Screening And Function Study On Molecules Associating With P.gingivalis Intracellular Survival

BackgroundPorphyromonas gingivalis is a Gram-negative,rod-shaped,obligate anaerobe,considered to be the key pathogen of chronic periodontitis.The bacterium can not only express a range of virulence factors to destroy the host,including lipopolysaccharide,fimbriae,gingipain and outer membrane vesicle,but also live in the host cells to evade immune defense mechanisms.In addition to the oral localization,P.gingivalis might have an impact on the development and regression of several systemic diseases,such as cardiovascular disease,adverse pregnancy outcomes and diabetes.It is reported that P.gingivalis could travel locally from the oral cavity into different tissues through the circulatory system,then invade and survive in the host cells as well as evade or suppress host immune defenses to aggravate tissue damage or affect tissue repair.As a facultative parasite,P.gingivalis can invade host cells by phagocytosis and endocytosis,and exhibit significant adaptability to the intracellular microenvironment to support its own intracellular lifestyle.Intracellular pathogens have evolved a variety of ways to control molecular signaling mechanisms of host cells,thus allowing them to use intracellular energy-rich host metabolites to sustain their growth;and to render the intracellular environment a shield for their evasion of immune surveillance.It has been shown that P.gingivalis invading human coronary artery endothelial cells could not only activate autophagy and inhibit the fusion of autophagosomes with lysosomes,but also spread through intercellular bridge to infect neighboring cells,which lead to persistent infection.Although P.gingivalis promotes its parasitism within host cells through a variety of strategies,another study has shown a precipitous decline in the number of intracellular P.gingivalis with prolonged infection.Moreover,the ability of P.gingivalis intracellular survive was greatly enhanced after multiple rounds of intracellular infection.It suggests that P.gingivalis which undergoes multiple rounds of intracellular infection could evolve different regulatory mechanisms on the host cells,thereby inhibiting host cell clearance mechanism of intracellular bacteria and making the intracellular microenvironment more adapted for their survival.How P.gingivalis entering the host cell adapts to the complex intracellular microenvironment of the host cell is the key to manipulate P.gingivalis infection.However,the exact mechanism of P.gingivalis intracellular survival has not been elucidated.In this study,based on a model of P.gingivalis intracellular survival in HUVECs,we aimed to preliminarily investigate the molecules and beneficial mechanism associating with P.gingivalis intracellular survival,and to provide a new theoretical experimental basis for the prevention and treatment strategies of periodontitis as well as systemic systemic diseases associated with P.gingivalis infection by transcriptomics.Materials and Methods1.Interactions of intracellular P.gingivalis and umbilical vein endothelial cellsPorphyromonas gingivalis was applied to invade HUVECs,and the cell lysates incubated anaerobically on blood agar plates were identified by 16S rRNA gene sequencing.Spread plate technique,bacterial growth curve and CCK8 cell proliferation were used to evaluate the interactions between intracellular P.gingivalis and HUVECs.2.Effect of autophagy on P.gingivalis intracellular survivalThe immunofluorescence was performed to observe the localization of intracellular P.gingivalis.Transmission electron microscope,stubRFP-sensGFP-LC3 lentivirus and Western blot were used to detect the effect of intracellular P.gingivalis on autophagic flux.Next,both Rapa and Baf A1 were used to further explore the effect of autophagy on P.gingivalis intracellular survival.3.Screening and preliminary validation of molecules associating with P.gingivalis intracellular survival based on transcriptomicsThe transcriptome analysis in the control,non-invasive P.g and invasive P.g groups was used to screen for molecules involved in P.gingivalis intracellular survival at the cellular level,and differential genes associating with P.gingivalis intracellular survival were initially validated by qRT-PCR.Results1.Interactions of intracellular P.gingivalis and umbilical vein endothelial cellsWe performed P.gingivalis intracellular survival in HUVECs and the cultured bacteria were demonstrated to be P.gingivalis standard strain ATCC 33277 by 16S rRNA gene sequencing.The P.gingivalis strains characterized as Round1-7 were harvested after the invasion into HUVECs.The colony counting on blood agar plates showed the number of intracellular P.gingivalis decreased significantly at 72h compared to 48h and colony numbers in Round 5 was higher than that in Round 1.Moreover,colony numbers increased significantly in both Round 4 and Round 7 when compared with Round 1,and Round 7 had more significantly upward than Round 4.The results of bacterial growth curve showed compared to non-invasive P.g,invasive P.g had a prolonged lag phase and there were no statistically significant differences between two groups reaching the plateau stage.The CCK-8 assay indicated that compared with the control group or non-invasive P.g group,invasive P.g significantly inhibited the proliferative activity of HUVECs at 3-7d.2.Effect of autophagy on P.gingivalis intracellular survivalThe immunofluorescence results showed that CFSE-labeled intracellular P.gingivalis had a colocalization phenomenon with lysosomes and autolysosomes.TEM showed autophagosomes appear as a double-membrane structures with P.gingivalis in HUVECs at 72 h.Dual fluorescence LC3 assay showed the number of autophagosomes increased significantly and autolysosomes decreased significantly at 48h,while the number of autophagosomes increased significantly and autolysosomes did not change significantly at 72h.In addition,qRT-PCR and Western blot results indicated that the long-term intracellular parasite P.gingivalis could activate autophagy upstream signals to promote autophagosome formation,but inhibit the fusion of autophagosomes with lysosomes.Then,we determined the effect of autophagy on P.gingivalis intracellular survival with 200nM Rapa,200nM Baf A1 or 200nM Rapa+200nM Baf A1.Colony counting on blood agar plates showed the number of intracellular P.gingivalis in both Rapa+non-invasive P.g group and Rapa+invasive P.g group showed a significant decrease when Rapa pretreatment was added and there were no statistical differences between Rapa+non-invasive P.g group and Rapa+non-invasive P.g group.The same result was obtained when Rapa and Baf A1 pretreatment were simultaneously added.Similarly,when Baf Al pretreatment was added,there was a significant reduction in Baf Al+non-invasive P.g group and Baf A1+invasive P.g group.However,compared with the Baf Al+non-invasive P.g group,the colony numbers in the Baf Al+invasive P.g group was significantly increased.3.Screening and preliminary validation of molecules associating with P.gingivalis intracellular survival based on transcriptomicsIntracellular P.gingivalis led to gene expression differences in HUVECs and GO enrichment and KEGG pathway enrichment analysis revealed that the differentially expressed genes after quality control were significantly enriched in the cell cycle.Besides,bioinformatics analysis focused on mTORCl signaling downregulation in HUVECs.qRT-PCR showed that the mRNA levels of the genes related to the mTORCl signaling,including SLC1A5,SLC7A5 and SLC7A11,were decreased in the noninvasive P.g and invasive P.g groups,consistent with the transcriptome sequencing.In addition,there were no significant differences between the non-invasive P.g group and the invasive P.g group.Conclusion1.The host cells could always generate several defense mechanisms to eliminate intracellular P.gingivalis.2.After multiple rounds of intracellular infection,P.gingivalis evolves sophisticated mechanisms to improve persistent intracellular survival.3.Intracellular P.gingivalis could not only affect the cell cycle progression,but also stimulate autophagy flux as well as inhibit the maturation of autophagosomes to manipulate its survival and proliferation.4.Intracellular P.gingivalis may hijack the autophagy pathway to promote its own intracellular survival by restructuring the host intracellular metabolism,leading to a state of cellular starvation,which in turn inhibits mTORC1 signaling.