Porphyromonas gingivalis (Pg) is one of the main pathogens in chronic periodontitis (CP). Studies on the immunogenicity of its virulence factors may contribute to understanding the host response to infection. The present study aimed to use in silico analysis as a tool to identify epitopes from Lys-gingipain (Kgp) and neuraminidase virulence factors of the Pg ATCC 33277 strain. Protein sequences were obtained from the NCBI Protein Database and they were scanned for amino acid patterns indicative of MHC II binding using the MHC-II Binding Predictions tool from the Immune Epitope Database (IEDB). Peptides from different regions of the proteins were chemically synthesized and tested by the indirect ELISA method to verify IgG immunoreactivity in serum of subjects with CP and without periodontitis (WP). T cell epitope prediction resulted in 16 peptide sequences from Kgp and 18 peptide sequences from neuraminidase. All tested Kgp peptides exhibited IgG immunoreactivity whereas tested neuraminidase peptides presented low IgG immunoreactivity. Thus, the IgG reactivity to Kgp protein could be reaffirmed and the low IgG reactivity to Pg neuraminidase could be suggested. The novel peptide epitopes from Pg were useful to evaluate its immunoreactivity based on the IgG-mediated host response. In silico analysis was useful for preselecting epitopes for immune response studies in CP.Electronic supplementary materialThe online version of this article (10.1186/s13568-019-0757-x) contains supplementary material, which is available to authorized users.
Background: Periodontitis is a progressive inflammatory process, and its pathogenesis is related to the presence of a dysbiotic subgingival biofilm that elicits the immune response. Porphyromonas gingivalis is a keystone pathogen, and its Lys-gingipain (Kgp) virulence factor is involved in the pathogen-host interaction through the production of cytokines by host cells, but the specific mechanisms of this interaction have not been elucidated. The present study evaluated the in vitro production of interferongamma (IFN-), interleukin (IL)-6, and IL-1 cytokines in response to antigenic stimulation of peripheral blood mononuclear cells (PBMCs) with novel Kgp synthetic peptides. Methods:Our previous in silico study predicted 16 immunogenic peptides from Kgp protein. Nine peptides derived from different regions of the protein were chemically synthesized. The synthetic peptides Kgp12, 17, and 18 were selected based on the immunoglobulin G immunoreactivity in the serum of patients with periodontitis (P) and individuals without periodontitis (WP), and they were used in in vitro stimulation of PBMC derived from groups P and WP. Enzyme-linked immunosorbent assay and microsphere-based flow cytometric assay were used to verify the levels of the cytokines produced in PBMC cultures after 48 hours.Results: Kgp12, 17, and 18 peptides induced lower production of IFN-. Kgp12 induced higher levels of IFN-in WP than in P individuals. Kgp12 induced higher production of IL-6 and IL-1 compared with the other stimuli. Conclusion:The novel Kgp synthetic peptides tested herein are immunogenic peptides (epitopes) since they induced the production of cytokines by PBMC and therefore may be useful tools in evaluating the pathogen-host interaction. K E Y W O R D Scytokines, immunology, microbiology, periodontitis J Periodontol. 2019;90:993-1001.
A periodontite é uma doença inflamatória que acomete os tecidos de suporte do dente,podendo levar à reabsorção do osso alveolar, destruição de fibras colágenas e, por fim, perdadentária (Lindhe, 1999). Esta enfermidade apresenta etiologia multifatorial, tendo como umdos principais agentes etiológicos a presença de um biofilme disbióticona região subgengival(Hajishengalis, 2014)).Dentre os microrganismos presentes no biofilme, destaca-se Porphyromonasgingivalis, que é um bacilo gram-negativo, anaeróbio estrito, imóvel, intensamenteproteolítico e um importante indutor de inflamação (Mayer et al., 2013). É um patógenochavena disbiose oral e é o microrganismo mais estudado em sua relação com a periodontitecrônica (Hajishengallis & Lambris, 2012).P. gingivalis possui uma ampla gama de fatores de virulência, como hemaglutininas(Gao et al., 2010), LPS, fímbrias, polissacarídeo de superfície resistente ao complemento eproteases que degradam moléculas sinalizantes e citocinas (Preshaw & Taylor, 2011).As neuraminidases são proteínas sintetizadas por microrganismos patogênicos,incluindo P. gingivalis, e são consideradas fatores de virulência, contribuindo para a suacapacidade de induzir a doença periodontal (Teughels et al., 2011; Li et al., 2012).Por outro lado, o reconhecimento desses fatores de virulência pelo sistema imune dohospedeiro ocorre por um sistema complexo de reconhecimento e sinalização da imunidadeinata, culminando na apresentação de antígenos para os linfócitos T e ativação da imunidadeinata. A maioria dos linfócitos T reconhece apenas peptídeos lineares curtos, pois seusreceptores de antígenos (TCR) são específicos para antígenos apresentados por moléculas doMHC presentes na superfície das células apresentadoras de antígeno (APC) e tais moléculasse ligam a peptídeos. Os linfócitos T CD4+ reconhecem peptídeos provenientes de proteínasextracelulares, apresentados pelas moléculas MHC de classe II (Abbas et al., 2011).Nos seres humanos, os genes que codificam o MHC localizam-se no braço curto docromossoma 6. O HLA (MHC humano) de classe II possui três loci de genes, denominadosHLA-DP, HLA-DQ e HLA-DR. Cada lócus contém vários genes separados denominados Aou B, que codificam as cadeias α ou β, respectivamente. A nomenclatura do alelo HLAconsidera o enorme polimorfismo (variação entre indivíduos) identificado por métodossorológicos e moleculares (Abbas et al., 2011).Diante do exposto, o presente trabalho buscou identificar os epítopos peptídicosimunogênicos com afinidade para HLA, em seguida sintetizar as sequências peptídicas paradepois utilizar em experimentos com cultivo celular para a compreensão do papel daneuraminidase na patogênese da periodontite crônica, bem como para posterior utilização emensaios imunoenzimáticos que auxiliem no diagnóstico da doença.
Introduction: Periodontitis is a multifactorial disease, characterized by an inflammatory response of the periodontal tissues to a dysbiotic biofilm in the subgingival surface. The presence of keystone pathogens, such as Porphyromonas gingivalis, is one of the main causes of dysbiosis, although the host response is preponderant in the beginning and the progression of the disease. The periodontal treatment is based on the mechanic scaling of the biofilm but using of chemicals adjuvants has been preconized. However, there are many restrictions related to the antibiotics and other chemical adjuvants usage, which makes the use of herbal medicines for this purpose very promising. In addition, many herbal medicines have been used in the folk medicine, with various biologic effects.
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