Studies On Adhesion Effects And Mechanism Of Lactobacillus SQ0048

Lactic acid bacteria is a recognized probiotic that has probiotic functions such as maintaining micro-ecological balance,inhibiting the adhesion and growth of pathogenic bacteria,and regulating host immunity.The adhesion strength Lactobacillus is an important indicator to assess whether it can be a probiotic,and that is a prerequisite for its subsequent probiotic effect.In this research,we investigated adhesion of two lactobacilli isolated from the bovine vagina with good growth and high acid-producing performance.Then,Lactobacillus SQ0048 as main research objiect,its antagonistic activity against pathogenic bacteria,and the transcriptomic sequencing of SQ0048 strain adhering to Bovine vaginal epithelial cells(BVECs)were investigated.We also further evaluated the effect of Lactobacillus SQ0048 on the protein processing in the endoplasmic reticulum of BVECs,aiming to analyze the ability and molecular mechamism of Lactobacillus SQ0048 adhesion and antagonize.The details are as follows:(1)Adhesion of Lactobacillus SQ0048 and its antagonistic on the adhesion of pathogensThe hydrophobicity,self-aggregation,biofilm formation and adhesion ability of SQ0048 strain and SQ0054 strain were tested using hydrocarbon adhesion,ultraviolet spectrophotometry,enzyme-linked and Giemsa stain.CCK8 was used to measure the BVEC viability treated with Lactobacillus SQ0048 and pathogens,and fluorescent labeling was evaluated their time and dose effect on adherence to BVECs.The method of fluorescent labeling and CCK8 were used to detect Lactobacillus SQ0048in antagonizing the adhesion of pathogens,and BVEC viability.Hydrophobicity results showed that Lactobacillus SQ0048 was significantly higher than that of SQ0054 under the same p H and hydrophobic solvent conditions(P<0.05).Under the same p H conditions,self-agglutination ability of Lactobacillus SQ0048 was significantly higher than that of SQ0054 at different time(P<0.05).Biofilm-forming capacity result showed that Lactobacillus SQ0048 was a strong biofilm-forming strain and SQ0054 was a feeble biofilm-forming strain,and under the same p H conditions,the biofilm-forming capacity of Lactobacillus SQ0048 was significantly higher than SQ0054(P<0.05).Adhesion ability assay in vitro showed that the ability Lactobacillus SQ0048 adhere to BVECs was significantly higher than SQ0054(P<0.01).At the concentration of 1×10~8CFU/m L,Lactobacillus SQ0048 was no significant effect on BVEC viability at different exposure times(P>0.05).The maximum adhesion of Lactobacillus SQ0048 was achieved at a concentration of 10~8CFU/m L and exposure 4 h,which was significantly higher than other exposure concentration and time.Both E.coli and S.aureus at different exposure concentrations and times significantly reduced the BVEC viability,compared withcontrol group(P<0.05).The maximum adhesion of E.coli and S.aureus was achieved at a concentration of 10~8CFU/m L and exposure 3 h(E.coli)or 4 h(S.aureus).In exclusion,competition,displacement test,compared with the control,Lactobacillus SQ0048 significantly reduced the E.coli and S.aureus adhesion rate(P<0.05).Compared with the BVEC viability infected with pathogens alone,Lactobacillus SQ0048 significantly increased the BVEC viability(P<0.05).(2)Transcriptome Profiling Analysis of BVECs Response to isolated Lactobacillus SQ0048We investigate the pathways involved in host cell responses using transcriptome sequencing(RNA-seq).The result showed 296 significantly altered differentially expressed genes(DEGs),of which 170 were upregulated and 126 downregulated.Gene Ontology(GO)enrichment analysis of the DEGs revealed significant enrichment of 424 GO terms throughout the differentiation process(P<0.05).Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed that the DEGs were successfully annotated as members of 171 pathways,with 23 significantly enriched KEGG pathways(P<0.05).GO and KEGG enrichment analysis obtained differentially significant signaling pathways associated with the immune response and adhesion of Lactobacillus SQ0048 were IL-17 signaling pathway,antigen processing and presentation signaling pathway,and protein processing in the endoplasmic reticulum.The pathways were enriched with a total of 15 significantly up-regulated DEGs(P<0.05);most of the DEGs participated in protein processing in the endoplasmic reticulum.The expression trends of 15 DEGs were found to be similar to those obtained by RNA-seq.(3)The mechanism of adhesion of Lactobacillus SQ0048 to BVECsEffects of Lactobacillus SQ0048 on the expression of Sec62 and HSP90AA1 in BVECs were detected by RT-qPCR and Westernblot.Using RNA interference technology to silence Sec62 gene,RT-qPCR and Westernblot verify the Sec62silenced BVECs,and tested the effect of Lactobacillus SQ0048 on the expression of HSP90AA1 within Sec62-silenced BVECs.Lentivirus-mediated CRISPR/CAS9gene-editing technique was used to knock down(Knock down,KD)the HSP90AA1gene in BVECs,and validated by RT-qPCR and Westernblot.The proliferative capacity of HSP90AA1-KD BVECs was determined by the CCK8 assay.Fluorescent labeling was evaluated Lactobacillus SQ0048 and pathogens on the adhesion of HSP90AA1-KD BVECs and Lactobacillus SQ0048 antagonizes the adhesion of pathogens to HSP90AA1-KD BVECs.The results showed that compared with BVECs group,Lactobacillus SQ0048significantly up-regulated the expression of Sec62 and HSP90AA1 in BVECs,and successfully screened si RNAs that specifically silenced Sec62 gene with 75%interference efficiency could be used in subsequent experiments.Lactobacillus SQ0048 significantly reduced the expression of HSP90AA1 in BVECs by Sec62-si RNA silencing(P<0.05),suggesting that up-regulation of HSP90AA1expression in BVECs by SQ0048 strain was achieved by inducing Sec62 gene expression.In this study,HSP90AA1-KD BVECs with 80%knockdown efficiency was successfully constructed.Compared with the BVECs,Lactobacillus SQ0048showed a significant decrease in the adhesion capacity of HSP90AA1-KD BVECs(P<0.05);there was no significant effect in pathogens,compared with BVECs;suggesting that the adhesion of Lactobacillus SQ0048 to BVECs was associated with its up-regulation of HSP90AA1 expression,while the pathogens were not significantly correlated with HSP90AA1.Compared with the control group,the adhesion of HSP90AA1-KD BVECs by the antagonistic pathogens of SQ0048 strain was significantly reduced(P<0.05),suggesting that antagonism of pathogens by SQ0048 strain was also achieved through up-regulation of HSP90AA1.From this study,we concluded that:(1)The ability of Hydrophobicity,self-aggregation,biofilm formation,and adhesion to BVECs of Lactobacillus SQ0048 was significantly higher than the SQ0054 strain.The exposure concentration of Lactobacillus SQ0048 was 1×10~8CFU/m L,and there was no significant effect at different exposure times on BVEC viability.Lactobacillus SQ0048 could significantly reduce the adhesion of E.coli and S.aureus to BVECs by exclusion,competition,and displacement.(2)Based on RNA-seq sequencing analysis,we obtained that the main signaling pathway that Lactobacillus SQ0048 adheres to BVECs and antagonizes the pathogens was the protein processing in the endoplasmic reticulum,in which Sec62 and HSP90AA1were the main DEGs in this signaling pathway.(3)The molecular mechanism of Lactobacillus SQ0048 in adhering to BVECs was related to its upregulation of Sec62and HSP90AA1 expression in BVECs.