Proliferation Mechanism And Pathogenesis Of Prevotella Intermedia In Chronic Periodontitis

Chronic periodontitis is an inflammatory disease characterized by periodontal tissue destruction, pocket formation, alveolar bone resorption and finally leading to tooth loss. A large line of evidence confirmed that chronic periodontitis is initiated by the colonization and proliferation of a group of gram-negative periodontal pathogens such as Porphyromonas gingivalis and Prevotella intermedia in the subgingival plaque biofilm. These pathogens can directly destroy the periodontal tissue by producing lipopolysaccharide, proteinase and metabolic products. Most importantly, they can elicit immune responses of immune cells and activated local resident cells to release inflammatory cytokines and proteinases, therefore leading to periodontal tissue destruction.P. intermedia, a gram negative black-pigmented obligate anaerobic rod, has been reported to be an important periodontal pathogen and it is associated with various forms of periodontal diseases, including pregnancy gingivitis, acute necrotizing ulcerative gingivitis and chronic periodontitis. Furthermore, it has also been implicated in extra-oral infections such as pulmonary infections and noma in children. Virulence factors of this bacterium include adhesins, fimbriae, hemagglutinin, hemalysin and hydrolytic enzymes.Iron plays an essential role in both bacterial growth and progression of diseases. It participates in many biological processes through incorporation into proteins. In human, iron availability is limited by intracellular containment within ferritin, hemosiderin and heme-containing proteins such as hemoglobin and myoglobin, and extracellular binding to transferrin and lactoferrin. Moreover, free heme is bound rapidly by hemopexin and albumin, whereas hemoglobin is bound by haptoglobin. Consequently, free iron is maintained at a far too low level to support bacterial growth. Therefore, the ability of an organism to scavenge iron from the iron-limited environment of its human host is a critical determinant of virulence.P. intermedia has an obligate requirement of iron for its growth. However, it lacks the ability to synthesize heme, an important iron source. Therefore, it must develop sufficient mechanisms through which to acquire iron in order to survive and proliferate in the host. Previous studies demonstrated that P. intermedia can utilize hemoglobin as an effective iron source, however, the mechanisms governing iron uptake from hemoglobin have not been elucidated, therefore the present work aimed to address this question.The successful colonization and proliferation is a prerequisite for a pathogen to initiate a disease. The host responses caused by the invasion of a pathogen is more important in the development of tissue destruction during chronic periodontitis. Matrix Metalloproteinases (MMPs) are a group of zinc and calcium dependent proteinases capable of degrading extracellular matrix (ECM) and basement membrane components. MMPs diversely participate in physiological events such as morphogenesis, tissue remodeling, wound healing, host defense and immunomodulatory reactions. They also play pivotal roles in pathological conditions such as cancer, rheumatoid arthritis and chronic periodontitis. MMPs produced by host cells, together with other proteinases, are responsible for the connective tissue destruction in periodontitis.P. intermedia induced proinflammatory cytokines expression in human gingival epithelial cells and human periodontal ligament cells (hPDLc). However, until present, the effects of P. intermedia on MMPs production in human periodontal tissues have not been explored. Since periodontal ligament cells are the major constituents of the periodontium and they are highly affected during periodontitis, in this study we also investigated the effects of P. intermedia on MMPs expression in hPDLc and explored the possible roles of mitogen activated protein kinase (MAPK) signaling pathway in the regulation of MMPs production.The following were the major findings of our study:1. Utilization of hemoglobin and underlying iron uptake mechanisms1.1 By using bacterial culture and dot blot analysis, we confirmed that P. intermedia can effectively utilize human hemoglobin as an iron source. P. intermedia whole cells can bind to hemoglobin in a pH dependent manner. The strongest binding activity was at pH 5.0.1.2 A hemoglobin-binding outer membrane protein was identified using batch affinity and SDS-PAGE method. Through a combination of anion exchange chromatography, affinity chromatography and hydroxyapatite chromatography, a 60 kDa hemoglobin-binding protein (HBP) was purified to homogeneity.1.3 The 60 kDa HBP bound to hemoglobin in a pH dependent manner and the strongest binding activity was found to be at pH 5.0. Surface plasmon resonance analysis showed that the dissociation constant (Kd) between HBP and hemoglobin was 1.48×10-8M, indicating the binding of HBP to hemoglobin was specific.1.4 Inhibition studies of HBP-binding to hemoglobin demonstrated that globin greatly decreased the binding activity, whereas hemin, catalase and cytochrome C exerted no inhibitory effects, suggesting that the globin moiety rather than the heme moiety of hemoglobin was involved in hemoglobin-binding.1.5 Amino acid sequence analysis of the internal sequences of HBP showed no apparent homology with known hemoglobin-binding protein/receptors in other bacteria, indicating that the 60 kDa HBP might be a new hemoglobin-binding protein.1.6 Iron restriction had no effect on the expression of HBP and on the binding of P. intermedia whole cells to hemoglobin, which suggested that HBP might be constitutively expressed in order to ensure iron uptake of P. intermedia.1.7 SDS-PAGE and scanning densitometry showed that P. intermedia can time-dependently degrade hemoglobin. In accordance with hemoglobin binding, hemoglobin degradation was also pH related, with the strongest degradation at pH 5.0. Inhibition studies demonstrated that a cysteine proteinase might be involved in hemoglobin degradation. Since iron restriction had no influence on the degradation effects, the proteinase might be constitutively expressed. 2. Effects of P. intermedia on MMPs production in hPDLc2.1 By RT-PCR method, it was found that hPDLc constitutively express MMP-1, -2, -3, -14, TIMP-1 and TIMP-2. P. intermedia diversely regulated MMP mRNA expression in hPDLc by inducing the expression MMP-9, up-regulating the expression of MMP-1 and TIMP-1 and down-regulating the expression of TIMP-2.2.2 Gelatin zymography revealed that P. intermedia supernatant time and dose dependently induced MMP-9 protein secretion by hPDLc.2.3 ELISA analysis showed that P. intermedia supernatant time and dose dependently enhanced MMP-1 and TIMP-1 secretion. In contrast, it inhibited TIMP-2 secretion. However, MMP-3 production was not influenced by P. intermedia stimulation.2.4 MAPK signaling pathways seemed to play different roles in the P. intermedia-regulated MMPs production in hPDLc. ERK, JNK and p38 specific inhibitors exerted no influence on MMP-9 and MMP-3 production. Whereas, ERK, JNK and p38 inhibitors demonstrated great inhibitory effects on P. intermedia-stimulated MMP-1 production. ERK and JNK inhibition showed significant reduction in P. intermedia-stimulated TIMP-1 production. However, the inhibitory effect of P. intermedia on TIMP-2 secretion was reversed by JNK and p38 inhibition.Conclusion:In this study, we confirmed that P. intermedia can utilize hemoglobin as an effective iron source. The mechanisms underlying iron uptake from hemoglobin by P. intermedia might be as follows: First, P. intermedia specifically binds to hemoglobin through a 60 kDa outer membrane protein. Then the bound hemoglobin is degraded by a cysteine proteinase, hence releasing the heme molecule contained within hemoglobin. Finally, the heme was captured by P. intermedia for its utilization. Effective iron uptake ensured the colonization and proliferation of P. intermedia in the host. After successful proliferation, P. intermedia can induce and stimulate MMPs production in hPDLls. The diverse regulation of MMPs production might contribute to periodontal tissue destruction during chronic periodontitis.