Characterization of the  - and  -Mannosidases of Porphyromonas gingivalis

Rangarajan, Minnie; Aduse‐Opoku, Joseph; Hashim, Ahmed; Paramonov, Nikolay A.; Curtis, Michael A.

doi:10.1128/jb.00898-13

Cited by 19 publications

(16 citation statements)

References 38 publications

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“…Inactivation of PG1711 in P. gingivalis has no effect on the synthesis of O-LPS, A-LPS, and the Arg gingipains (20). In this study, therefore, we focused upon the influence of PG0129 on core OS biosynthesis and O-and A-LPS formation.…”

Section: Discussionmentioning

confidence: 99%

“…Complementation of the ⌬PG0129 mutant strain was performed by initial cloning of PG0129 possessing an additional 550 bp of DNA at the 5= end into the NotI-BglII sites of pUCET1 (20) via high-fidelity DNA amplification, manipulation, and cloning. Integration of the appropriate part of the resulting plasmid (pExp129) into the genome of PG0129 was confirmed by PCR using a combination of primer sets (see Table S1 in the supplemental material) (6).…”

Section: Methodsmentioning

confidence: 99%

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Identification of the Linkage between A-Polysaccharide and the Core in the A-Lipopolysaccharide of Porphyromonas gingivalis W50

et al. 2015

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Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. The structure of the core oligosaccharide (OS) of O-LPS and the attachment site of the O-polysaccharide (O-PS) repeating unit [¡3)-␣-D-Galp-(1¡6)-␣-D-Glcp-(1¡4)-␣-L-Rhap-(1¡3)-␤-D-GalNAcp-(1¡ Porphyromonas gingivalis, a Gram-negative anaerobe, is frequently isolated from the subgingival plaque of periodontitis patients and is considered to be an important etiologic agent in periodontal disease. Among the major virulence factors of the organism are the cysteine proteases Arg gingipains (Rgps) and Lys gingipain (Kgp), specific for Arg-X and Lys-X peptide bonds, respectively, which are capable of degrading several host proteins (1), and lipopolysaccharide (LPS), which has the potential to alter the responses of the host innate defense system specifically through its lipid A component (2, 3).P. gingivalis W50 synthesizes two LPSs, namely O-LPS and A-LPS (4-6). Monoclonal antibody (MAb) 1B5, raised against one (RgpA cat ) of the isoforms of Arg gingipain, which are differentially glycosylated proteins, cross-reacts with A-LPS, suggesting that they share an epitope (7). Our group has identified the Man␣1-2Man␣1-phosphate fragment present in A-polysaccharide (A-PS) as part of the epitope recognized by MAb 1B5 (5). Thus, there appear to be common steps in the biosynthesis of A-LPS and the glycosylation of Arg gingipains in P. gingivalis.Analysis of an R-type LPS isolated from the P. gingivalis ⌬PG1051 mutant strain (waaL, encoding O-antigen ligase) enabled us to solve the structure of the core region of LPS (Fig. 1) (8).The inner core region lacks L-(D)-glycero-D(L)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-D-mannooctulosonic acid (Kdo), which is attached to a glycerol (Gro) residue in the outer core via a monophosphodiester bridge (8). The outer region of the "uncapped core" is composed of a linear ␣-(1¡3)-linked D-Man OS with four or five members, one half of which are modified by phosphoethanolamine (PEA) at position O-6, which is attached to the glycerol at position O-2. In addition,

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification of the Linkage between A-Polysaccharide and the Core in the A-Lipopolysaccharide of Porphyromonas gingivalis W50

et al. 2015

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“…A conserved core oligosaccharide is linked to lipid A via 3-deoxy-D-manno-octulosonic acid, and in the case of P. gingivalis this core has been shown to consist of mannose, allosamine, glycerol, and phosphoethanolamine (7). Attached to this core is a high-molecular-weight polysaccharide of one of two clearly distinguishable types, generating two classes of LPS molecules (8,9).…”

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confidence: 99%

Deletion of a 77-Base-Pair Inverted Repeat Element Alters the Synthesis of Surface Polysaccharides in Porphyromonas gingivalis

et al. 2015

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Bacterial cell surface glycans, such as capsular polysaccharides and lipopolysaccharides (LPS), influence host recognition and are considered key virulence determinants. The periodontal pathogen Porphyromonas gingivalis is known to display at least three different types of surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown that PG0121 (in strain W83) encodes a DNABII histone-like protein and that this gene is transcriptionally linked to the K-antigen capsule synthesis genes, generating a large ϳ19.4-kb transcript (PG0104-PG0121). Furthermore, production of capsule is deficient in a PG0121 mutant strain. In this study, we report on the identification of an antisense RNA (asRNA) molecule located within a 77-bp inverted repeat (77bpIR) element located near the 5= end of the locus. We show that overexpression of this asRNA decreases the amount of capsule produced, indicating that this asRNA can impact capsule synthesis in trans. We also demonstrate that deletion of the 77bpIR element and thereby synthesis of the large 19.4-kb transcript also diminishes, but does not eliminate, capsule synthesis. Surprisingly, LPS structures were also altered by deletion of the 77bpIR element, and reactivity to monoclonal antibodies specific to both O-LPS and A-LPS was eliminated. Additionally, reduced reactivity to these antibodies was also observed in a PG0106 mutant, indicating that this putative glycosyltransferase, which is required for capsule synthesis, is also involved in LPS synthesis in strain W83. We discuss our finding in the context of how DNABII proteins, an antisense RNA molecule, and the 77bpIR element may modulate expression of surface polysaccharides in P. gingivalis. IMPORTANCEThe periodontal pathogen Porphyromonas gingivalis displays at least three different types of cell surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown using Northern analysis that the K-antigen capsule locus encodes a large transcript (ϳ19.4 kb), encompassing a 77-bp inverted repeat (77bpIR) element near the 5= end. Here, we report on the identification of an antisense RNA (asRNA) encoded within the 77bpIR. We show that overexpression of this asRNA or deletion of the element decreases the amount of capsule. LPS structures were also altered by deletion of the 77bpIR, and reactivity to monoclonal antibodies to both O-LPS and A-LPS was eliminated. Our data indicate that the 77bpIR element is involved in modulating both LPS and capsule synthesis in P. gingivalis. E ncapsulation is a well-known mechanism that protects pathogenic bacteria from clearance by host immune defenses (1). This reduction in clearance can lead to persistent survival and thereby long-term interplay between the bacterium and host. Capsules not only reduce the ability of the host effectors to gain access to the bacterial cell but also mask the cell surface and thereby modulate the host's response to the bacterium. This model is supported by studies showing that encapsulated bacteria have an advantage in immune evasion (2). Synthesis of a...

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“…The P. gingivalis Δ PG902 and Δ PG1711 mutant strains have been described elsewhere (24). Growth of P.…”

Section: Methodsmentioning

confidence: 99%

LptO (PG0027) Is Required for Lipid A 1-Phosphatase Activity in Porphyromonas gingivalis W50

et al. 2017

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Porphyromonas gingivalis produces outer membrane vesicles (OMVs) rich in virulence factors, including cysteine proteases and A-LPS, one of the two lipopolysaccharides (LPSs) produced by this organism. Previous studies had suggested that A-LPS and PG0027, an outer membrane (OM) protein, may be involved in OMV formation. Their roles in this process were examined by using W50 parent and the ΔPG0027 mutant strains. Inactivation of PG0027 caused a reduction in the yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and the ΔPG0027 mutant strains were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Lipid A from W50 cells contained bis-P-pentaacyl, mono-P-pentaacyl, mono-P-tetraacyl, non-P-pentaacyl, and non-P-tetraacyl species, whereas lipid A from ΔPG0027 mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl (m/z 1,688) and mono-P-tetraacyl (m/z 1,448) lipid A from ΔPG0027 showed that both contained lipid A 1-phosphate, suggesting that the ΔPG0027 mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the ΔPG0027 mutant strains were similar, whereas the phosphatase activity in the periplasm of the ΔPG0027 mutant was lower than that in W50, supporting a role for PG0027 in lipid A dephosphorylation. W50 OMVs were enriched in A-LPS, and its lipid A did not contain nonphosphorylated species, whereas lipid A from the ΔPG0027 mutant (OMVs and cells) contained similar species. Thus, OMVs in P. gingivalis are apparently formed in regions of the OM enriched in A-LPS devoid of nonphosphorylated lipid A. Conversely, dephosphorylation of lipid A through a PG0027-dependent process is required for optimal formation of OMVs. Hence, the relative proportions of nonphosphorylated and phosphorylated lipid A appear to be crucial for OMV formation in this organism.IMPORTANCE Gram-negative bacteria produce outer membrane vesicles (OMVs) by “blebbing” of the outer membrane (OM). OMVs can be used offensively as delivery systems for virulence factors and defensively to aid in the colonization of a host and in the survival of the bacterium in hostile environments. Earlier studies using the oral anaerobe Porphyromonas gingivalis as a model organism to study the mechanism of OMV formation suggested that the OM protein PG0027 and one of the two lipopolysaccharides (LPSs) synthesized by this organism, namely, A-LPS, played important roles in OMV formation. We suggest a novel mechanism of OMV formation in P. gingivalis involving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs.

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Characterization of the - and -Mannosidases of Porphyromonas gingivalis

Cited by 19 publications

References 38 publications

Identification of the Linkage between A-Polysaccharide and the Core in the A-Lipopolysaccharide of Porphyromonas gingivalis W50

Identification of the Linkage between A-Polysaccharide and the Core in the A-Lipopolysaccharide of Porphyromonas gingivalis W50

Deletion of a 77-Base-Pair Inverted Repeat Element Alters the Synthesis of Surface Polysaccharides in Porphyromonas gingivalis

LptO (PG0027) Is Required for Lipid A 1-Phosphatase Activity in Porphyromonas gingivalis W50

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