Study On The Mechanism Of Andrographolide Intefrering Quorum Sensing Of Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli (APEC) is an important pathogen associatedwith a wide range of localized or systemic diseases in avian species and can induce avariety of symptoms, including septicemia, air sacculitis and salpingitis. Among the171serotypes of APEC, O78is one of the most frequently detected serogroups in Asia.APEC contains a number of virulence factors, including iron uptake chelate gene D(iucD), iron regulatory protein2(irp-2), type1pili fimA gene (fimA); type1pili fimCgene (fimC), pyelonephritis-associated pili papC (papC), temperature-sensitivehemagglutinin gene (tsh), hemolysin E (hlyE), serum survival gene (iss), outermembrane proteins A (ompA), and vacuolating autotransporter toxin gene (vat),among others. These virulence factors are associated with bacterial iron acquisition,metabolism, adhesion and invasion, and serum survival to attack the host. Quorumsensing (QS) of E.coli act as a global regulatory system that controls genes involvedin pathogenesis, bacterial metabolism and protein biosynthesis, et al. Therefore, theexpression of above virulence factors may be also associated with quorum sensing.APEC infection begins in the upper respiratory tract and breaches the blood-air barrierto induce septicemia. Type II pneumocytes play an important role of maintaining thefunction of blood–air barrier. Andrographolide is a major active constituent ofAndrographis paniculata (Burm. F) Nees. Andrographolide exhibitsanti-inflammatory, antibacterial, and antiviral activities. It has been reported thatandrographolide possesses QS-inhibiting activity in Pseudomonas aeruginosa.Therefore, in this study, we set out to investigate the association betweenautoinducer-2(AI-2) and cell damage caused by APEC-O78, and to investigatewhether andrographolide inhibits the injury of APEC-O78to chicken type IIpneumocytes through regulation of the QS system. In this study, the supernatant of APEC-O78were collected. Then the supernatantwas diluted10-fold with V. harveyi BB170dilution to performe AI-2bioluminescenceassay. The results showed that APEC-O78has the ability to produce AI-2,supernatants of APEC-O78induce the luminescence has a positive correlation withthe inoculation and time. The level of luminescence induced by supernatants ofAPEC-O78changed during different growth phases, the AI-2activity increasedmarkedly during log phase and reaches the maximum value in the late log phase, anddeclined precipitously in the stationary phase.On the basis of the injury model of avian pathogenic E. coli O78adhesion tochicken type II pneumocytes, the association between AI-2and cell damage caused byAPEC-O78was investigated by Lactate dehydrogenase (LDH) activity detection,trypan blue staining assay and RT-PCR. Our results showed that APEC-O78(107-109CFU/mL) have the ability to induce chicken type II pneumocytes death, andAPEC-O78at108CFU/mL caused significantly more cell death of chicken type IIpneumocytes than at107and109CFU/mL. Cytotoxicity assays and trypan blueexclusion study informed that the damage caused by APEC-O78was the bacterialdensity-dependent. Subsequent investigations demonstrated virulence factors weredecreased significantly in107CFU/mL and109CFU/mL compared with108CFU/mL(p<0.05). These results showed that cell damage and cell death caused by APEC-O78,LDH release by chicken type II pneumocytes and the expression of mRNA ofvirulence factors were correlated with the AI-2activity of APEC-O78.To investigate whether andrographolide inhibits the injury of APEC-O78tochicken type II pneumocytes through regulation of the QS system. We usedexperimental methods of Lactate dehydrogenase (LDH) activity detection, trypan bluestaining assay, scanning electron microscope (SEM), laser scanning confocal andRT-PCR in this study. The results showed that sub-MIC of andrographolidesignificantly reduced the release of LDH, F-actin cytoskeleton polymerization, andthe degree of the adherence to chicken type II pneumocytes induced by APEC-O78.We also found that andrographolide remarkably decreased the secretion of AI-2ofAPEC-O78. Furthermore, Chicken type II pneumocytes damage on cell ultrastructure, cell death induced by APEC-O78, and the expression of mRNA of virulence factors ofAPEC-O78were consistent with the result of inhibitory effect of andrographolide (10μg/mL) on the release of AI-2.In conclusion, the current study suggested that AI-2of QS is a global regulatoryfactor play a decisive role on the cell damage caused by APEC-O78. Andrographolideworked as quorum sensing inhibitor of APEC-O78, remarkably decreased thesecretion of AI-2, and its effects on reducing chicken type II pneumocytes damageinduced by APEC-O78were in accordance with the secretion of AI-2. These resultswill provide a basic research for andrographolide to prevent colibacillosis of chickens.