scholarly journals Identification of a Second Lipopolysaccharide in Porphyromonas gingivalis W50

2008 ◽  
Vol 190 (8) ◽  
pp. 2920-2932 ◽  
Author(s):  
Minnie Rangarajan ◽  
Joseph Aduse-Opoku ◽  
Nikolay Paramonov ◽  
Ahmed Hashim ◽  
Nagihan Bostanci ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Lipid A ◽  
Fatty Acid Analysis ◽  
Chemical Extraction ◽  
Resonance Spectroscopy ◽  
Cell Integrity ◽  
O Antigen ◽  
Flight Mass Spectrometry ◽  
Anchoring System ◽  
A Cell

ABSTRACT We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction. 1H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.

scholarly journals Identification of a Gingipain-Sensitive Surface Ligand of Porphyromonas gingivalis That Induces Toll-Like Receptor 2- and 4-Independent NF-κB Activation in CHO Cells

2009 ◽  
Vol 77 (10) ◽  
pp. 4414-4420 ◽  
Author(s):  
Koki Haruyama ◽  
Atsutoshi Yoshimura ◽  
Mariko Naito ◽  
Mami Kishimoto ◽  
Mikio Shoji ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Periodontal Pathogen ◽  
Mass Spectrometry Analysis ◽  
Deletion Mutants ◽  
Toll Like Receptor ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Virulence Potential ◽  
A Cell ◽  
Toll Like Receptor 2

ABSTRACT Porphyromonas gingivalis is a major periodontal pathogen that has the pathogenic proteinases Arg-specific gingipain and Lys-specific gingipain. We previously found that a cell surface component on P. gingivalis is able to induce Toll-like receptor 2 (TLR2)- and TLR4-independent signaling in 7.19 cells and that this component can be degraded by gingipains. In this study, we purified this component from the P. gingivalis gingipain-null mutant KDP136 and obtained two candidate proteins. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis showed that the proteins, with molecular masses of 123 and 43 kDa, were encoded by PGN_0748 and PGN_0728 (pgm6), respectively, in the P. gingivalis ATCC 33277 genome sequence. The PGN_0748-encoded protein, which we refer to as gingipain-sensitive ligand A (GslA), reacted with antiserum that could effectively inhibit the activity of KDP136 to induce NF-κB activation in 7.19 cells, but Pgm6 did not. To further determine what protein is responsible for the NF-κB activation, we constructed gslA, pgm6, and pgm6 pgm7 deletion mutants from KDP136. When 7.19 cells were exposed to those mutants, the gslA deletion mutant did not induce NF-κB activation, whereas the pgm6 and pgm6 pgm7 deletion mutants did. Furthermore, NF-κB activation in 7.19 cells induced by KDP136 was partially inhibited by antiserum against a recombinant protein expressed from the 5′-terminal third of gslA. These results indicate that GslA is one of the factors that induce NF-κB activation in 7.19 cells. Interestingly, the gslA gene was present in four of seven P. gingivalis strains tested. This restricted distribution might be associated with the virulence potential of each strain.

scholarly journals Structural Analysis of the Core Region of O-Lipopolysaccharide of Porphyromonas gingivalis from Mutants Defective in O-Antigen Ligase and O-Antigen Polymerase

2009 ◽  
Vol 191 (16) ◽  
pp. 5272-5282 ◽  
Author(s):  
Nikolay A. Paramonov ◽  
Joseph Aduse-Opoku ◽  
Ahmed Hashim ◽  
Minnie Rangarajan ◽  
Michael A. Curtis
Keyword(s):  
Porphyromonas Gingivalis ◽  
Inner Core ◽  
Outer Region ◽  
Outer Core ◽  
Core Region ◽  
Resonance Spectroscopy ◽  
Core Oligosaccharide ◽  
Unusual Structure ◽  
The Core ◽  
O Antigen

ABSTRACT Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. Here, we elucidate the structure of the core oligosaccharide (OS) of O-LPS from two mutants of P. gingivalis W50, ΔPG1051 (WaaL, O-antigen ligase) and ΔPG1142 (O-antigen polymerase), which synthesize R-type LPS (core devoid of O antigen) and SR-type LPS (core plus one repeating unit of O antigen), respectively. Structural analyses were performed using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy in combination with composition and methylation analysis. The outer core OS of O-LPS occurs in two glycoforms: an “uncapped core,” which is devoid of O polysaccharide (O-PS), and a “capped core,” which contains the site of O-PS attachment. The inner core region lacks l(d)-glycero-d(l)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-d-manno-octulosonic acid, which is attached to a glycerol residue in the outer core via a monophosphodiester bridge. The outer region of the “uncapped core” is attached to the glycerol and is composed of a linear α-(1→3)-linked d-Man OS containing four or five mannopyranosyl residues, one-half of which are modified by phosphoethanolamine at position 6. An amino sugar, α-d-allosamine, is attached to the glycerol at position 3. In the “capped core,” there is a three- to five-residue extension of α-(1→3)-linked Man residues glycosylating the outer core at the nonreducing terminal residue. β-d-GalNAc from the O-PS repeating unit is attached to the nonreducing terminal Man at position 3. The core OS of P. gingivalis O-LPS is therefore a highly unusual structure, and it is the basis for further investigation of the mechanism of assembly of the outer membrane of this important periodontal bacterium.

scholarly journals Identification of Proteus mirabilisMutants with Increased Sensitivity to Antimicrobial Peptides

2001 ◽  
Vol 45 (7) ◽  
pp. 2030-2037 ◽  
Author(s):  
Andrea J. McCoy ◽  
Hongjian Liu ◽  
Timothy J. Falla ◽  
John S. Gunn
Keyword(s):  
Antimicrobial Peptides ◽  
Lipid A ◽  
Bacterial Species ◽  
Polymyxin B ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Swarming Motility ◽  
Spectrometry Analysis ◽  
O Antigen ◽  
Flight Mass Spectrometry

ABSTRACT Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example,Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilismutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility.

scholarly journals Analyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 197
Author(s):  
Nobuyuki Okahashi ◽  
Masahiro Ueda ◽  
Fumio Matsuda ◽  
Makoto Arita
Keyword(s):  
Mass Spectrometry ◽  
Immune Response ◽  
Liquid Chromatography ◽  
Mammalian Cells ◽  
Lipid A ◽  
Acyl Chain ◽  
Time Of Flight ◽  
Intestinal Bacteria ◽  
Gram Negative ◽  
Flight Mass Spectrometry

Lipid A is a characteristic molecule of Gram-negative bacteria that elicits an immune response in mammalian cells. The presence of structurally diverse lipid A types in the human gut bacteria has been suggested before, and this appears associated with the immune response. However, lipid A structures and their quantitative heterogeneity have not been well characterized. In this study, a method of analysis for lipid A using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was developed and applied to the analyses of Escherichia coli and Bacteroidetes strains. In general, phosphate compounds adsorb on stainless-steel piping and cause peak tailing, but the use of an ammonia-containing alkaline solvent produced sharp lipid A peaks with high sensitivity. The method was applied to E. coli strains, and revealed the accumulation of lipid A with abnormal acyl side chains in knockout strains as well as known diphosphoryl hexa-acylated lipid A in a wild-type strain. The analysis of nine representative strains of Bacteroidetes showed the presence of monophosphoryl penta-acylated lipid A characterized by a highly heterogeneous main acyl chain length. Comparison of the structures and amounts of lipid A among the strains suggested a relationship between lipid A profiles and the phylogenetic classification of the strains.

scholarly journals Synthetic tetra-acylated derivatives of lipid A from Porphyromonas gingivalis are antagonists of human TLR4

2008 ◽  
Vol 6 (18) ◽  
pp. 3371 ◽  
Author(s):  
Yanghui Zhang ◽  
Jidnyasa Gaekwad ◽  
Margreet A. Wolfert ◽  
Geert-Jan Boons
Keyword(s):  
Porphyromonas Gingivalis ◽  
Lipid A ◽  
Derivatives Of

Antimicrobial activity and active compounds of a Rhus verniciflua Stokes extract

2018 ◽  
Vol 73 (11-12) ◽  
pp. 457-463
Author(s):  
Jinfeng Yang ◽  
Yong Soo Kwon ◽  
Myong Jo Kim
Keyword(s):  
Antimicrobial Properties ◽  
Antimicrobial Activities ◽  
Penicillium Oxalicum ◽  
Resonance Spectroscopy ◽  
Trichoderma Virens ◽  
Traditional Herbal Medicine ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Rhus Verniciflua Stokes ◽  
Rhus Verniciflua

Abstract The Rhus verniciflua Stokes (RVS) extract is used as a traditional herbal medicine in Southeast Asian countries such as Korea and China. In the present study, one phenolic acid and six flavonoids were isolated from an 80% ethanol RVS extract to examine their antimicrobial activities. These compounds were identified as 3′,4′,7-trihydroxyflavone (1), methyl gallate (2), gallic acid (3), fusti (4), fisetin (5), butin (6), and sulfuretin (7) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. The antimicrobial activities of compounds 5 and 6 (at a dose of 16 μg/mL each) were superior to that of the control, cycloheximide (at a dose of 25 μg/mL), against Hypocrea nigricans; additionally, the activities of compounds 1 and 2 (at a dose of 8 μg/mL each) were superior to the control against Penicillium oxalicum. Also, chemical compounds 1 and 5 (at a dose of 16 μg/mL each) had higher activities than the control (25 μg/mL) against Trichoderma virens. Chemical compound 1 (at a dose of 8 μg/mL) had a similar activity to that of the control against Bacillus subtilis. The obtained results suggest that the RVS extract could be a promising food and nutraceutical source because of the antimicrobial properties of its phenolic compounds.

Ruthenium red: a highly efficient and versatile cell staining agent for single-cell analysis using inductively coupled plasma time-of-flight mass spectrometry

The Analyst ◽  
2021 ◽  
Author(s):  
Wen Qin ◽  
Hans-Joachim Stärk ◽  
Thorsten Reemtsma
Keyword(s):  
Inductively Coupled Plasma ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Ruthenium Red ◽  
Cell Analysis ◽  
Inductively Coupled ◽  
Flight Mass Spectrometry ◽  
Cell Staining ◽  
A Cell ◽  
Tof Ms

A new method for determining the concentration of elements in single cells by the SC-ICP-TOF-MS method based on a metal-containing stain as a cell volume proxy has been developed and validated.

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