Effect Of Type I Quorum Sensing Molecule AHL On Acid Resistance Of Pathogenic E.coli O157:H7

Enterohemorrhagic Escherichia coli(EHEC)O157:H7 is a kind of human pathogen worldwide that can induce mild clinical case such as diarrhea and intestinal inflammation,or lead to serious symptoms such as hemolytic colitis and hemolytic uremic syndrome.We notice that the carrier hosts of E.coli O157:H7 such as cattle and pigs can not be infected by itself,food-borne infection is the main pathway for E.coli O157:H7 to infect humans.It is worthwhile noticed that E.coli O157:H7 infected through food in low-does can overcome extreme acid stress in stomach and then successfully enter into the host intestines,which is mainly due to its strong acid resistance(AR).Over the past few decades,E.coli AR mechanisms was established,including the global stress regulator RpoS-dependent AR system(AR-I)and amino acid-dependent AR systems(AR-Ⅱ).Besides,some potential regulatory systems and molecules that can modulate the AR response have also been discovered in recent years,such as type I quorum sensing(QS)signaling molecule acyl homoserine lactone(AHL),which can regulate glutamate decarboxylase encoding gene gadA,one of the AR-Ⅱ genes.However,regulatory mechanisms of AHL remain to be known.In this study,we investigated the effects of AHL on the AR of intestinal pathogenic E.coli O157:H7 EDL933 and the potential regulatory mechanism.First,we examined the effects of AHL on the survival ability of O157:H7 in extreme acid environments(pH=2.5)by exogenously adding AHL and synthesizing AHL.The results of AR experiments showed that exogenous AHL(100μM and 10μM)can significantly improve the survival rate of O157:H7 EDL933.We further analyzed the effect of AHL on AR in terms of different categories and concentrations,and found that 3-oxo-C6-HSL,an AHL molecule containing a carbonyl group at the 3-carbon position,can increase survival rate of E.coli EDL933 obviously,which is 1.5 to 2.5 times higher than C4-HSL and C6-HSL molecules under low pH condition.Different AHL concentrations also have noteworthy differences in AR activation of O157:H7 EDL933,100uM 3-oxo-C6 can significantly increase the survival rate of E.coli relative to 10μM 3-oxo-C6-HSL.Since E.coli lack the AHL synthetase LuxI,we cloned the AHL synthase gene yenI of Yersinia enterocolitica into O157:H7 and its AHL-synthesizing ability was then testified.The survival rate of recombinant EDL933/pyenI strain was also enhanced in AR assay.However,exogenous AHL could not enhance the acidic survival rate of non-intestinal pathogenic E.coli APEC.These results indicated that AHL has a regulatory effect on the AR response of enteropathogenic O157:H7,while may have no effect on non-enteropathogenic E.coli.However,lack of related studies limted further explaination for the AR difference between EHEC EDL933 and APEC CE129 under AHL.According to their different survival niches and hosts,we speculate that it may be related to differences in genome evolution caused by distinct living environments.Subsequently,we exploited RT-PCR and Western bolt technology to further explore whether the AHL can enhance the AR of EHEC EDL933 by activating AR-I system regulatory gene rpoS and the AR-Ⅱ system glutamate decarboxylase encoding gene gadA.Under normal environment(pH=7.0)and acid stress condition(pH=2.5),AHL can significantly enhance the expression of rpoS and gadA gene,indicating AHL can regulate RpoS and GadA to improve the AR of EHEC EDL933.Under normal physiological pH condition,the AHL receptor SdiA’s transcription level was slightly enhanced by AHL(～1.5-fold),while under low pH condition,sdiA transcription was significantly up-regulated(～7-fold).And acidic stimulation(pH=2.5)alone(without AHL)decreased sdiA expression of EHEC EDL933.These resultes demonstrated that the functional activation of sdiA gene requires the coordination of acidic pH and AHL.In order to further examine the regulatory mechanism of AHL-SdiA on RpoS and GadA,we constructed a sdiA mutant strain EDL933 ΔsdiA.AR test showed that the acidic survival rate of EDL933ΔsdiA was significantly reduced,and the expressions of gadA and rpoS were also reduced,which indicated that AHL can activate gadA and rpoS through sdiA under acid stress.However,exogenous addition of AHL could reactivate the gadA and rpoS gene of EDL933ΔsdiA strains,which revealed that in addition to SdiA,unknown factors may also be involved in AHL’s enhancement on AR response of enteropathogenic E.coli.Meanwhile,by constructing EDL933 ΔrpoS,the role of RpoS in the AR pathway regulated by AHL was examined.In AR assay,the survival rate of ΔrpoS decreased to 42%,and addition of exogenous AHL could not effectively restore its survival ability,which indicates that RpoS probably is crucial for the AR response regulated by AHL.The expression of gadA droped to 74.4%in ΔrpoS under acid stress,but the addition of AHL restored gadA transcription to above normal physiological levels,suggesting that AHL does not activate gadA expression by exclusively activating rpoS.In this study,we investigated the effect of AHL in terms of various kinds and concentrations on the AR of enteropathogenic E.coli EDL933,and found that AHL can enhance AR of pathogenic E.coli via SdiA receptor,global stress regulator RpoS and glutamate decarboxylase GadA.More importantly,this study indicated that mixed infection of EHEC with other bacteria in the stomach may help EHEC to resist acid stress or other stress by eavesdropping AHL molecules.