Study On The Mechanism Of Porphyromonas Gingivalis Outer Membrane Vesicles In Inhibiting The Invasion Of Fusobacterium Nucleatum Into Oral Epithelial Cells

Objective: Periodontitis is a chronic infectious disease of periodontal tissue caused by dental plaque biofilm.It can cause the destruction of periodontal supporting tissues,leading to inflammation,periodontal pocket formation,progressive attachment loss,alveolar bone resorption,and even cause teeth to loosen and fall off,which is one of the main diseases that endanger human oral health.Porphyromonas gingivalis(P.gingivalis)and F.nucleatum(F.nucleatum)play vital roles in the occurrence and development of periodontitis.P.gingivalis is a melanin-producing gram-negative anaerobe that forms a "red complex" with Treponema denticola and Tannerella forsythia,which is one of the main pathogens of periodontal disease.It can invade into host cells through endocytosis,affect the synthesis and secretion of certain cytokines,regulate cell proliferation,apoptosis and other biological behaviors,thereby exerting its pathogenic effects.F.nucleatum(F.nucleatum)is a long fusiform gram-negative anaerobe and is largely detected in patients with periodontal disease.It acts as a "bridge bacteria" connecting the early and late colonizers in the oral cavity and plays an important role in the formation of plaque biofilm and the progression of mixed infections.The expression of a large number of adhesins on the surface of F.nucleatum mediates important biofilm aggregation behavior and interaction with host cells.The interaction between different microbial populations affects the occurrence and development of host diseases.Therefore,it is necessary to explore the characteristics of different pathogenic bacteria and the virulence mechanism of their interaction.As two common oral pathogens,the interaction between P.gingivalis and F.nucleatum has gradually attracted attention.During the growth of P.gingivalis,double-layer spherical membranous vesicles with a diameter of about 50-250 nanometers are continuously secreted from the surface of the bacteria,which called outer membrane vesicles(OMVs).Due to the protection of the vesicle membrane structure,high concentrations of pathogenic factors can avoid being degraded and achieve long-distance transmission.These characteristics make P.gingivalis OMVs more toxic than the parent bacteria.Therefore,P.gingivalis OMVs may represent P.gingivalis to interact with other oral bacteria and affect the functions of them.At present,there have been some experiments in vivo and in vitro to explore the impact of P.gingivalis and F.nucleatum on the host after mixed infection,but there are few reports on the internal mechanism.Therefore,this experiment further established a model of F.nucleatum treated with P.gingivalis OMVs to explore the internal mechanism of P.gingivalis affecting F.nucleatum’s invasion into human immortalized oral epithelial cells(HIOECs).Methods: 1.P.gingivalis and F.nucleatum were mixed to infect HIOECs,and flow cytometry was used to detect the invasion rate of P.gingivalis and F.nucleatum respecttively to observe whether the mixed infection of P.gingivalis and F.nucleatum affects the ability of the two bacteria to invade HIOECs.2.P.gingivalis OMVs were extracted with kit and identified with transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA).A model of F.nucleatum treated with P.gingivalis OMVs before and after heat inactivation was established.Flow cytometry and laser confocal scanning microscope were used to detect the invasion of F.nucleatum into HIOECs after treatment.3.The changes in the morphology of F.nucleatum were observed by scanning electron microscopy(SEM),the changes in the proliferation ability of F.nucleatum were detected with a multifunctional microplate reader.4.The expression of F.nucleatum surface adhesion factors Fad A and Fom A were detected by q RT-PCR and Western blot.5.The changes in the degree of auto-aggregation of F.nucleatum were observed through an optical microscope.Results: 1.After P.gingivalis and F.nucleatum were mixed to infected HIOECs,the invasion rate of P.gingivalis increases,and the invasion rate of F.nucleatum decreases(P<0.05).2.P.gingivalis OMVs were extracted successfully.Compared with the control group,P.gingivalis OMVs can cause a decrease in the invasion rate of F.nucleatum(P<0.05),while heat-inactivated P.gingivalis OMVs cannot cause similar changes.3.Both P.gingivalis OMVs before and after heat inactivation did not cause changes in the morphology and proliferation of F.nucleatum.4.P.gingivalis OMVs can reduce the protein expression of F.nucleatum surface adhesion-related proteins Fad A and Fom A(P<0.05),while P.gingivalis OMVs treated with heat inactivation cannot cause the similar changes.5.P.gingivalis OMVs can reduce the degree of auto-aggregation of F.nucleatum,suggesting that P.gingivalis OMVs reduce the adhesion ability of F.nucleatum,but P.gingivalis OMVs treated with heat inactivation did not cause similar change.Conclusions: P.gingivalis OMVs can reduce the protein expression of F.nucleatum surface adhesion-related proteins Fad A and Fom A without causing changes in the morphology and proliferation of F.nucleatum,and weaken the auto-aggregation of F.nucleatum,thereby inhibiting F.nucleatum from invading into oral epithelial cells.