Separation of novel phosphoproteins of Porphyromonas gingivalis using phosphate-affinity chromatography.

Phosphorylation of serine, threonine and tyrosine is a central mechanism for regulating the structure and function of proteins in both eukaryotes and prokaryotes. However, the action of phosphorylated proteins present in Porphyromonas gingivalis, a major periodontopathogen, is not fully understood. Here, six novel phosphoproteins that possess metabolic activities were identified, namely PGN_0004, PGN_0375, PGN_0500, PGN_0724, PGN_0733 and PGN_0880, having been separated by phosphate-affinity chromatography. The identified proteins were detectable by immunoblotting specific to phosphorylated Ser (P-Ser), P-Thr, and/or P-Tyr. These results imply that novel phosphorylated proteins might play an important role for regulation of metabolism in P. gingivalis.

Effect of the Antimicrobial Peptide LL-37 on Gene Expression of Chemokines and 29 Toll-like Receptor-Associated Proteins in Human Gingival Fibroblasts Under Stimulation with Porphyromonas gingivalis Lipopolysaccharide.

The antimicrobial peptide LL-37 neutralizes the biological activity of lipopolysaccharide (LPS), while it upregulates the expression of several immune-related genes. We investigated the effect of LL-37 on gene regulation of human gingival fibroblasts (HGFs), stimulated with or without Porphyromonas gingivalis-derived LPS, a ligand for Toll-like receptor (TLR). LL-37 was non-toxic to HGFs up to a concentration of 10 µg/ml. P. gingivalis LPS upregulated the expression of IL8, CXCL10, and CCL2, whereas LL-37 reduced this upregulation. In absence of LPS, LL-37 itself upregulated the expression of IL8 and CCL2. LL-37 increased the expression of P2X7, which was constitutively expressed in HGFs. The P2X7 antagonist A-438079 suppressed the cytotoxicity and upregulatory effect of LL-37 on chemokine response, but not its downregulatory effect on P. gingivalis LPS-induced chemokine response. Whether LL-37 alters the expression of 29 genes that encode TLR-associated proteins, including TLRs, co-receptors, signaling molecules, and negative regulators, in HGFs, under stimulation with LPS, was examined. Among TLRs, P. gingivalis LPS upregulated the level of TLR4, whereas LL-37 reduced it. In co-receptors, LL-37 downregulated the level of CD14. Among signaling molecules, LL-37 augmented the LPS-upregulated expression of IRAK1. Similar effects were observed in the specific negative regulators TNFAIP3, RNF216, TOLLIP, and SIGIRR. Our results suggest that LL-37 exerts cytotoxicity and upregulation of chemokine response via the P2X7 receptor, while it induces downregulation of P. gingivalis LPS-induced chemokine response through alteration in the expression of 7 specific TLR-associated genes: downregulation of TLR4 and CD14 and upregulation of IRAK1, TNFAIP3, RNF216, TOLLIP, and SIGIRR.

OmpA-Like Proteins of Porphyromonas gingivalis Mediate Resistance to the Antimicrobial Peptide LL-37.

Subgingival bacteria are continually exposed to gingival crevicular fluids that are derived from serum, which contain various bactericidal agents. The periodontopathic bacterium Porphyromonas gingivalis has been demonstrated to possess a variety of abilities to resist bactericidal agents, due to which it is able to propagate in the subgingival environment. We previously demonstrated that the major surface glycoproteins of P. gingivalis-Pgm6 and Pgm7, also called outer membrane protein A-like proteins (OmpALPs)-mediate resistance to the bactericidal activity of human serum, but their precise role remains unknown. In this study, we investigated the sensitivity of the wild-type and Pgm6/Pgm7-deficient P. gingivalis strains toward major antimicrobial peptides in the oral cavity, human ß-defensins (hBDs) 1-3, and human cathelicidin LL-37. hBDs showed a considerably weak bactericidal activity against both bacterial strains. LL-37 also showed a weak activity against the wild-type strain; however, it showed a significant activity against the Pgm6/Pgm7-deficient strain. In the Pgm6/Pgm7-deficient strain, LL-37 remarkably accumulated on the bacterial cell surface, which may result in the destruction of the outer membrane. Additionally, the bactericidal activity of hBDs against the Pgm6/Pgm7-deficient strain was found to be synergistically promoted in the presence of LL-37. Our results suggest that OmpALPs specifically protect P. gingivalis from the bactericidal activity of LL-37; thus, P. gingivalis may adeptly survive in LL-37-producing subgingival environments.

OmpA-like proteins of Porphyromonas gingivalis contribute to serum resistance and prevent Toll-like receptor 4-mediated host cell activation.

Porphyromonas gingivalis possesses various abilities to evade and disrupt host immune responses, by which it acts as an important periodontal pathogen. P. gingivalis produces outer membrane protein A (OmpA)-like proteins (OmpALPs), Pgm6 and Pgm7, as major O-linked glycoproteins, but their pathological roles in P. gingivalis infection are largely unknown. Here, we report that OmpALP-deficient strains of P. gingivalis show an enhanced stimulatory activity in coculture with host cells. Such an altered ability of the OmpALP-deficient strains was found to be due to their impaired survival in coculture and the release of LPS from dead bacterial cells to stimulate Toll-like receptor 4 (TLR4). Further analyses revealed that the OmpALP-deficient strains were inviable in serum-containing media although they grew normally in the bacterial medium. The wild-type strain was able to grow in 90% normal human serum, while the OmpALP-deficient strains did not survive even at 5%. The OmpALP-deficient strains did not survive in heat-inactivated serum, but they gained the ability to survive and grow in proteinase K-treated serum. Of note, the sensitivity of the OmpALP-deficient strains to the bactericidal activity of human ß-defensin 3 was increased as compared with the WT. Thus, this study suggests that OmpALPs Pgm6 and Pgm7 are important for serum resistance of P. gingivalis. These proteins prevent bacterial cell destruction by serum and innate immune recognition by TLR4; this way, P. gingivalis may adeptly colonize serum-containing gingival crevicular fluids and subgingival environments.

Basal autophagy prevents autoactivation or enhancement of inflammatory signals by targeting monomeric MyD88.

Autophagy, the processes of delivery of intracellular components to lysosomes, regulates induction of inflammation. Inducible macroautophagy degrades inflammasomes and dysfunctional mitochondria to downregulate inflammatory signals. Nonetheless, the effects of constitutive basal autophagy on inflammatory signals are largely unknown. Here, we report a previously unknown effect of basal autophagy. Lysosomal inhibition induced weak inflammatory signals in the absence of a cellular stimulus and in the presence of a nutrient supply, and their induction was impaired by MyD88 deficiency. During lysosomal inhibition, MyD88 was accumulated, and overabundant MyD88 autoactivated downstream signaling or enhanced TLR/IL-1R-mediated signaling. MyD88 is probably degraded via basal microautophagy because macroautophagy inhibitors, ATG5 deficiency, and an activator of chaperone-mediated autophagy did not affect MyD88. Analysis using a chimeric protein whose monomerization/dimerization can be switched revealed that monomeric MyD88 is susceptible to degradation. Immunoprecipitation of monomeric MyD88 revealed its interaction with TRAF6. In TRAF6-deficient cells, degradation of basal MyD88 was enhanced, suggesting that TRAF6 participates in protection from basal autophagy. Thus, basal autophagy lowers monomeric MyD88 expression, and thereby autoactivation of inflammatory signals is prevented. Given that impairment of lysosomes occurs in various settings, our results provide novel insights into the etiology of inflammatory signals that affect consequences of inflammation.

Identification of OmpA-Like Protein of Tannerella forsythia as an O-Linked Glycoprotein and Its Binding Capability to Lectins.

Bacterial glycoproteins are associated with physiological and pathogenic functions of bacteria. It remains unclear whether bacterial glycoproteins can bind to specific classes of lectins expressed on host cells. Tannerella forsythia is a gram-negative oral anaerobe that contributes to the development of periodontitis. In this study, we aimed to find lectin-binding glycoproteins in T. forsythia. We performed affinity chromatography of wheat germ agglutinin, which binds to N-acetylglucosamine (GlcNAc) and sialic acid (Sia), and identified OmpA-like protein as the glycoprotein that has the highest affinity. Mass spectrometry revealed that OmpA-like protein contains O-type N-acetylhexosamine and hexose. Fluorometry quantitatively showed that OmpA-like protein contains Sia. OmpA-like protein was found to bind to lectins including E-selectin, P-selectin, L-selectin, Siglec-5, Siglec-9, Siglec-10, and DC-SIGN. The binding of OmpA-like protein to these lectins, except for the Siglecs, depends on the presence of calcium. N-acetylneuraminic acid (NeuAc), which is the most abundant Sia, inhibited the binding of OmpA-like protein to all of these lectins, whereas GlcNAc and mannose only inhibited the binding to DC-SIGN. We further found that T. forsythia adhered to human oral epithelial cells, which express E-selectin and P-selectin, and that this adhesion was inhibited by addition of NeuAc. Moreover, adhesion of an OmpA-like protein-deficient T. forsythia strain to the cells was reduced compared to that of the wild-type strain. Our findings indicate that OmpA-like protein of T. forsythia contains O-linked sugar chains that can mediate interactions with specific lectins. This interaction is suggested to facilitate adhesion of T. forsythia to the surface of host cells.