its survival and pathogenesis. Maturation/activation of gingipains, a major protease, in P. gingivalis involves networks of poorly understood processes. We have previously shown that genes of the vim locus including vimF, a putative glycosyltransferase (GTase) are involved in glycosylation/maturation of gingipains. Objective: To further characterize the putative glycosyltansferase activity of VimF by testing its ability to transfer glycans to P. gingivalis proteins including the gingipains. Methods: The purified rVimF was used in a calorimetric assay to estimate glycosyltransferase activity in the presence of UDP sugars including UDP-galactose, UDP-glucose, UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine as the donor substrate and glucose, galactose, lactose, N-acetyleglucosamine, N-acetylegalactosamine and mannose as acceptor substrate. Results...
Introduction: To survive in the inflammatory environment of the periodontal pocket would suggest ... more Introduction: To survive in the inflammatory environment of the periodontal pocket would suggest Porphyromonas gingivalis can adapt to and protect itself from the deleterious effects of oxidative stress. Transcriptome analysis identified a 2.7 fold up-regulation of PG2212 in P. gingivalis W83 exposed to hydrogen peroxide. Bioinformatics analysis showed PG2212 to be a putative regulatory protein with a Cys2-His2 zinc-finger DNA binding domain. Objective: To evaluate the regulatory transcription properties of PG2212 and assess its role in oxidative stress resistance in P. gingivalis. Method: The PG2212 gene in P. gingivalis was inactive by allelic exchange mutagenesis using the ermF cassette. DNA-microarray and Real-time quantitative PCR were used to evaluate differentially gene expression in the PG2212-defective mutant. EMSA was used to study protein binding to the promoter region of the PG2212 regulated genes. Result: Compared to the parent strain, the PG2212-defective isogenic muta...
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