| Objectives: Periodontitis is an infectious disease associated with pathogenic microorganisms and mediated by host with multiple factors.It is characterized by inflammation of periodontal soft tissue,periodontal pocket formation,attachment loss and alveolar bone resorption,which can lead to tooth loosening and loss in severe cases.Porphyromonas gingivalis(P.gingivalis)is the main pathogen of periodontitis.It can use a variety of virulence factors produced by it to interact with other microorganisms to promote the imbalance of periodontal microenvironment homeostasis.Results in excessive immune and inflammatory response and further aggravate the destruction of periodontal tissue.Histatin 5 is a 24-amino acid fragment(DSHAKRHHGYKRKFHEKHHSHRGY)naturally occurring cationic antimicrobial peptide found in saliva.In our study,we found that the concentration of histatin 5 was negatively correlated with the content of P.gingivalis in suand subgingival plaque,suggesting that histatin 5 had antibacterial activity against P.gingivalis,but the exact mechanism remains unclear.Most studies on the antibacterial mechanism of histatin 5 have been conducted in Candida albicans,while P.gingivalis is rarely reported.In this study,we focused on the antibacterial activity and mechanism of histatin 5 at low concentrations(< MIC)against P.gingivalis,which was closer to the actual effective concentration in oral cavity.As a kind of heme auxotrophic bacteria(HAB),P.gingivalis lacks the ability to synthesize protoporphyrin-IX by itself and needs to capture the essential nutrient heme from the environment.Heme is an iron porphyrin compound containing ferrous ions,which is mainly formed by the oxidative hydrolysis of hemoglobin in erythrocytes.The process of heme uptake by P.gingivalis from erythrocytes is as follows: first,P.gingivalis adheres to erythrocyte directly through the Hemagglutinin(HA)protein;Subsequently,P.gingivalis secrete proteases such as gingipains to cleave the erythrocyte membrane,releasing hemoglobin and oxidative hydrolysis to heme.Finally,the heme binding protein Hmu Y,which is present on the outer membrane and outer membrane vesicles of P.gingivalis,traps heme and transports it from the bacterial outer membrane to the cytoplasm through its cognate outer membrane transport receptor Hmu R.hag A,hag B,and hag C are genes encoding hemagglutinin proteins.Among them,gene hag A encodes the adhesin domain of P.gingivalis together with rgp A and kgp,which can adhere and agglutinate erythrocytes.Hmu Y protein is a unique heme binding protein of P.gingivalis that is regulated by iron level.It can bind to free heme in the environment and protect heme from degradation by other substances in the environment by changing the molecular conformation.In the process of uptake of heme from erythrocytes by P.gingivalis,hemagglutinin and Hmu Y play an important role in agglutination of erythrocytes and heme capture.When the functions of hemagglutinin and Hmu Y change,the ability of P.gingivalis to uptake heme will be affected.In this study,we aimed to explore the mechanism of physiological concentration of histatin 5 inhibiting the growth of planktic P.gingivalis from the perspective of heme uptake.Research methods:1.The minimum inhibitory concentration(MIC)of histatin 5 against the growth of P.gingivalis W83 was determined by broth microdilution method.2.P.gingivalis W83 was co-cultured with histatin 5 at 1/4 MIC and 1/2 MIC,and the absorbance at 600 nm at different time points was detected and the growth curve was drawn.3.CCK-8 assay was used to detect the cell viability of P.gingivalis cells after coculturing with 1/4 MIC,1/2 MIC,and MIC of histatin 5 for 48 hours.4.The outer membrane structure of P.gingivalis W83 after co-culture with 25 μg/m L histatin 5 for 24 hours was detected by transmission electron microscopy.5.Pull-down assay was used to detect the protein-protein interaction between histatin 5and P.gingivalis W83,and liquid chromatography mass spectrometry(LC-MS)was used to detect the binding amino acids.6.The concentration of histatin 5 in P.gingivalis was detected by ELISA after 12 h,24 h,48 h and 72 h of co-culture.7.The hemagglutinin activity of P.gingivalis was detected by erythrocyte agglutination test after co-culture with 1/4 MIC and 1/2 MIC of histatin 5 for 24 hours.8.The heme uptake ability of P.gingivalis was detected by heme uptake assay after 24 h of co-culture with 1/4 MIC and 1/2 MIC of histatin 5.9.The m RNA expression levels of P.gingivalis Hag A/B/C and Hmu Y were detected by q RT-PCR after co-culture with 1/4 MIC and 1/2 MIC histatin 5 for 24 h.10.P.gingivalis Hmu Y rabbit polyclonal antibody was prepared,and Western blot was used to detect the expression of P.gingivalis Hmu Y protein after co-culture with 1/4 MIC and 1/2 MIC histatin 5 for 24 hours.Results:1.Histatin 5 inhibited the growth of P.gingivalis W83 at a minimum concentration(MIC)of 100 μg/m L,a quarter MIC of 25 μg/m L,and a half MIC of 50 μg/ ml.The 1/4MIC value was close to the physiological concentration of histatin 5 detected in the saliva of periodontal healthy people.2.The growth curve showed that 25 μg/m L and 50 μg/m L histatin 5 inhibited the growth of planktic P.gingivalis.3.CCK-8 results showed that 25 μg/m L and 50 μg/m L histatin 5 could significantly reduce the cell viability of P.gingivalis(P < 0.05).4.Transmission electron microscopy results showed that 25 μg/m L histatin 5 did not cause damage to bacterial membrane structure.5.Pull-down assay and liquid chromatography mass spectrometry showed that histatin5 could bind to amino acid fragments of Rag A and Rag B proteins of P.gingivalis.6.The results of ELISA showed that the concentration of histatin 5 did not change significantly at 12 h,24 h,48 h,and 72 h(P > 0.05),suggesting that P.gingivalis and its growth metabolites could not degrade histatin 5.7.The results of hemagglutination test showed that the hemagglutination activity of P.gingivalis was inhibited after co-culture with 25 μg/m L and 50 μg/m L histatin 5 for 24 h.8.The uptake of heme by P.gingivalis was significantly lower in the 25 μg/m L and 50μg/m L histatin 5 groups than in the control group(P < 0.05).9.The results of q RT-PCR showed that 25 μg/m L and 50 μg/m L histatin 5 downregulated the m RNA expression levels of Hag A/B/C and Hmu Y in P.gingivalis(P <0.05).10.Western blot results showed that 25 μg/m L and 50 μg/m L histatin 5 significantly down-regulated the protein expression of P.gingivalis Hmu Y(P < 0.05).Conclusions: Histatin 5 at physiological concentration could inhibit the uptake of heme by P.gingivalis through inhibiting hemagglutinin activity and down-regulating the protein expression of Hmu Y. |
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