Fusobacterium Nucleatum Reduces Antibiotic Susceptibility Of Pseudomonas Aeruginosa And Aggravates Inflammation Of Pulmonary Epithelial Cells

Objective:Chronic obstructive pulmonary disease（COPD）is a chronic respiratory disease characterized by persistant airflow limitation and progression in relation to the repetition of its exacerbation.COPD has become the third leading cause of death worldwide with a global incidence of 15.5%.Bacterial respiratory infection,especially Pseudomonas aeruginiosa,is the most important cause of acute exacerbation and even death in patients with COPD.Antibiotics are currently the most commonly therapeutic strategy against respiratory infections.P.aeruginosa usually forms a biofilm after colonization on the surface of tissue mucosa.P.aeruginosa is embedded in exopolysaccharide matrixes containing a large number of alginate,polysaccharide coding locus（Pel）and polysaccharide synthesis locus（Psl）,that protect P.aeruginosa from antibiotics exposure.This not only increases the difficulty of treatment,but also increases the risk of exacerbation and death of COPD patients.Periodontitis is a chronic infectious disease that occurs in periodontal support tissues mainly caused by dental plaque biofilms,and is closely related to respiratory diseases such as COPD.Studies have confirmed that dental plaque is a reservoir of respiratory pathogens.Direct inhalation of the the shedding dental plaque colonized by respiratory pathogens into the lung is one of the most possible mechanisms involved in the association between oral bacteria and respiratory diseases.Fusobacterium nucleatum is an oral commensal and periodontal pathogen.Recent studies have found that F.nucleatum can colonize the lower respiratory tract and is a respiratory pathogen.Tan et al.found that the lung function of patients with COPD gradually weakened as their periodontal health deteriorated.Improving oral care through mechanical or chemical control of dental-plaque biofilm formation reduces the risk of respiratory infection and exacerbation of COPD.Therefore,we speculate that the periodontal pathogen F.nucleatum may change the structure,pathogenicity and antibiotic sensitivity of the respiratory tract microbiota after colonization in the respiratory tract,and then aggravate the respiratory tract infection.The typical characteristic of F.nucleatum is its ability to adhere to almost all oral bacteria and a variety of mammalian cells via its outer membrane proteins,also called adhesins.Fusobacterium adhesin A（FadA）is a unique adhesin of oral Fusobacteria,and exists in two forms,the non-secreted pre-FadA anchored in the inner membrane and the secreted mature FadA（mFadA）exposed on the bacterial surface.Nevertheless,only the high molecular-weight complex FadAc consisting of mFadA and pre-FadA is required for attachment and invasion of host cells.FadA directly binds to vascular endothelial cadherin,changes the cell localization of cadherin and increases endothelial permeability to allow bacteria to cross endothelium and to disseminate systemically.Moreover,FadA binds to E-cadherin,activatesβ-catenin signaling to promote colorectal carcinogenesis.However,it is not clear whether FadA participate in mediating the interaction between F.nucleatum and other bacteria.Therefore,we believe that FadA plays an important regulatory role in the process of F.nucleatum initiating or exacerbating respiratory infections.At the same time,FadA may mediate the interspecies interaction between F.nucleatum and P.aeruginosa.Based on the above background and thinking,this study first verified from clinical studies whether there was a co-infection with F.nucleatum and P.aeruginosa in patients with acute exacerbation of COPD,and whether the presence and content of F.nucleatum would affect the lung function of patients with COPD.Secondly,the mechanism of F.nucleatum affected the biofilm formation and antibiotic sensitivity of P.aeruginosa,and the effect of these two bacteria on the biological activity of pulmonary epithelial cells were described from two aspects of the interaction between bacteria（F.nucleatum and P.aeruginosa）and the interaction between bacteria and respiratory epithelial cells（F.nucleatum and/or P.aeruginosa and lung epithelial cells）.Finally,the role of FadA in F.nucleatum regulating P.aeruginosa antibiotic sensitivity and inducing inflammatory response in pulmonary epithelial cells was clarified.The aim of this study is to provide a new anti-infection strategy for the prevention and treatment of respiratory tract infections in patients with COPD by revealing the interaction between bacteria and its effect on biological characteristics of pulmonary epithelial cells.Methods:1.We collected tracheal aspirate from patients with acute exacerbation of COPD,detected the colonization and content of F.nucleatum and P.aeruginosa in tracheal aspirate from COPD patients by amplifying bacterial 16S rRNA using qPCR method,and evaluated the correlation between pulmonary function index of COPD patients（FEV₁%）and the number of F.nucleatum in tracheal aspirates.2.We cultured F.nucleatum,collected F.nucleatum supernatant and purifyed FadA protein to incubate P.aeruginosa biofilm.We evaluated the bacterial proliferation ability using colony counting method,measured the change of pH in the both with a pH meter,observed bacterial biofilm structure and biomass by scanning electron microscope and crystal violet staining.RT-PCR method was used to detect the expressions of P.aeruginosa extracellular polysaccharides algD,pelB and pslA.The bacterial antibiotic sensitivity was evaluated by K-B diffusion method and measurement of the minimum inhibitory concentration and minimum bactericidal concentration of antibiotics including imipenem,meropenem,gentamicin,acamicin,ciprofloxacin,and levofloxacin.3.We established an in vitro model of F.nucleatum and P.aeruginosa infecting A549cells alone or together,detected bacterial adhesion and invasion efficiency,and observed bacterial adhesion/invasion formation and cellular morphological changes use light microscope,transmission electron microscope and scanning electron microscope.The cell viability and cytotoxicity were detected by CCK8 and LDH,respectively.Calcein-AM/PI double stain was used to observe the number of live and dead cells.Flow cytometry was used to analyze cell cycle distribution.The secretions of IL-1β,IL-6,IL-8 and TNF-αwere evaluated using enzyme-linked immunosorbent assay.The protein expressions of p-NF-κB,NF-κB,p-STAT3,STAT3,p-AKT and AKT were detected by western blot.4.The differentially expressed mRNAs and miRNAs were screened by RNA sequencing,and the GO and KEGG enrichment analysis of differentially expressed mRNAs were performed using webgestalt software.STRING database combined with Cytoscape software was used to build protein-protein interaction（PPI）network and screen the important PPI functional modules and hub genes.The core transcription factors were obtained using hTFtarget database,and a transcription factor-mRNA regulatory network was constructed.The target genes of differentially expressed miRNAs were predicted by three softwares including mi RWalk,mi RDB and TargetScan,and the miRNA-mRNA and transcription factor-mi RNA regulator networks were built.5.We treated A549 cells with the purified FadA protein to verify whether FadA is the important virulence factor of F.nucleatum to induce dysregulation of miRNA and mRNA in the pulmonary epithelial cells.Results:1.45.3%of patients with acute exacerbation of COPD had a coinfection of F.nucleatum and P.aeruginosa,and lung function weakened following the number of F.nucleatum increased in patients coinfected with P.aeruginosa.2.F.nucleatum interacted with P.aeruginosa to shift broth pH from weakly alkaline to weakly acidic,promoted bacterial proliferation in the planktonic and biofilm conditions,and form a dense and complex biofilm surrounding by excessive extracellular matrix and to reduce susceptibility of dual-species biofilm to meropenem,amikacin,gentamicin and ciprofloxacin because of over-expressions of pelB and pslA.3.FadA enhanced the biofilm formation ability of P.aeruginosa,induced the high expressions of pelB and pslA,and reduced the sensitivity of P.aeruginosa biofilm to amikacin,gentamicin and ciprofloxacin.4.P.aeruginosa and F.nucleatum could adhere and invade into A549 cells,and their adhesion and invasion efficiencies increased following bacterial multiple of infection increased.Coinfection with these two bacteria could significantly increase the invasion efficiency of each bacterium.5.P.aeruginosa attached and destroyed cellular junctions,made cell shrinking and round.F.nucleatum aggregated to form a network-like structure and attached on the cell surface,and A549 cell was shaped like a long spindle.When A549 cells were infected with P.aeruginosa and F.nucleatum,these two bacteria coaggregated and adhered to cellular junction and surface,and cell morphology became round.6.P.aeruginosa infection alone（MOI 10,50 and 100）inhibited the proliferation of pulmonary epithelial cells,induced cell death and enhanced the secretions of IL-1βand IL-6.F.nucleatum infection alone（MOI 100）did not affect cell toxicity and death,but promoted cell proliferation and enhanced the secretions of IL-1β,IL-6 and TNF-α.The cytotoxicity induced by P.aeruginosa and F.nucleatum coinfection was lower than that of P.aeruginosa alone group,and the secretions of IL-6 and TNF-αwere higher than that of P.aeruginosa alone group.7.RNA sequencing combined with bioinformatics analysis suggested that F.nucleatum bound to CDH11 to adhere and invade into lung epithelial cells via FadA,and activated CDH11/EGFR/MAPK13/JUN/miR-27b-5p signaling pathway to amplify pulmonary inflammation.Meanwhile,F.nucleatum blocked AURKA/STAT3/E2F signaling pathway,inhibited the expressions of MCM3-7,CCNA2 and CCNB1,and induced S and G2/M phase arrest of pulmonary epithelial cells.Conclusions:1.F.nucleatum is a biomarker for lung function decline in AECOPD patients with P.aeruginosa infection,F.nucleatum reduces the antibiotic susceptibility of P.aeruginosa through upregulating pelB and pslA via FadA.Therefore,targeting FadA to interfere with biofilm structure or inhibit exopolysaccharide synthesis will be a new treatment strategy for AECOPD patients coinfected with F.nucleatum and P.aeruginosa.2.F.nucleatum inhibits the cytotoxic damage induced by P.aeruginosa and enhances the inflammatory response in pulmonary epithelial cells.This may contribute to the exacerbation of respiratory infection and difficult treatment caused by F.nucleatum.3.F.nucleatum binds to CDH11 to adhere and invade into lung epithelial cells via FadA,and activates CDH11/EGFR/MAPK13/JUN/miR-27b-5p signaling pathway to amplify pulmonary inflammation.Meanwhile,F.nucleatum blocks AURKA/STAT3/E2F signaling pathway,inhibits the expressions of MCM3-7,CCNA2 and CCNB1,and induces S and G2/M phase arrest of pulmonary epithelial cells.Therefore,target FadA or blocking CDH11/EGFR/MAPK13/JUN/miR-27b-5p signaling pathway or AURKA/STAT3/E2F signaling pathway,could be a new strategy for controlling respiratory infection in AECOPD patients.