DOI: https://doi.org/10.21203/rs.3.rs-1363677/v1
Background: To analyze the distribution of Porphyromonas gingivalis (P. gingivalis) kgp genotype in subgingival plaque of patients with diabetic periodontitis. The genetic polymorphism of the kgp gene in diabetic periodontitis was explored, and the correlation between different genotypes and diabetic periodontitis was obtained.
Methods: Subgingival plaque samples were collected from 49 patients with periodontitis (21 with diabetes, 28 with non-diabetic periodontitis) and 11 healthy periodontal subjects. DNA was extracted, and the positive samples of P. gingivalis were screened by PCR with specific primers of 16S rRNA gene of P. gingivalis, and the fragment of kgp catalytic domain was obtained by PCR with specific primers of kgp. The genotype of kgp was determined by Mse I digestion of the catalytic domain fragment of kgp, and the experimental data were analyzed by the Pearson χ2 method.
Results: Among the 11 healthy periodontal population samples collected in this study, 4 cases (36.4 %) of P. gingivalis were detected. Among 49 subgingival plaque samples of periodontitis, 47 patients (95.9 %) were detected with P. gingivalis. There was a statistical difference between the two. Among the 47 samples with P. gingivalis, the detection rate of kgp I was 57.4 %, the detection rate of kgp II was 31.9 %, and the detection rate of kgp I + II was 10.6 %. The kgp genotype was not associated with diabetes. There was no significant difference in the kgp genotypes between the two groups with different PD depths.
Conclusion: P. gingivalis is associated with periodontitis. P. gingivalis kgp gene polymorphism exists in patients with diabetic periodontitis subgingival plaque.
Periodontitis is a common disease of the oral cavity, one of the most common causes of tooth loss [1] . In periodontitis, oral bacterial biofilm is the most important pathogenic factor, and its main pathogenic mechanism is that various bacteria cause damage to the host tissue directly or indirectly, ultimately leading to periodontitis [2] . Risk factors for periodontitis (local or systemic) play an important role in the progression of the disease [1] , especially in poorly controlled diabetes[3,4].
Diabetes is a heterogeneous metabolic disorder characterized by elevated blood glucose, the most common of which is type 2 diabetes that develops as a result of insulin resistance in body tissues[3]. Epidemiological studies have shown that the periodontitis susceptibility of diabetic patients is three times that of non-diabetic patients due to the fact that diabetes accelerates the production of advanced glycation end products (AGEs) [4] , which can increase the risk of periodontal bacterial red complexes (including Porphyromonas gingivalis, P. gingivalis) [5] that can damage periodontal tissues by increasing the levels of the red complex of periodontal bacteria [3] .
P. gingivalis is a gram-negative, melanin-producing obligate anaerobic bacterium, the main pathogen of chronic periodontitis. It is also a marker of periodontitis disease progression [6, 7] . Pathogenicity mainly hydrolyzes proteins and degrade collagen to produce volatile fatty acids. P. gingivalis can resist the immune response of the animal organism to some extent, reducing the lethality of antibodies in serum to bacteria, inhibiting the phagocytosis of bacteria by phagocytes, and weakening the chemotaxis of leukocytes to P. gingivalis. It also has the ability to cause sepsis in animals and promote the production of local abscesses or the occurrence and development of localized abscesses. Carter et al. [8] found that P. gingivalis can affect human gene expression, increasing the expression of genes associated with Alzheimer's disease, diabetes, and cardiovascular disease in the human body, thus affecting the susceptibility of related diseases. This suggests that P. gingivalis may be associated with diabetes.
Recent studies have confirmed that gingivalin, fimbriae, and lipopolysaccharides are important virulence factors of P. gingivalis. Among them, gingivalin is one of the most important virulence factors of P. gingivalis and plays a vital role in mediating the interaction between bacteria and hosts [9, 10] . It destroys the human immune system (e.g., complement molecules, immunoglobulins, and cytokines) by decomposing, thereby affecting the antibacterial activity of mucosal peptides and promoting the inhibition of the inflammatory response of human gingival fibroblasts [11, 12] .
Gingipain can be divided into two main categories: the first is arginine-specific cysteine proteases (gingipain R, Rgp), and the second is lysine-specific cysteine proteases (gingipain K, Kgp). Among them, Kgp has the most significant effect on virulence [13, 14] . Compared with other virulence factors, researchers found that Kgp can make human cells produce higher levels of IL-6 and IL-1β in vitro experiments [15] . Nadkarni et al. [16] studied the kgp gene of the standard strain P. gingivalisW83, hg66,w83v, and 381. The results revealed that the kgp gene of P. gingivalis was divided into three regions: the propeptide region, the catalytic domain, and the adhesion region. The catalytic domain, also known as the protease region, is the main site that encodes lysine-gingivalin. Beikler et al. [14] isolated and cultured 23 strains of P. gingivalis from clinical samples and sequenced the kgp gene. The results showed that the catalytic domain (kgp catalytic domain, kgp-cd) of the kgp gene was quite different, while the prepeptide region and adhesion region were relatively conservative. It was also found that the lysine-gingivalin activity of different P. gingivalis clinical isolates was different, suggesting that the gene polymorphism of kgp-cd may be related to the difference of lysine-gingivalin activity. According to the difference in enzyme digestion sites of the catalytic domain fragment, P. gingivalis can be divided into type 2 (kgp-cd I-II). In a review of diabetes and periodontal disease, Ângelo et al. [17] stated that periodontitis is one of the major oral manifestations of diabetes. Moreover, periodontal destruction is more severe in diabetic patients (type 1 or type 2) than in non-diabetic patients. However, the historical literature has only examined P. gingivalis kgp in periodontitis, and the specific distribution of different kgp genotypes of P. gingivalis in patients with diabetic periodontitis has not been reported. Therefore, it is of great significance to study the kgp genotypes of P. gingivalis strains associated with diabetes to reveal the pathogenic mechanism of P. gingivalis in diabetic patients with periodontitis. In this study, the distribution of P. gingivalis kgp genotype in periodontitis with diabetes was detected by catalytic domain primer specific PCR and enzyme digestion, and Pearson χ2 test was used to study the correlation between different genotypes and clinical symptoms.
In this study, the distribution of P. gingivalis kgp gene polymorphism in subgingival plaque of patients with diabetic periodontitis was observed by cross-sectional study. The Ethical Committee approved the study of the Institute of Dental Disease Control and Prevention of Xuhui District [Shanghai and Xufang Colombian (2020) No.1]. Each participant read and signed the informed consent before sampling.
Subgingival plaque samples were collected from patients with diabetic periodontitis treated in the Department of Periodontal Medicine of the Institute of Dental Control in Xuhui District from January 2020 to November 2020, and 49 subgingival plaque samples were obtained, which were divided into the diabetic periodontitis group (21 cases) and the non-diabetic periodontitis group (28 cases). The dental plaque samples of 11 healthy periodontal subjects were analyzed.
Samples were taken from 49 periodontitis patients aged 27-68 years in the Department of Periodontology, Xuhui District, Shanghai, China.
a. Diabetic periodontitis group: extensive periodontitis, with the diagnostic criteria defined by the European Federation of Periodontal Diseases and the American Society of Periodontal Diseases ; ≥ 40 years old; complete residual teeth I > 15; no smoking or smoking cessation for more than one year; patients who were diagnosed with type 2 diabetes for more than two years, or whose general health did not exclude individual abnormal blood lipid indicators, but were not diagnosed with metabolic syndrome; non-communicable diseases such as active tuberculosis; sign informed consent.
b. Non-diabetic periodontitis group: extensive periodontitis, whose diagnostic criteria were defined by the European Federation of Periodontal Diseases and the American Society of Periodontal Diseases; ≥ 40 years old; complete residual teeth I > 15; no smoking or smoking cessation for more than one year; hbAlc (glycosylated hemoglobin ) < 7 %; non-communicable diseases such as active tuberculosis ; sign informed consent.
c. Inclusion criteria for periodontal health group: negative bleeding on probing; probing depth < 3mm; no attachment loss; no imaging bone loss; hbAlc < 7 %; non-communicable diseases such as active tuberculosis; sign informed consent.
A history of periodontal system treatment within one year; continuous administration of antibiotics for more than one week within six months before the examination; having multiple bad restorations in the mouth affecting the examiner; women pregnancy or lactation
One mesiobuccal site of anterior and posterior teeth with the most obvious gingival swelling was selected, and the subgingival plaque was collected to avoid adjacent sites of missing teeth and subgingival sites with prosthesis. After aseptic and moisturizing teeth, the subgingival plaque of the tested tooth was taken with a sterile subgingival scraper, suspended in 1 mL PBS buffer, and frozen at -20 °C for DNA extraction. At the same time, periodontal doctors used periodontal probes to record periodontal parameters at six sites of the tested tooth, including plaque index (PI), probing bleeding (BOP), probing depth (PD), gingival retraction (GR), and clinical attachment loss CAL).
DNA was extracted from 49 clinical samples by a low-dose bacterial genomic DNA extraction kit (Kaijie Biotechnology (Shanghai) Co., Ltd.), and the obtained DNA was frozen at -20 °C.
The 16S rRNA primers of P. gingivalis [18] were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (the primer sequences are shown in Table 1). The polymerase chain reaction amplification system: Ex Hot Start Version 25 μL (Shanghai Sangon Bioengineering Technology Service Co., Ltd.); 2 μL each primer (10 μM); DNA template 2μL, add ddH2O to 50μL. The reaction conditions were as follows: pre-denaturation at 94 °C for 10 min, denaturation at 94 °C for 60 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, 35 cycles, extension at 72 °C for 5 min. In the amplification reaction, the standard strains P. gingivalis W83 and ATCC 33277 were used as a positive control, and the same volume of sterile double distilled water was used as blank control. Take 5μL polymerase chain reaction mixture 1.2 % agarose gel electrophoresis, gel imaging analyzer imaging analysis (electrophoresis Figure 1).
The specific primers for the catalytic domain of the kgp gene were designed by reference to Beikler [14] (see Table 1 for primer sequences and Figure 2 for electrophoresis diagrams). The polymerase chain reaction amplification system: Ex Hot Start Version 25 μL (Shanghai Sangon Bioengineering Technology Service Co., Ltd.); 2 μL each primer (10 μM); DNA template 2μL, add ddH2O to 50μL. The reaction conditions were as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, 30 cycles, extension at 72 °C for 10 min. In the amplification reaction, the standard strains P. gingivalis W83 and ATCC 33277 were used as a positive control, and the same volume of sterile double distilled water was used as blank control. Take 5μL polymerase chain reaction mixture 1 % agarose gel electrophoresis, gel imaging analyzer imaging analysis.
Table 1
Specific primer sequence of P. gingivalis 16S rRNA and kgp catalytic domain
primer |
sequence |
size/bp |
P. gingivalis 16SrRNA |
F 5’- TGTAGATGACTGATGGTGAAAACC-3’ |
197 |
R 5’-ACGTCATCCACACCTTCCTC-3' |
||
kgp cd |
F 5’-GAACTGACGAACATCATTG-3’ |
890 |
R 5’-GCTGGCATTAGCAACACCTG-3’ |
Restriction endonuclease Mse I was designed by Beikler [14] (see Table 2 for enzyme sites). The PCR products of the catalytic domain of the kgp gene were directly digested by Mse I and detected by 1.2 % agarose gel electrophoresis (see table 3 for typing basis and test results and figure 3 for electrophoresis chart). 50 μL enzyme digestion system: 1 μL of MseI (Shanghai Development Laboratory Reagent Co., Ltd.); 10xNEBuffer (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) 5 μL; polymerase chain reaction product 1μg, add ddH2O to 50μL. The reaction conditions were as follows: enzyme digestion at 37 °C for 15 min. In the enzyme digestion reaction, the standard strains P. gingivalis W83 and ATCC 33277 were used as the positive control, and the same volume of sterile double distilled water was used as the blank control. Take 5 μL enzyme reaction mixture 1.2 % agarose gel electrophoresis, gel imaging analyzer imaging analysis.
Table 2
Mse I restriction site
restriction enzyme cutting site |
5’-3’ |
135 |
125 CAGCCAACCAT▼TAAATATGGTATGCA |
423 |
412 AATAACTCGCGT▼TAAGGAGAAAGG |
Table 3
Classification of two kgp cd genotypes of P. gingivalis
genotype |
Mse I |
|
result of survey |
restriction enzyme cutting site |
segments |
segment size/bp |
|
Type-Ⅰ |
+ + |
3 |
447+288+135 |
Type-Ⅱ |
- - |
1 |
890 |
According to the experimental data, the detection rate of P. gingivalis, the detection rate of kgp, and the detection rate of the two genotypes of kgp were analyzed. Then we further investigated whether there was a statistical correlation between the detection of different kgp genotypes and diabetes. If all theoretical frequencies in the four tables are greater than or equal to 5, the Pearson χ2 test method is used for analysis. If the number of lattices with theoretical frequency 1 ≤ T < 5 in the table exceeds 1 / 5 of the total lattices, the Fish exact probability method is used for analysis. The data analysis was completed by SAS 8.2 statistical software, and the test level was α = 0.05.
In this study, a total of 60 samples were collected, including 21 cases of diabetic periodontitis, 28 cases of non-diabetic periodontitis, and 11 cases of healthy periodontal population. A total of 51 cases of P. gingivalis were detected (21 cases of diabetic periodontitis, 26 cases of non-diabetic periodontitis, and 4 cases of periodontal healthy people), and the total detection rate was 85 % (100 % in diabetic periodontitis, 92.9 % in non-diabetic periodontitis and 36.4 % in periodontal healthy people). In addition, in 49 periodontitis samples (including diabetic periodontitis and non-diabetic periodontitis), a total of 47 cases of P. gingivalis were detected, with a detection rate of 95.9 %. In the statistical analysis, we found that the detection rate of P. gingivalis in periodontitis patients was significantly higher than that in periodontal healthy people, and there was a statistical difference (see table 4).
In 51 samples with P. gingivalis detected, the catalytic domain fragment of the kgp gene was obtained by PCR and digested by Mse I. Finally, the kgp genotype was observed by 1.2 % agarose gel electrophoresis. PCR and enzyme digestion results showed that the detection rate of kgp was 100 % in 51 samples with P. gingivalis. The detection of kgp genotype in 47 patients with periodontitis was as follows: type I 27 / 47 (57.4 %), type II 15 / 47 (31.9 %), type I + II 5 / 47 (10.6 %). Among them, type I 10 / 21 (47.6 %), type II 9 / 21 (42.9 %), type I + II 2 / 21 (9.5 %) in diabetic periodontitis group. Non-diabetic periodontitis group type I 17 / 26 (65.4 %), type II 6 / 26 (23.1 %), type I + type II 3 / 26 (11.5 %). (The specific detection results are shown in Table 5). In addition, the detection of kgp genotype in four periodontal healthy people was 1 / 4 (25 %) of type I and 3 / 4 (75 %) of type II.
In the 47 samples of periodontitis patients with positive kgp obtained in this study, Pearson χ2 test analysis showed no significant correlation between kgp genotype and diabetes. In periodontal pockets of different depths, there was no significant difference in kgp genotypes between the diabetic periodontitis group and the non-diabetic periodontitis group (P > 0.05) (see Table 6, Table 7).
Table 4
Analysis of correlation between detection of P. gingivalis and periodontitis
|
P. gingivalis positive |
P. gingivalis negative |
Total |
Periodontitis group |
47* |
2 |
49 |
Periodontal healthy people |
4 |
7 |
11 |
Total |
51 |
9 |
60 |
*: Statistically relevant,P<0.05
Table 5
Correlation analysis between kgp genotype and periodontitis patients with diabetes mellitus group
|
Type kgp I |
Type kgp II |
Type kgp I+II |
Total |
Diabetic periodontitis group |
10 |
9 |
2 |
21 |
Non-diabetic periodontitis group Periodontal healthy people |
17 1 |
6 3 |
3 0 |
26 4 |
Total |
28 |
18 |
5 |
51 |
Table 6
Table of genotyping distribution in periodontitis with diabetes mellitus
|
genotyping |
Total |
|||
Type I |
Type II |
Type I+II |
|||
PD |
<3mm |
2 |
0 |
0 |
2 |
3-5mm |
4 |
5 |
1 |
10 |
|
>5mm |
4 |
4 |
1 |
9 |
|
Total |
10 |
9 |
2 |
21 |
Table 7
Table graph of genotyping distribution in periodontitis without diabetes
|
genotyping |
Total |
|||
Type I |
Type II |
Type I+II |
|||
PD |
<3mm |
5 |
2 |
1 |
8 |
3-5mm |
6 |
3 |
1 |
10 |
|
>5mm |
6 |
1 |
1 |
8 |
|
Total |
17 |
6 |
3 |
26 |
P. gingivalis is the main pathogen of periodontitis. Its virulence factors include fimbriae, capsule, extracellular vesicles, lipopolysaccharide, gingipain, and endotoxin, playing an important role in the progression of periodontitis. In addition, P. gingivalis can help bacteria adhere, colonize on periodontal tissue and subgingival plaque surface, and further invade tissue cells and play a toxic role [2] . Makiura et al.[19] studied the changes in blood glucose in patients with type 2 diabetes after subgingival curettage and found that the presence of P. gingivalis in the periodontal pocket will affect blood glucose levels. Carter et al. found that P. gingivalis can cause up-regulation of gene expression related to Alzheimer's disease, diabetes, and cardiovascular disease in the human body, thereby affecting the susceptibility of related diseases [8] . P. gingivalis may be associated with diabetes. In previous literature, researchers analyzed the distribution of P. gingivalis in periodontitis, but the detection of Pg in different populations was different. Kulkarni et al. [20] found that the detection rate of P. gingivalis was 93.3 % in periodontitis patients with periodontal pocket 5mm by PCR. Only 1 case of P. gingivalis was detected in periodontal healthy people. Mahendra et al. [21] detected subgingival plaque in patients with periodontitis after coronary artery bypass grafting and found that the detection rate of P. gingivalis was 64.71 % in sites with periodontal pocket > 5mm. Similarly, Marin et al. [22] used PCR to detect subgingival plaque results showed that the detection rate of P. gingivalis in patients with periodontal pockets > 5mm was 90 %; the detection rate of P. gingivalis in periodontal healthy people was 83.3 %. Mínguez et al. [23] used the bacterial culture method to culture P. gingivalis in dental plaque of 45 patients with chronic periodontitis in Morocco, and the detection rate was 84.4 %. Kumawat et al. [24] performed PCR detection on subgingival plaque samples from healthy people, patients with chronic periodontitis, and aggressive periodontitis. The detection rates were 10% in the healthy periodontal population, 73.3% in patients with chronic periodontitis, and 80% in patients with aggressive periodontitis, respectively. In 2019, Sen et al. collected subgingival plaque from patients with periodontitis and isolated and cultured P. gingivalis. They found that the detection rate of P. gingivalis in patients with diabetic periodontitis (68 %) was higher than that in patients with non-diabetic periodontitis (42 %), which was statistically significant [25] . In this study, the detection rate of P. gingivalis in diabetic periodontitis was 100 %, and the detection rate in non-diabetic periodontitis was 92.9 %. Compared with previous studies, it may be related to patients' deeper overall probing depth. The detection rate of P. gingivalis in healthy periodontal people was 36.4 %, similar to previous studies. There was a statistical difference in the detection rate of P. gingivalis between the periodontitis group and periodontal healthy people, indicating that P. gingivalis was associated with periodontitis.
Ozmeric et al. [26] found 11 lysine residues and three arginine residues in human heme by molecular typing. As the preferred substrate of kgp, heme can combine with hemoglobin to degrade hemoglobin, condense hemoglobin and produce melanin. When P. gingivalis was cultured on the blood agar plate, P. gingivalis had the ability to produce melanin, and P. gingivalis often showed black colonies, while P. gingivalis could cause heme agglutination on the cell surface. Previous studies have proved that P. gingivalis strain with kgp gene defect cannot produce melanin, and its blood cell agglutination ability is significantly weakened. This demonstrated that kgp played an important role in the colonization of P. gingivalis and affected its virulence and growth. Haraguchi et al. found that gingival protease K played an important role in the downregulation of biofilm-associated with Actinobacillus in the oral cavity. The competitive relationship between gingivalis Actinobacillus was closely related to plaque maturation on the surface of oral teeth [27] . At the same time, gingival protease K is involved in producing an enzyme (unknown structure), which can destroy the aggregation process of actinobacteria and make P. gingivalis gain a competitive advantage. Michael et al. pointed out that RgpA / B and Kgp were the main virulence factors of P. gingivalis, which played a decisive role in colonization and penetration of bacteria into host tissues and establishing ecological disorders and diseases [28] . Pomowski et al. [29]also suggested that Kgp and Rgp are responsible for 85 % of the extracellular protein hydrolysis activity and have higher concentrations in the gingival crevicular fluid of periodontitis patients. Among them, Kgp has the highest activity, which is involved in the survival of bacteria and the development of periodontitis. At the same time, the authors found that Kgp, as a multi-domain protein, contains a signal peptide, N-terminal propeptide domain (NPD), catalytic domain (CD), immunoglobulin incomplete family domain (IgSF), adhesion domain, C-terminal domain (CTD) and so on. Liu et al. found in the mouse experiment that Rgp and Kgp had a synergistic effect on P. gingivalis-mediated cell migration and the expression of pro-inflammatory mediators [30]. In previous studies, we found that there were mainly two methods for genotyping of kgp. One is that the specific sequences in the catalytic domain were processed by restriction endonuclease MseI proposed by Beikler in 2003 to obtain gene fragments with different lengths, and they were divided into type I and type II [14] according to the size of the fragments. One is the classification based on the C-terminal DNA sequence differences of kgp [17]. Because the classification method proposed by Beikler has many applications and good repeatability, this paper uses this method as the standard for classification.
Chen et al. found that the detection rate of type I and type II of kgp gene in the gingivitis group was 79 % and 21.1 %, respectively. In the gingival health group, the detection rate of kgp gene type I was 22.2 %, and the detection rate of type II was 77.8 %. The statistical test showed that the distribution difference of kgp genotype was statistically significant [31] . In this study, through the analysis of clinical samples, we found that the gene polymorphism and distribution of kgp genotype in P. gingivalis with diabetic periodontitis, type I accounted for 57.4 %, type II accounted for 31.9 %, type I + II accounted for 10.6 %. Among them, type I accounted for 47.6 %, type II accounted for 42.9 %, and type I + II accounted for 9.5 % in the group with diabetic periodontitis. In the non-diabetic periodontitis group, type I accounted for 65.4 %, type II accounted for 23.1 %, type I + II accounted for 11.5 %; among the healthy periodontal population, type I accounted for 25 %, and type II accounted for 75 %. Although there was no significant difference in statistics, the detection rate of kgp I was generally higher than that of type II in patients with periodontitis, but in periodontal healthy people, the opposite was consistent with previous studies. In addition, it is worth noting that the detection rate of the kgp gene in periodontitis with diabetes and periodontitis group was 100 %, of which 42 cases were detected alone, and 5 cases were detected in combination. In previous studies on dental plaque, it was found that in the plaque of a single host, the detected P. gingivalis was often only one clone of the kgp gene [14, 32, 33] . Our results are different, which needs further study.
Beikler detected the kgp gene in 102 clinical samples. The results showed no significant difference in kgp type I and type II detection rate under different periodontal pocket depths and probing bleeding. At the same time, there was no significant difference in proteolytic activity between Kgp-I and Kgp-II [14] . Chen et al. also used the same method and found that kgp gene type I was associated with gingivitis [31]. This study used P. gingivalis W83 strain and ATCC33277 strain as controls. It was found that the virulent strain W83 was classified as kgp I type, while the non-virulent strain ATCC33277 was classified as kgp II type. Previous studies have found that the detection rate of kgp I P. gingivalis in periodontally ill people is much higher than that in healthy people [14, 16, 31] . This study also confirms this conclusion. In addition, the results of this study showed no correlation between the kgp genotype and periodontal probing depth and diabetes, which may be related to the insufficient sample size, which needs to be further proved in the future. However, we can see from the existing results that the kgp genotype may be different in P. gingivalis with different virulence, and its distribution may have certain rules. Therefore, follow-up studies need to focus on individuals carrying kgp type I P. gingivalis, which may increase periodontal disease incidence. The relationship between kgp genotype and the occurrence and progression of periodontal diseases remains to be further studied
In recent years, some scholars have studied specific inhibitors for Kgp, namely KYT-1 ((Cbz) -Lys-Arg-CO-Lys-N (NH3) 2). On this basis, Kataoka et al. designed a drug KYT-41 with a strong antibacterial ability and confirmed its efficacy and safety through animal experiments [34] . In this study, according to the design of Beikler, whether the two kgp genotypes have the same efficacy in the use of the inhibitor will be further verified in future experiments. The future research will focus on the correlation of kgp genes with different structures in the pathogenesis of diseases, which lays the foundation for improving the research and development of Kgp inhibitors in the future.
This study has shown that P. gingivalis is associated with periodontitis which consistent with previous studies. Our study findings highlight the presence of P. gingivalis kgp gene polymorphism in patients with diabetic periodontitis subgingival plaque. This is the first attempt testing presence of P. gingivalis kgp gene polymorphism in patients with diabetic periodontitis. In the future, further experiments will be needed to demonstrate these mechanisms of kgp gene polymorphism.
Kgp: lysine-specific cysteine proteases; Rgp: arginine-specific cysteine proteases; PCR: polymerase chain reaction.
The authors would like to extend their sincere gratitude to their colleagues and the staff at the Shanghai Xuhui District Dental Institute and University Hospital Regensburg for their support.
DG and XM designed and planned the study; WQ supervised the study; DG contributed to data collection and interpretation; DG performed the statistical analysis; DG and XM wrote the manuscript, and RJ and WQ reviewed the manuscript. All authors approved the final version of the manuscript.
This research was funded by Shanghai Medical Key Specialty (ZK2019B12), Scientific Research Project of Xuhui Provincial Commission of Health and Family Planning (SHXH201706), and Shanghai Xuhui District Dental Institute Medical Research Project(SHXYF201902). Shanghai Medical Key Specialty (ZK2019B12) contributed to the design of the study; Scientific Research Project of Xuhui Provincial Commission of Health and Family Planning (SHXH201706) contributed to collection, analysis, and interpretation of data; Shanghai Xuhui District Dental Institute Medical Research Project (SHXYF201902) contributed to the writing of manuscript.
The datasets generated during this study will be available from the corresponding author on reasonable request.
Written informed consent for publication of individual details and accompanying images will be obtained from the trial participants. The consent forms are in the possession of the authors and are available for review by the Editor-in-Chief.
The authors declare that they have no competing interests.
The Ethical Committee approved the study of the Institute of Dental Disease Control and Prevention of Xuhui District [Shanghai and Xufang Colombian (2020) No.1]. Each participant read and signed the informed consent before sampling.
1 Department of Stomatology, Shanghai Xuhui District Dental Institute, 500 Fenglin Road, Shanghai 200032, People’s Republic of China, email: [email protected] . 2 Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Regensburg, Germany, email: [email protected]. 3 Department of Stomatology, Shanghai Xuhui District Dental Institute, 500 Fenglin Road, Shanghai 200032, People’s Republic of China, email: [email protected] . 4 Department of Stomatology, Shanghai Xuhui District Dental Institute, 500 Fenglin Road, Shanghai 200032, People’s Republic of China, email: [email protected]