Role of Streptococcus gordonii cell wall components in the bacterial biofilm formation and chemokine induction

Abstract

Objectives Streptococcus gordonii, a Gram-positive, oral, and commensal bacterium, can be a notorious life-threatening pathogen causing endocarditis. It is frequently isolated from the periapical lesion of patients with apical periodontitis. Serine-rich repeat (SRR) adhesins of S. gordonii such as gordonii surface protein B (GspB) are associated with bacterial colonization and cell-associated virulence factors like lipoteichoic acid (LTA) and lipoprotein are representative virulence factors to induce pro-inflammatory cytokines and chemokines in host cells. Although they seemingly contribute to biofilm formation and the inflammatory responses during apical periodontitis, little is known about the pathogenic mechanisms by S. gordonii and key virulence factors of S. gordonii to be responsible for biofilm formation on root canal dentin and the induction of inflammatory responses. The aim of this study was to investigate the role of S. gordonii cell wall-associated virulence factors on biofilm formation and interleukin (IL)-8 induction in human periodontal ligament (PDL) cells.

Methods The effect of S. gordonii GspB on biofilm formation was investigated using wild-type and GspB-deficient mutant S. gordonii strains. Confocal microscopy and crystal violet assay were performed to determine biofilm formation. Bacterial growth was examined by measuring optical density at 600 nm with spectrometry. Bacterial adherence and biofilm formation on the culture plate and human dentin slices were visualized with a scanning electron microscope. The role of S. gordonii cell wall-associated virulence factors on IL-8 induction in human PDL cells was investigated using ethanol-inactivated S. gordonii wild-type, LTA-deficient mutant (ΔltaS), and lipoprotein-deficient mutant (Δlgt). IL-8 mRNA expression and production were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Toll-like receptor 2 (TLR2) activation was determined using transient transfection followed by a reporter gene assay in the human embryonic kidney 293 (HEK293) cells overexpressing TLR2. The cells were pretreated with inhibitors of MAP kinases for 1 h followed by stimulation with S. gordonii or its lipoproteins to characterize the intracellular signaling pathways required for IL-8 induction by S. gordonii.

Results The GspB-deficient S. gordonii mutant strain was less potent than the wild-type strain in the biofilm formation. Of note, there was no difference in bacterial growth rate between the mutant and the wild-type strains. Differences in biofilm-forming ability between the wild-type and mutant strains were more distinct in the sucrose-supplemented media. Furthermore, the GspB-deficient mutant exhibited attenuated formation of aggregates on the surface of the culture plate and human dentin slices. On the other hands, S. gordonii wild-type induced IL-8 expression at both protein and mRNA levels in human PDLs in a dose- and time-dependent manner. Transient transfection followed by a reporter gene assay in the HEK293 cells overexpressing TLR2 demonstrated that S. gordonii wild-type and purified lipoproteins induced substantial activation of NF-κB, whereas purified LTA showed a minimal activation. Additionally, IL-8 production induced by S. gordonii wild-type or purified lipoproteins was significantly inhibited by anti-TLR2 neutralizing antibody. S. gordonii wild-type and ΔltaS induced IL-8 production but such response was not observed when the cells were stimulated with the Δlgt. Concordantly, lipoproteins purified from S. gordonii induced IL-8 production in PDLs in a dose-dependent manner whereas LTA purified from S. gordonii failed to induce it. Furthermore, S. gordonii lipoprotein-induced IL-8 production was decreased by inhibitors for p38, ERK, and JNK.

Conclusions Taken together, the results in this study show that GspB is closely involved in the initial adherence and biofilm formation of S. gordonii, especially on human dentin. Furthermore, lipoprotein of S. gordonii plays a critical role in the IL-8 production in human PDL cells. Therefore, this current results suggest that GspB promotes S. gordonii biofilm formation and lipoprotein of S. gordonii leads to inflammatory responses by promoting IL-8 production of human PDL cells, which may cause apical periodontitis.