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 β-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 β-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

OmpA-like protein is a major glycoprotein isolated by WGA affinity chromatography.

<p>(a) Lectin-binding glycoproteins were isolated from wild-type (WT) <i>T</i>. <i>forsythia</i> and a <i>T</i>. <i>forsythia</i> deletion mutant that lacked OmpA-like protein (Δ<i>1331</i>) using WGA affinity chromatography. Isolated proteins were subjected to SDS-PAGE. The gels were stained with CBB. M, molecular marker; P, whole cell lysate prior to application to the WGA affinity column; B, proteins bound to the WGA column; arrowhead, OmpA-like protein. (b) Glycoproteins isolated from <i>T</i>. <i>forsythia</i> WT were subjected to SDS-PAGE. The gels were stained with Pro-Q emerald. MG, CandyCane glycoprotein molecular weight standards; P, whole-cell lysates prior to application to the WGA affinity column; B, proteins bound to the WGA column; arrowhead, OmpA-like protein.</p

Sugar chains of OmpA-like protein contribute to lectin binding.

<p>The wells of 96-well microtiter plates were coated with 5 μg/ml OmpA-like protein for 18–24 h. After blocking, 200 mM GlcNAc, 200 mM NeuAc, or 10 mM mannose was added to the wells for 2 h before addition of 10 μg/ml Fc-conjugated recombinant proteins for 3 h. Binding was determined by an Fc-specific ELISA. The results are expressed as the mean ± SD (n = 3). *, <i>P</i> < 0.01.</p

Enzymatic deglycosylation of OmpA-like protein.

<p>Fetuin (a, b) and OmpA-like protein (c, d) were treated with <i>N</i>-glycanase. The samples were then subjected to SDS-PAGE and stained with Pro-Q emerald and SyproRuby. M, molecular marker; MG; CandyCane glycoprotein molecular weight standards.</p

Identification of OmpA-Like Protein of <i>Tannerella forsythia</i> as an <i>O</i>-Linked Glycoprotein and Its Binding Capability to Lectins

<div><p>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. <i>Tannerella forsythia</i> is a gram-negative oral anaerobe that contributes to the development of periodontitis. In this study, we aimed to find lectin-binding glycoproteins in <i>T</i>. <i>forsythia</i>. We performed affinity chromatography of wheat germ agglutinin, which binds to <i>N</i>-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 <i>O</i>-type <i>N</i>-acetylhexosamine and hexose. <a href="http://lsd-project.jp/weblsd/c/begin/fluorometry" target="_blank">Fluorometry</a> 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. <i>N</i>-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 <i>T</i>. <i>forsythia</i> 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 <i>T</i>. <i>forsythia</i> strain to the cells was reduced compared to that of the wild-type strain. Our findings indicate that OmpA-like protein of <i>T</i>. <i>forsythia</i> contains <i>O</i>-linked sugar chains that can mediate interactions with specific lectins. This interaction is suggested to facilitate adhesion of <i>T</i>. <i>forsythia</i> to the surface of host cells.</p></div

Detection of Sia in OmpA-like protein.

<p>(a, b) The wells of 96-well microtiter plates were coated with 5 μg/ml OmpA-like protein, sialidase-treated OmpA-like protein, fetuin, or asialofetuin for 18–24 h. After blocking, the wells were incubated with 5 μg/ml control (C)-biotin, WGA-biotin, succinylated WGA (Suc-WGA)-biotin, or MAM-biotin for 3 h. Binding was determined by ELISA using streptavidin-HRP. Data are expressed as the mean ± SD (n = 3). *, <i>P</i> < 0.01 for comparison with control-biotin. ^†, <i>P</i> < 0.01 for comparison with WGA-biotin. (c) OmpA-like protein, sialidase-treated OmpA-like protein, fetuin, and asialofetuin were analyzed by dot blotting with anti-Siaα2–3 antibody.</p

Sugar chains of OmpA-like protein mediate adhesion of <i>T</i>. <i>forsythia</i> to human oral epithelial cells.

<p>(a) Total RNA (1 μg) was prepared from Ho-1-n-1 cells and HUVECs to assess the expression of <i>SELE</i> (E-selectin), <i>SELP</i> (P-selectin), and <i>ACTB</i> (β-actin) by RT-PCR. HUVECs, which express E-selectin and P-selectin mRNA, were used as positive controls. The PCR products were visualized on 1.5% agarose gels stained with ethidium bromide and photographed under UV light. The results are representative of three independent experiments. (b) WT, Δ<i>1331</i> or Δ<i>1331</i> complemented with <i>tf1331</i> (<i>tf1331</i> comp) was added to Ho-1-n-1 cells at a m.o.i. of 100 for 3 h. At the end of the incubation period, the cells were washed three times with PBS and lysed by incubation in sterile water for 20 min. Cell lysates were serially diluted in sterile PBS and cultured on supplemented blood agar plates. The number of viable adherent bacteria was determined by counting the colonies that appeared. The results are expressed as the mean ± SD (n = 3). *, <i>P</i> < 0.05 for comparison with WT. ^†, <i>P</i> < 0.05 for comparison with Δ<i>1331</i>. (c) Ho-1-n-1 cells were preincubated with and without 200 mM GlcNAc, 200 mM NeuAc, and 10 mM mannose for 2 h. Then, WT or Δ<i>1331</i> was added at a m.o.i. of 100 for 3 h, and the number of viable adherent bacteria was determined. The results are expressed as the mean ± SD (n = 3). *, <i>P</i> < 0.05.</p