Role of Porphyromonas gingivalis HmuY in Immunopathogenesis of Chronic Periodontitis.

Periodontitis is a multifactorial disease, with participation of bacterial, environmental, and host factors. It results from synergistic and dysbiotic multispecies microorganisms, critical "keystone pathogens," affecting the whole bacterial community. The purpose of this study was to review the role of Porphyromonas gingivalis in the immunopathogenesis of chronic periodontitis, with special attention paid to HmuY. The host response during periodontitis involves the innate and adaptive immune system, leading to chronic inflammation and progressive destruction of tooth-supporting tissues. In this proinflammatory process, the ability of P. gingivalis to evade the host immune response and access nutrients in the microenvironment is directly related to its survival, proliferation, and infection. Furthermore, heme is an essential nutrient for development of these bacteria, and HmuY is responsible for its capture from host heme-binding proteins. The inflammatory potential of P. gingivalis HmuY has been shown, including induction of high levels of proinflammatory cytokines and CCL2, decreased levels of IL-8, and increased levels of anti-HmuY IgG and IgG1 antibodies in individuals with chronic periodontitis. Therefore, the HmuY protein might be a promising target for therapeutic strategies and for development of diagnostic methods in chronic periodontitis, especially in the case of patients with chronic periodontitis not responding to treatment, monitoring, and maintenance therapy.

Induction of interleukin (IL)-1ß, IL-10, IL-8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis, an anaerobic gram-negative bacterium, is associated with chronic periodontitis. This study was undertaken to evaluate the production of interleukin (IL)-1ß, IL-8 and IL-10 by human peripheral blood mononuclear cells (PBMC) stimulated with P. gingivalis antigens and to assess the levels of serum immunoglobulin (Ig)G, IgA and IgG subclasses raised against P. gingivalis HmuY protein. MATERIAL AND METHODS: PBMC from patients with chronic periodontitis (CP) and from nonperiodontitis (NP) control subjects were stimulated with P. gingivalis antigens, and the cytokine levels in the culture supernatants were determined by ELISA. The specificity of serum antibodies raised against HmuY was analyzed by Western blotting and by ELISA. RESULTS: Compared with the NP controls, the CP patients produced higher levels of total serum IgG and IgG1 specific for P. gingivalis HmuY. No differences were found between CP and NP groups in the production of IL-1ß and IL-8 by PBMC stimulated with total P. gingivalis antigens. Only P. gingivalis lipopolysaccharide (LPS) induced higher levels of IL-10 in the CP group. Higher levels of IL-1ß and IL-10 were induced by HmuY than by other antigens derived from the wild-type P. gingivalis strains. In contrast, total antigens derived from the hmuY-deletion mutant strain induced the production of significantly higher levels of IL-8 and significantly lower levels of IL-1ß. CONCLUSION: Our data suggest that P. gingivalis HmuY may be considered an immunogenic protein associated with host-pathogen interactions.

High diversity in mucin genes and mucin molecules in Trypanosoma cruzi.

Mucins are highly O-glycosylated molecules which in mammalian cells accomplish essential functions, like cytoprotection and cell-cell interactions. In the protozoan parasite Trypanosoma cruzi, mucin-related glycoproteins have been shown to play a relevant role in the interaction with and invasion of host cells. We have previously reported a family of mucin-like genes in T. cruzi whose overall structure resembled that of mammalian mucin genes. We have now analyzed the relationship between these genes and mucin proteins. A monoclonal antibody specific for a mucin sugar epitope and a polyclonal serum directed to peptide epitopes in a MUC gene-encoded recombinant protein, detected identical bands in three out of seven strains of T. cruzi. Immunoprecipitation experiments confirmed these results. When expressed in eukaryotic cells, the MUC gene product is post-translationally modified, most likely, through extensive O-glycosylation. Gene sequencing showed that the central domains encoding the repeated sequences with the consensus T8KP2, varies in number from 1 to 10, and the number of Thr residues in each repeat could be 7, 8, or 10. A run of 16 to 18 Thr residues was present in some, but not all, MUC gene-derived sequences. Direct compositional analysis of mucin core proteins showed that Thr residues are much more frequent than Ser residues. The same fact occurs in MUC gene-derived protein sequences. Molecular mass determinations of the 35-kDa glycoproteins further extend the heterogeneity of the family to the natural mucin molecules. Difficulties in assigning each of the several MUC genes identified to a mucin product arise from the high diversity and partial sequence conservation of the members of this family.

Structural characterization of the major glycosylphosphatidylinositol membrane-anchored glycoprotein from epimastigote forms of Trypanosoma cruzi Y-strain.

We have investigated the structure of the glycosylphosphatidylinositol (GPI) anchor and the O-linked glycan chains of the 40/45-kDa glycoprotein from the cell surface of the protozoan parasite Trypanosoma cruzi. This glycoconjugate is the major acceptor for sialic acid transferred by trans-sialidase of T. cruzi Y-strain, epimastigote form. The GPI anchor was liberated by treatment with hot alkali, and the phosphoinositol-oligosaccharide moiety was characterized and shown to have the following structure. [formula: see text] Unusually the glucosamine was 6-O-substituted with 2-aminoethylphosphonate, and 2-aminoethylphosphonate was also present on the third mannose residue distal to glucosamine, partially replacing the ethanolamine phosphate. The beta-eliminated reduced oligosaccharide chains showed that two novel classes of O-linked N-acetylglucosamine oligosaccharide were present. The first series had the structures Galp beta 1-3GlcNAc-ol; Galp beta 1-6(Galp beta 1-3)GlcNAc-ol; and Galp beta 1-2Galp beta 1-6(Galp beta 1-3)GlcNAc-ol, whereas the other series had a 1-4 linkage to N-acetylglucosaminitol and had structures Galp beta 1-4GlcNAc-ol, Galp beta 1-6(Galp beta 1-4)GlcNAc-ol, and Galp beta 1-2Galp beta 1-6(Galp beta 1-4)GlcNAc-ol. We have also investigated the kinetics of in vitro sialylation of these O-linked oligosaccharides by the T. cruzi transsialidase and have shown that incorporation of one molecule of sialic acid hinders entry of a second molecule when two potential acceptor sites are present.

The presence of galactofuranose and ribose units in asparagine-linked oligosaccharides of the digenetic trypanosomatid Endotrypanum schaudinni.

It was found that the digenetic trypanosomatid Endotrypanum schaudinni transferred Man7GlcNAc2 in protein N-glycosylation. Endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides were identified as Man7GlcNAc2, Man6GlcNAc2, Rib1Man6GlcNAc2 and/or Gal1Man6GlcNAc2, Man5GlcNAc containing two galactose or ribose units or one of each residues, Rib1Man5GlcNAc2 and Gal1Man5GlcNAc2. The galactoses were in the furanose configuration. Endo-beta-N-acetylglucosaminidase H-resistant glycopeptides that were retained by concanavalin A-Sepharose and eluted with alpha-methylmannoside were found to contain mannose, galactofuranose and ribose units. The presence of galactofuranoses in N-glycoproteins has been reported previously in several monogenetic trypanosomatids but only in one digenetic parasite (Trypanosoma cruzi). This and a recent publication on the structure of Blastocrithidia culicis N-linked oligosaccharides are the first reports on the presence of ribose in eukaryotic glycoconjugates.

The presence in Trypanosoma cruzi microsomes of alpha(1,2), alpha(1,3) and alpha(1,6) mannosidase activities not involved in protein-linked Man9GlcNAc2 processing.

Trypanosoma cruzi microsomes were found to possess membrane-bound alpha(1,2), alpha(1,3) and alpha(1,6) mannosidase activities that had an almost neutral optimum pH value, did not require CaCl2 for activity and were inhibited by swainsonine but not by deoxymannojirimycin. A mannosidase activity that degraded p-nitrophenylmannoside and that was inhibited by swainsonine was also present in the parasite microsomes. Experiments performed with intact cells showed that processing of protein-linked Man9GlcNAc2 was inhibited by deoxymannojirimycin but not by swainsonine. It was concluded that the activities detected were not involved in protein-linked Man9GlcNAc2 processing.

Novel (rhamnosyl and ribosyl) and uncommon (xylosyl) monosaccharide residues are present in asparagine-linked oligosaccharides of the trypanosomatid Blastocrithidia culicis.

Blastocrithidia culicis is a trypanosomatid protozoon that transfers Man6GlcNAc2 in protein N-glycosylation. Compounds containing mannosyl, xylosyl, and rhamnosyl residues were found among the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of whole cell glycoproteins of this parasite. The compositions of some of them were as follows: Man5GlcNAc2, Man6GlcNAc2, Rha1Man5GlcNAc2, Rha2Man6GlcNAc2, Xyl1Rha2Man6-GlcNAc2, Xyl1Rha3Man6GlcNAc2, and Xyl2Rha3Man6-GlcNAc2. On the other hand, oligosaccharides containing mannosyl, xylosyl, rhamnosyl, and ribosyl units were liberated from endo-beta-N-acetylglucosaminidase-resistant glycopeptides upon treatment with N-glycanase. This is the first report on the presence of ribosyl units in eukaryote glycoconjugates, of rhamnosyl residues in asparagine-linked oligosaccharides, and of xylosyl units in high mannose-type compounds.

Primary structure of the oligosaccharide chain of lipopeptidophosphoglycan of epimastigote forms of Trypanosoma cruzi.

The lipopeptidophosphoglycan of epimastigote forms of Trypanosoma cruzi is composed of a glycan linked through a non-N-acetylated glucosamine residue to an inositol phosphorylceramide. Using conventional analysis techniques, including 1H, 13C, and 31P NMR spectroscopy and negative ion fast atom bombardment mass spectroscopy, the structure of the carbohydrate-containing part of the molecule is determined as: (Sequence: see text). There is uncertainty as to which 2-O-substituted alpha-D-Manp unit is attached the side chain or whether it is distributed between the two units. Some of the structures lack the Galf side chain. The inositol unit is linked to ceramide via a phosphodiester bridge. The major aliphatic components of the ceramide portion were lignoceric acid and sphinganine.

Novel 17-kilodalton Leishmania antigen revealed by immunochemical studies of a purified glycoprotein fraction recognized by murine T lymphocytes.

Recently, a glycoprotein fraction, designated gp10/20, purified from Leishmania mexicana amazonensis was shown to induce a cellular immune response mediated by murine L3T4+ T lymphocytes. This fact led us to pursue further the characterization of this fraction. The present study demonstrated that gp10/20 is a degradation product of a 17-kilodalton antigen present in promastigotes and amastigotes of L. mexicana amazonensis. This antigen was easily detected in promastigotes of L. mexicana mexicana, L. donovani, L. chagasi, L. major, and L. tropica. However, culture forms of L. braziliensis complex expressed either low amounts of the 17-kilodalton antigen or an antigenically unrelated antigen. The recognition of gp10/20 by several serum samples of patients with kala-azar was also shown.

Chemical structures of a galactose-rich glycoprotein of Leishmania tarentolae.

1. Aqueous phenol treatment of water extracted disrupted cells of Leishmania tarentolae (LV-414) provided a glycoprotein mixture which was purified by gel filtration chromatography, and Concanavalin A-Sepharose column. 2. The bound fraction on Concanavalin A-Sepharose column (protein 74%, and carbohydrate, 26%) had [alpha]D + 9 degrees and contained mannose (18%), galactose (60%), and glucose (22%), and some of the galactose residues were resistant to periodate oxidation. 3. Treatment of the phenol extract with hot aqueous NaBH4 containing NaOH gave a preparation having mannose (12%), galactose (82%), and glucose (6%). 4. Methylation analysis showed the presence of a mainly linear structure with non-reducing end-units of mannopyranose (6%), 3-O-substituted galactopyranosyl (64%), 2-O- (11%), and 6-O- (5%) substituted mannopyranosyl, and 4-O- (9%), and 4,6-di-O- (3%) substituted glycopyranosyl units. 5. The specific rotation of the preparation, +20 degrees, indicated beta-linked galactopyranosyl units.

Characterization of cellular immune response to chemically defined glycoconjugates from Leishmania mexicana subsp. amazonensis.

Two defined glycoconjugates (GP-10/20 and FR II Phe) purified from Leishmania mexicana subsp. amazonensis were analyzed with respect to their ability to induce cellular responses in immunized and infected mice. Each glycoconjugate was recognized by specific immune cells, as assessed by the proliferative response of lymph node cells of immunized mice. The response to GP-10/20 depended on helper T cells and antigen-presenting cells and was restricted by a major histocompatibility complex class II gene product. A specific anti-GP-10/20 T-cell line was established, and it was able to transfer a delayed-type hypersensitivity (DTH) response to normal mice. Both antigens were also recognized during an ongoing disease, as assessed by DTH response of infected mice. By this response, it was possible to distinguish susceptible from resistant strains of mice. In the course of the disease in resistant mice a correlation between the size of the primary lesion and the DTH response to GP-10/20 was observed. The presence of the glycoproteins on both promastigote and amastigote forms of the parasite, the antigenic similarities between both fractions, and the distribution of the GP-10/20 antigen in other trypanosomatids were studied. The results showed that both antigens were present on promastigotes and amastigotes. GP-10/20 shared no epitopes with FR II Phe, was included as part of the crude preparation leishmanin, and had some cross-reactive determinants with Leishmania donovani and Crithidia deanei.