The central region of the msp gene of Treponema denticola has sequence heterogeneity among clinical samples, obtained from patients with periodontitis
© Gaibani et al; licensee BioMed Central Ltd. 2010
Received: 11 August 2010
Accepted: 7 December 2010
Published: 7 December 2010
Treponema denticola is an oral spirochete involved in the pathogenesis and progression of periodontal disease. Of its virulence factors, the major surface protein (MSP) plays a role in the interaction between the treponeme and host. To understand the possible evolution of this protein, we analyzed the sequence of the msp gene in 17 T. denticola positive clinical samples.
Nucleotide and amino acid sequence of MSP have been determined by PCR amplification and sequencing in seventeen T. denticola clinical specimens to evaluate the genetic variability and the philogenetic relationship of the T. denticola msp gene among the different amplified sequence of positive samples. In silico antigenic analysis was performed on each MSP sequences to determined possible antigenic variation.
The msp sequences showed two highly conserved 5' and 3' ends and a central region that varies substantially. Phylogenetic analysis categorized the 17 specimens into 2 principal groups, suggesting a low rate of evolutionary variability and an elevated degree of conservation of msp in clinically derived genetic material. Analysis of the predicted antigenic variability between isolates, demonstrated that the major differences lay between amino acids 200 and 300.
These findings showed for the first time, the nucleotide and amino acids variation of the msp gene in infecting T. denticola, in vivo. This data suggested that the antigenic variability found in to the MSP molecule, may be an important factor involved in immune evasion by T. denticola.
Periodontitis is a chronic inflammatory condition that is characterized by the progressive destruction of periodontal tissue . This common infection is caused by polymicrobial flora, comprising several anaerobic, gram-negative bacteria. The oral spirochete Treponema denticola is often isolated from the affected sites and plays an important role in the polymicrobial pathogenesis of acute and chronic periodontal disease [2, 3]. The outer membrane of T. denticola bears several antigens that control the interaction with host cells and tissues thus contributing to the pathogenesis of periodontal disease. In particular, the major surface protein (MSP) has been recently reported to alter the normal homeostasis of endothelial cells in vitro . Further, MSP mediates the adhesion to and cytopathic effects of T. denticola on host cells [5, 6]. MSP is a porin-like protein that has pore-forming activity, similar to other porins in the outer membrane of Gram-negative bacteria. MSP exists in an oligomeric form in the cell membrane of T. denticola and is homologous to the T. pallidum subsp. pallidum repeat (Tpr) proteins, which is a target of the antibody response during syphilis [6, 7]. Recently, we demonstrated that specific polyclonal antibodies against MSP have strong opsonizing effects on the phagocytosis of T. denticola by murine macrophages in vitro . Several studies have shown the ability of MSP to mediate the attachment to extracellular matrix (ECM) components, to induce the release of proteinase from human polymorphonuclear leukocytes [9, 10], and to up regulate pro-inflammatory cytokines in different cells in vitro [11, 12]. MSP complexes with chymotrypsin-like protein (CTLP) in the outer membrane of living T. denticola to form an oligomeric complex that has an apparent molecular mass of approximately 150 kDa . The apparent molecular mass of isolated MSP ranges from 53 to 64 kDa, depending on the strain of T. denticola . MSP is 543 residues long in ATCC 35405 strain and composed by 547 amino acids in the ATCC 33520 strain, which share an identical sequence homology  between the 3' and 5' ends and a low degree of homology in the central region. In the OTK strain of T. denticola, the amino acid sequences of MSP differ significantly with that of ATCC35405 and ATCC33520 strains . Recently, Edwards and colleagues demonstrated that the central region of MSP mediates its binding to host extracellular matrix (ECM) components and that this area is the preferential target of host immune responses. These findings are consistent with the proposed cellular localization of the MSP antigen, wherein the central region is the only area of the molecule that is exposed on the surface of living T. denticola cells [10, 15]. Because the central region of MSP mediates the effects of the entire protein during its interaction with the host, it is likely that differences in the amino acid sequence of this region affect its function differentially during infection. Aim of this study was to investigated for the first time, the nucleotide and amino acids sequence of the msp gene among infected T. denticola obtained from periodontal patients. As hypothesized for the Tprk antigen of T. pallidum that undergoes nucleotide variation following serial passages of T. pallidum in rabbit , we analyzed the nucleotide and amino acids variation of the msp gene in, in vivo infecting T. denticola. Based on these considerations, we analyzed the sequence of the msp gene in 17 clinical samples from acute periodontitis patients who were positive for T. denticola by real-time PCR .
Clinical samples selection
Seventeen subjects with chronic periodontitis were selected from a pool of patients who sought dental treatment at the Department of Oral and Dental Sciences, University of Bologna, Bologna. In addition, ten periodontally healthy volunteers participated in the study as controls. Periodontitis patients showed at least two sites with probing pocket depth and clinical attachment level of ≥4 mm and gingival recession. Healthy controls showed no sites with a probing pocket depth of >3 mm and clinical attachment level of <4 mm. Exclusion criteria included pregnancy, systemic conditions that could affect the progression or treatment of periodontal diseases, and use of antibiotics 6 months prior to entry into the study. All patients were informed about the nature of the study, and a signed consent form was obtained from each individual. The study was conducted following the ethical rules of University of Bologna. The sampling procedure has been reported .
Specific T. denticol a primers sets and cycling condition used in this study.
Nucleic acid extraction
Bacterial DNA was purified from paper conical tip swabs with the automated NucliSENS EasyMAG (Biomerieux, France) extractor according to the manufacturer's instructions.
PCR amplification and sequencing
The amplification of msp was performed using the Kx14 and Kx04 primers, located in the 5' and 3' end flanking regions of the msp open reading frame (ORF), using reported conditions . Individual nested PCR reactions were used to amplify regions of msp separately (5'end, 3' end, and the central region), using the Kx14 primer with Kx09 for the 5'end, the primer set Td03 and Td06 for the central fragment, and the Td05 and Td04 primers for the 3'end. For a complete and detailed description of the primer sets, see Table 1. The amplification mixture (50 μl final volume) contained 2 μM dNTPs, 0.2 mM of each primer, 3 mM MgCl2 , 1 UI of Taq DNA polymerase (Fermentas Life Science, Canada), and 5 μl of template DNA. Cycling conditions are reported in Table 1. The amplicons were purified using the PureLink Quick Gel Extraction Kit (Invitrogen, Italy) and sequenced (PRIMM, Italy).
Phylogenetic and predicitive antigenicity variability analysis
Multiple alignment was generated with ClustalW software (version 2, available at: http://www.ebi.ac.uk/clustalw2), and a phylogenetic tree was constructed with bootstrap resempling analysis (1000 interactions) by using neighbor-joining algoritm implemented in MEGA 4 software . All 17 msp gene sequences were deposited in GenBank with the following accession numbers: B5: GU946067; B6: GU946068; B14: GU946069; B16: GU946070; B20: GU946071; B21: GU946072; B22: GU946073; B23: GU946074; B30: GU946075; B34: GU946076; B37: GU946077; B40: GU946078; B46: GU946079; B52: GU946080; B66: GU946081; B69: GU946082; B74: GU946083. The putative antigenic variability in the 17 msp sequences was evaluated in silico by analyzing entire individual amino acid MSP sequences with CLC Bio Workbench .
Nucleotides and amino acids variability of the Major Surface Protein coding sequence
Outer membrane-exposed antigens regulate many interactive processes between T. denticola and host cells and tissues. In this study, we demonstrated that the central region of the msp gene experiences the highest degree of variability compared with the 5'and 3' ends in PCR-positive clinical samples from patients who suffer from acute periodontal disease. These data confirm the high conservation in the terminal ends of msp that has been reported for T. denticola strains that have been cultured under laboratory conditions for long periods .
The central area of the MSP polypeptide has recently been demonstrated to be exposed to the surface of living T. denticola cells and act as the primary target for immune responses. In particular, Edwards and colleagues reported that this region contains epitopes that bind to immune serum against recombinant MSP from the homologous strain ATCC 35405 . We recently reported that specific immune responses against whole native MSP affects the efficacy of T. denticola phagocytosis by isolated murine macrophages under anaerobic conditions in vitro .
The variability in MSP amino acid sequences in the clinical samples in this study could influence the evolution of periodontitis, because the efficacy of a patient's immune response during in vivo infections might be hampered. The interaction between T. denticola and the host immune response could influence sequence variations into the surface-exposed portion of MSP, corresponding to the central region of msp gene. This immune-driven sequence variability in one of the most prominent virulence factors of T. denticola might underlie the pathogenic mechanisms that govern the development of periodontal diseases in vivo. Further, T. denticola migrates from the oral cavity to other anatomical sites . This property of wild-type infectious treponemes could be linked to variations in the surface-exposed portion of MSP. In addition, amino acid variations that were detected in 7 clinical specimens led to antigenic modification of the surface-exposed portion of MSP. The antigenic variation in surface-exposed polypeptides is a common pathogenic feature of spirochetes. In particular, the outer membrane lipoprotein P66 of Borrelia spp. has a surface-exposed loop that is subject to selective pressure by antibodies, resulting in sequence and size variations . The antigenic differences in the most prominent surface-exposed protein of 2 strains of the avian pathogenic Borrelia anserina impede strain-specific antibody-mediated protection against infections . MSP shares high homology with the Tprk antigen of T. pallidum subsp. pallidum . The TprK protein is a target of opsonizing antibodies and protective immunity and is subject to immunologically driven sequence variation . Centurion-Lara and colleagues demonstrated that the tprK gene undergoes nucleotide variation following serial passages of T. pallidum in rabbit . tprK gene is highly variable within T. pallidum strains, and the evidence that its V regions elicit variant-specific antibody responses supports the hypothesis that TprK variants help organisms avoid immune responses in infected individuals, contributing to the ability of T. pallidum to effect chronic infection .
Before our study, no sequence or related antigenic variations had been reported in T. denticola msp between patients. The differences in amino acid sequences of MSP that were predicted in this study can consistently modify its function during the pathogenesis of periodontal disease with regard to binding capacity to cells and extracellular matrix and to evasion of host responses by T. denticola. Further studies are necessary to demonstrate that the predicted antigenic variability of MSP regulates the pathogenesis of chronic periodontal disease, as has been hypothesized for the Tprk antigen of T. pallidum during the development of syphilis.
This work was supported by grant "RFO 2009" from the University of Bologna to VS
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