Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane β-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.     

Characterization of the α- and β-Mannosidases of Porphyromonas gingivalis

Mannose is an important sugar in the biology of the Gram-negative bacterium Porphyromonas gingivalis. It is a major component of the oligosaccharides attached to the Arg-gingipain cysteine proteases, the repeating units of an acidic lipopolysaccharide (A-LPS), and the core regions of both types of LPS produced by the organism (O-LPS and A-LPS) and a reported extracellular polysaccharide (EPS) isolated from spent culture medium. The organism occurs at inflamed sites in periodontal tissues, where it is exposed to host glycoproteins rich in mannose, which may be substrates for the acquisition of mannose by P. gingivalis. Five potential mannosidases were identified in the P. gingivalis W83 genome that may play a role in mannose acquisition. Four mannosidases were characterized in this study: PG0032 was a β-mannosidase, whereas PG0902 and PG1712 were capable of hydrolyzing p-nitrophenyl α-d-mannopyranoside. PG1711 and PG1712 were α-1→3 and α-1→2 mannosidases, respectively. No enzyme function could be assigned to PG0973. α-1→6 mannobiose was not hydrolyzed by P. gingivalis W50. EPS present in the culture supernatant was shown to be identical to yeast mannan and a component of the medium used for culturing P. gingivalis and was resistant to hydrolysis by mannosidases. Synthesis of O-LPS and A-LPS and glycosylation of the gingipains appeared to be unaffected in all mutants. Thus, α- and β-mannosidases of P. gingivalis are not involved in the harnessing of mannan/mannose from the growth medium for these biosynthetic processes. P. gingivalis grown in chemically defined medium devoid of carbohydrate showed reduced α-mannosidase activity (25%), suggesting these enzymes are environmentally regulated.     

Molecular Strategies Underlying Porphyromonas gingivalis Virulence

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is considered the keystone of periodontitis diseases, a set of inflammatory conditions that affects the tissues surrounding the teeth. In the recent years, the major virulence factors exploited by P. gingivalis have been identified and characterized, including a cocktail of toxins, mainly proteases called gingipains, which promote gingival tissue invasion. These effectors use the Sec pathway to cross the inner membrane and are then recruited and transported across the outer membrane by the type IX secretion system (T9SS). In P. gingivalis, most secreted effectors are attached to anionic lipopolysaccharides (A-LPS), and hence form a virulence coat at the cell surface. P. gingivalis produces additional virulence factors to evade host immune responses, such as capsular polysaccharide, fimbriae and outer membrane vesicles. In addition to periodontitis, it is proposed that this broad repertoire of virulence factors enable P. gingivalis to be involved in diverse human diseases such as rheumatoid arthritis, and neurodegenerative, Alzheimer, and cardiovascular disorders. Here, we review the major virulence determinants of P. gingivalis and discuss future directions to better understand their mechanisms of action.     

Structural and functional analyses of the Porphyromonas gingivalis type IX secretion system PorN protein

Porphyromonas gingivalis, the major human pathogen bacterium associated with periodontal diseases, secretes virulence factors through the Bacteroidetes-specific type IX secretion system (T9SS). Effector proteins of the T9SS are recognized by the complex via their conserved C-terminal domains (CTDs). Among the 18 proteins essential for T9SS function in P. gingivalis, PorN is a periplasmic protein that forms large ring-shaped structures in association with the PorK outer membrane lipoprotein. PorN also mediates contacts with the PorM subunit of the PorLM energetic module, and with the effector’s CTD. However, no information is available on the PorN structure and on the implication of PorN domains for T9SS assembly and effector recognition. Here we present the crystal structure of PorN at 2.0-Å resolution, which represents a novel fold with no significant similarity to any known structure. In agreement with in silico analyses, we also found that the N- and C-terminal regions of PorN are intrinsically disordered. Our functional studies showed that the N-terminal disordered region is involved in PorN dimerization while the C-terminal disordered region is involved in the interaction with PorK. Finally, we determined that the folded PorN central domain is involved in the interaction with PorM, as well as with the effector’s CTD. Altogether, these results lay the foundations for a more comprehensive model of T9SS architecture and effector transport.     

The outer membrane protein LptO is essential for the O-deacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) essential for secretion and attachment to the cell surface. Inactivation of lptO (PG0027) or porT produced mutants that lacked surface protease activity and an electron-dense surface layer. Both mutants showed co-accumulation of A-LPS and unmodified CTD proteins in the periplasm. Lipid profiling by mass spectrometry showed the presence of both tetra- and penta-acylated forms of mono-phosphorylated lipid A in the wild-type and porT mutant, while only the penta-acylated forms of mono-phosphorylated lipid A were found in the lptO mutant, indicating a specific role of LptO in the O-deacylation of mono-phosphorylated lipid A. Increased levels of non-phosphorylated lipid A and the presence of novel phospholipids in the lptO mutant were also observed that may compensate for the missing mono-phosphorylated tetra-acylated lipid A in the outer membrane (OM). Molecular modelling predicted LptO to adopt a β-barrel structure characteristic of an OM protein, supported by the enrichment of LptO in OM vesicles. The results suggest that LPS deacylation by LptO is linked to the co-ordinated secretion of A-LPS and CTD proteins by a novel secretion and attachment system to form a structured surface layer.     

Sequence‐independent processing site of the C‐terminal domain (CTD) influences maturation of the RgpB protease from Porphyromonas gingivalis

The Gram‐negative periodontal pathogen Porphyromonas gingivalis produces a family of outer membrane‐anchored proteases, the gingipains, shown to play an essential role in virulence of the organism. The C‐terminal domain (CTD) of gingipains and other secreted proteins is known to be the targeting signal for maturation and translocation of the protein through the outer membrane. The CTD is subsequently cleaved during the secretion process. Multiple alignment of various CTDs failed to define a consensus sequence at the putative CTD processing site. Using mutagenesis, we were able to show that cleavage at the site is not dependent on a specific residue and that recognition of the site is independent of local sequence. Interestingly, length of the junction between the CTD and adjacent Ig‐like subdomain has a critical influence on post‐translational glycan modification of the protein, whereby insertion of additional residues immediately N‐terminal to the cleavage site results in failure of glycan modification and release of soluble protease into the culture medium. Various hypotheses are presented to explain these phenomena. Knowledge of the role CTDs play in maturation of gingipains has broader application for understanding maturation of CTD homologues expressed by bacteria of the Bacteriodetes phylum.     

Protective Role of the PG1036-PG1037-PG1038 Operon in Oxidative Stress in Porphyromonas gingivalis W83

As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H₂O₂ induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H₂O₂ and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H₂O₂-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.     

PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ～70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.     

Flavobacterium johnsoniae PorV Is Required for Secretion of a Subset of Proteins Targeted to the Type IX Secretion System

Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.     

Flavobacterium johnsoniae Chitinase ChiA Is Required for Chitin Utilization and Is Secreted by the Type IX Secretion System

Flavobacterium johnsoniae, a member of phylum Bacteriodetes, is a gliding bacterium that digests insoluble chitin and many other polysaccharides. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding motility and for chitin utilization. Five potential chitinases were identified by genome analysis. Fjoh_4555 (ChiA), a 168.9-kDa protein with two glycoside hydrolase family 18 (GH18) domains, was targeted for analysis. Disruption of chiA by insertional mutagenesis resulted in cells that failed to digest chitin, and complementation with wild-type chiA on a plasmid restored chitin utilization. Antiserum raised against recombinant ChiA was used to detect the protein and to characterize its secretion by F. johnsoniae. ChiA was secreted in soluble form by wild-type cells but remained cell associated in strains carrying mutations in any of the T9SS genes, gldK, gldL, gldM, gldNO, sprA, sprE, and sprT. Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses suggested that ChiA was proteolytically processed into two GH18 domain-containing proteins. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families TIGR04131 and TIGR04183. ChiA does not exhibit strong similarity to these sequences and instead has a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside cells. Fusion of the ChiA CTD to recombinant mCherry resulted in secretion of mCherry into the medium. The results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS.     

Por secretion system-dependent secretion and glycosylation of Porphyromonas gingivalis hemin-binding protein 35

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study.     

Antibodies That Detect O-Linked β-d-N-Acetylglucosamine on the Extracellular Domain of Cell Surface Glycoproteins

The transfer of N-acetylglucosamine (GlcNAc) to Ser or Thr in cytoplasmic and nuclear proteins is a well known post-translational modification that is catalyzed by the O-GlcNAc transferase OGT. A more recently identified O-GlcNAc transferase, EOGT, functions in the secretory pathway and transfers O-GlcNAc to proteins with epidermal growth factor-like (EGF) repeats. A number of antibodies that detect O-GlcNAc in cytosolic and nuclear extracts have been described previously. Here we compare seven of these antibodies (CTD110.6, 10D8, RL2, HGAC85, 18B10.C7(#3), 9D1.E4(#10), and 1F5.D6 (#14) for detection of the O-GlcNAc modification on extracellular domains of membrane or secreted glycoproteins that may also carry various N- and O-glycans. We found that CTD110.6 binds not only to O-GlcNAc on proteins but also to terminal β-GlcNAc on the complex N-glycans of Lec8 Chinese hamster ovary (CHO) cells that lack UDP-Gal transporter activity and express GlcNAc-terminating, complex N-glycans. We show that CTD110.6, #3, and #10 antibodies can be used to detect cell surface glycoproteins bearing O-GlcNAc. Cell surface glycoproteins recognized by CTD110.6 antibody included NOTCH1 that possesses many EGF repeats with a consensus site for EOGT. Knockdown of CHO Eogt reduced binding of CTD110.6 to Lec1 CHO cells, and expression of a human EOGT cDNA increased the O-GlcNAc signal on Lec1 cells and the extracellular domain of NOTCH1. Thus, with careful controls, antibodies CTD110.6 (IgM), #3 (IgG), and #10 (IgG) can be used to detect membrane and secreted proteins modified by O-GlcNAc on EGF repeats.     

Deletion of the Cytophaga hutchinsonii type IX secretion system gene sprP results in defects in gliding motility and cellulose utilization

Cytophaga hutchinsonii glides rapidly over surfaces and employs a novel collection of cell-associated proteins to digest crystalline cellulose. HimarEm1 transposon mutagenesis was used to isolate a mutant with an insertion in CHU_0170 (sprP) that was partially deficient in gliding motility and was unable to digest filter paper cellulose. SprP is similar in sequence to the Porphyromonas gingivalis type IX secretion system (T9SS) protein PorP that is involved in the secretion of gingipain protease virulence factors and to the Flavobacterium johnsoniae T9SS protein SprF that is needed to deliver components of the gliding motility machinery to the cell surface. We developed an efficient method to construct targeted nonpolar mutations in C. hutchinsonii and deleted sprP. The deletion mutant was defective in gliding and failed to digest cellulose, and complementation with sprP on a plasmid restored both abilities. Sequence analysis predicted that CHU_3105 is secreted by the T9SS, and deletion of sprP resulted in decreased levels of extracellular CHU_3105. The results suggest that SprP may function in protein secretion. The T9SS may be required for motility and cellulose utilization because cell surface proteins predicted to be involved in both processes have C-terminal domains that are thought to target them to this secretion system. The efficient genetic tools now available for C. hutchinsonii should allow a detailed analysis of the cellulolytic, gliding motility, and protein secretion machineries of this common but poorly understood bacterium.     

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

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.Porphyromonas gingivalis is a gram-negative anaerobe which is strongly implicated in the etiology of periodontal disease. Several putative virulence factors are produced by this organism. These virulence factors include 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 (56), and lipopolysaccharide (LPS), which has the potential to cause an inflammatory response in the periodontal tissues of the host. These factors are important antigens in patients with periodontal disease and may account for a considerable proportion of the immune response directed against P. gingivalis (58).LPS is a major constituent of the outer membrane of gram-negative bacteria and facilitates interactions with the external environment. It consists of three regions: a hydrophobic lipid A embedded in the outer leaflet of the outer membrane, a core oligosaccharide (OS), and the O-polysaccharide (O-PS) side chain composed of several repeating units. The hydrophobic lipid A serves as an anchor for the LPS and consists of β-1,6-linked d-glucosamine disaccharide, which is usually phosphorylated at the 1 and/or 4′ positions and N and/or O acylated at positions 2, 3, 2′, and 3′ with various amounts of fatty acids. The rest of the LPS molecule projects from the surface. The core region is attached to lipid A and is composed of ∼10 sugars in most bacteria studied to date and can be further subdivided into an inner core and an outer core. The inner core usually contains l(d)-glycero-d-(l)-manno-heptose and 3-deoxy-d-manno-octulosonic acid (Kdo) residues, whereas the outer core is usually composed of hexoses. Attached to the outer core are the repeating units of O antigen (O-PS), which vary in composition, stereochemistry, and the sequence of O-glycosidic linkages between bacterial strains and thereby give rise to O-serotype specificity within bacterial species. Attachment of O antigen to core lipid A results in “smooth” LPS (S-type LPS), whereas LPS lacking O antigen is “rough” LPS (R-type LPS). Attachment of one repeating unit of O-PS to core lipid A results in SR-LPS (core-plus-one repeating unit) (41, 47, 48). In addition, the outer core OS region can be either “uncapped” or “capped.” The “uncapped” core OS is devoid of O-PS repeating units, whereas the “capped” core OS contains attached O-PS repeating units (47, 53) due to modifications in the outer core region.P. gingivalis W50 was originally thought to synthesize a single LPS composed of a tetrasaccharide repeating unit in the O-PS, [→6)-α-d-Glcp-(1→4)-α-l-Rhap-(1→3)-β-d-GalNAc-(1→3)-α-d-Galp-(1→], which is modified by phosphoethanolamine (PEA) at position 2 of Rha in a nonstoichiometric manner (43). However, a second LPS in this organism, namely A-LPS (49), which has a phosphorylated mannan-containing anionic polysaccharide (A-PS), was identified in our laboratory. The A-PS repeating unit is built up of a phosphorylated branched d-Man-containing oligomer composed of an α1→6-linked d-mannose backbone to which α1→2-linked d-Man side chains of different lengths (one or two residues) are attached at position 2. One of the side chains contains Manα1→2-Manα-1-phosphate linked via phosphorus to a backbone Man residue at position O-2. Although A-LPS is predominantly composed of α-d-mannose residues, it cannot be referred to as a homopolymer due to the presence of Manα1→2Manα1-phosphate-containing OS side chains forming a nonglycosidic linkage between the backbone α-mannose and side chains. Hence, it is likely that the synthesis of A-PS (A-LPS) occurs via a “wzy-dependent” pathway in which repeating units formed on the cytoplasmic face of the inner membrane are polymerized at the periplasmic face following transport or flipping across the cytoplasmic membrane. A-LPS cross-reacts with monoclonal antibody (MAb) 1B5 raised against one of the isoforms of Arg-gingipains, a family of differentially glycosylated cysteine proteases (14, 19). Deglycosylation of the cross-reacting Rgps with anhydrous trifluoromethane sulfonic acid abolishes their immunoreactivity to MAb 1B5, indicating that this antibody recognizes a carbohydrate-containing epitope also present in A-LPS (14, 44). Hence, there appear to be common elements in the biosynthesis of A-LPS and the Arg-gingipains of this organism.Inactivation of P. gingivalis waaL (PG1051, O-antigen ligase) abolishes the synthesis of both O-LPS and A-LPS (49). Hence, the WaaL O-antigen ligase appears to have dual specificity and is capable of ligating both O-PS and A-PS chains to core lipid A. The dual specificity of WaaL in the final step of LPS biosynthesis has also been demonstrated in the synthesis of Escherichia coli O-LPS and M_LPS (38) and for Pseudomonas aeruginosa A-band and B-band LPSs (1).However, the linkage between O-PS and A-PS and core OS has not been identified in P. gingivalis. In this paper, we describe a structural investigation of the core OS of O-LPS in which we used R-LPS prepared from ΔPG1051 (49) and ΔPG1142 (putative O-antigen polymerase), which we hypothesized would synthesize an SR-LPS (core plus one repeating unit) (60). The putative O-antigen polymerase encoded at PG1142 (42) is a phenylalanine-rich membrane protein consisting of 347 amino acids which shows 46% similarity over 297 amino acids to EpsK of Lactobacillus delbrueckii subsp. bulgaricus. EpsK is proposed to be a polymerase on the basis of homology and topological similarity to the O-antigen polymerase (Wzy) of E. coli and is required for the synthesis of an exopolysaccharide composed of Gal, Glc, and Rha (5:1:1) containing repeating units in L. delbrueckii (32). Application of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and methylation and monosaccharide analyses using gas chromatography-mass spectrometry (GC-MS) to purified core-containing OSs isolated from LPS from ΔPG1051 and ΔPG1142 mutants enabled us to solve the LPS core structure of an oral gram-negative bacterium for the first time.     

Intraspecies Variability Affects Heterotypic Biofilms of Porphyromonas gingivalis and Prevotella intermedia: Evidences of Strain-Dependence Biofilm Modulation by Physical Contact and by Released Soluble Factors

It is well known that strain and virulence diversity exist within the population structure of Porphyromonas gingivalis. In the present study we investigate intra- and inter-species variability in biofilm formation of Porphyromonas gingivalis and partners Prevotella intermedia and Prevotella nigrescens. All strains tested showed similar hydrophobicity, except for P. gingivalis W83 which has roughly half of the hydrophobicity of P. gingivalis ATCC33277. An intraspecies variability in coaggregation of P. gingivalis with P. intermedia was also found. The association P. gingivalis W83/P. intermedia 17 produced the thickest biofilm and strain 17 was prevalent. In a two-compartment system P. gingivalis W83 stimulates an increase in biomass of strain 17 and the latter did not stimulate the growth of P. gingivalis W83. In addition, P. gingivalis W83 also stimulates the growth of P. intermedia ATCC25611 although strain W83 was prevalent in the association with P. intermedia ATCC25611. P. gingivalis ATCC33277 was prevalent in both associations with P. intermedia and both strains of P. intermedia stimulate the growth of P. gingivalis ATCC33277. FISH images also showed variability in biofilm structure. Thus, the outcome of the association P. gingivalis/P. intermedia seems to be strain-dependent, and both soluble factors and physical contact are relevant. The association P. gingivalis-P. nigrescens ATCC33563 produced larger biomass than each monotypic biofilm, and P. gingivalis was favored in consortia, while no differences were found in the two-compartment system. Therefore, in consortia P. gingivalis-P. nigrescens physical contact seems to favor P. gingivalis growth. The intraspecies variability found in our study suggests strain-dependence in ability of microorganisms to recognize molecules in other bacteria which may further elucidate the dysbiosis event during periodontitis development giving additional explanation for periodontal bacteria, such as P. gingivalis and P. intermedia, among others, to persist and establish chronic infections in the host.     

Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils

Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)–tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells.     

Attenuation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Porphyromonas gingivalis Gingipains RgpA,RgpB, and Kgp

Porphyromonas gingivalis is a major pathogen of periodontal diseases, including periodontitis. We have investigated the effect of P. gingivalis infection on the PI3K/Akt (protein kinase B) signaling pathway in gingival epithelial cells. Here, we found that live P. gingivalis, but not heat-killed P. gingivalis, reduced Akt phosphorylation at both Thr-308 and Ser-473, which implies a decrease in Akt activity. Actually, PI3K, which is upstream of Akt, was also inactivated by P. gingivalis. Furthermore, glycogen synthase kinase 3α/β, mammalian target of rapamycin, and Bad, which are downstream proteins in the PI3K/Akt cascade, were also dephosphorylated, a phenomenon consistent with Akt inactivation by P. gingivalis. However, these events did not require direct interaction between bacteria and host cells and were independent of P. gingivalis invasion into the cells. The use of gingipain-specific inhibitors and a gingipain-deficient P. gingivalis mutant KDP136 revealed that the gingipains and their protease activities were essential for the inactivation of PI3K and Akt. The associations between the PI3K regulatory subunit p85α and membrane proteins were disrupted by wild-type P. gingivalis. Moreover, PDK1 translocation to the plasma membrane was reduced by wild-type P. gingivalis, but not KDP136, indicating little production of phosphatidylinositol 3,4,5-triphosphate by PI3K. Therefore, it is likely that PI3K failed to transmit homeostatic extracellular stimuli to intracellular signaling pathways by gingipains. Taken together, our findings indicate that P. gingivalis attenuates the PI3K/Akt signaling pathway via the proteolytic effects of gingipains, resulting in the dysregulation of PI3K/Akt-dependent cellular functions and the destruction of epithelial barriers.