Effect Of CyaA Gene On The Virulence Of Sheep Lung ExPEC XJ10

Extraintestinal pathogenic E.coli?ExPEC?can cause meningitis,sepsis,urinary tract infection,pneumonia of humans and animals and higher mortality in animals.The virulence of ExPEC is inseparable from the virulence factors it carries.Adenylate Cyclase?AC?encoded by the cyaA gene can catalyze the ATP to form Cyclic Adenosine Monophosphate?cAMP?,the binding of Cyclic Adenosine Monophosphate?cAMP?to its receptor protein regulates the transcription of nearly 50%of E.coli genes including virulence factors.10 strains of ExPEC?XJ01-XJ10?were isolated and identified from lung tissues of sheep with severe respiratory infection,and the cyaA gene was detected in XJ10 strain in the laboratory of the author,.Objective:In order to study the effect of cyaA gene on XJ10 virulence and other characteristics.Gene deletion strain and complementary strain were constructed to analyze the effects of cyaA gene on XJ10 BF formation ability,cell adhesion and invasion ability,LD50 and so on.Methods:?1?The homologous recombination method was used to construct the cyaA gene deletion strain of XJ10;the full length of the cyaA gene was linked to pHSG396 and the recombinant plasmid was electrotransferred into competent deletion strain,and the complementary strain was selected.The colony morphology and growth curve of the 3 strains were carried out.?2?The real-time PCR was used to detect the transcriptional regulation of the cyaA gene on XJ10 related to carbon source utilization and adhesion-invasive;96-well cell culture and crystal violet staining were used to detect BF formation ability quantitatively and qualitatively;LD₅₀0 of 3 strains were determined with Coriolis Standard Method.?3?The bacterial count was used to detect the effect of cyaA gene on adhesion and invasion of XJ10 to TC-1.Real-time PCR was used to detect the genes expression of the related to tight junction in lung and basement membrane in lung of mice infected by 3 strains.Results:?1?XJ10 gene deletion strain XJ10-?cyaA and complement strain cyaA⁺/?cyaA were successfully constructed;the morphology of the 3 strains was consistent,but the fermentation lactose ability of XJ10-?cyaA decreased and the growth rate was significantly lower than that of XJ10;Although cyaA⁺/?cyaA has lower utilization lactose and growth rate than that of XJ10,it is higher than that of XJ10-?cyaA.?2?Compared with XJ10,the transcription levels of 8 genes related to carbon source utilization and adhesion-invasion of XJ10-?cyaA were down-regulated,the expression levels of gltA,fabB and pap genes were significantly different from those of XJ10?P<0.05?,sucC,acs,fyuA,luxS and motA genes were not significantly different?P>0.05?;At the same culture conditions,the BF of XJ10-?cyaA were fragments only at 12 and 24 h,no obvious BF network structure;BF from cyaA⁺/?cyaA was consistent with XJ10,but it was more loose and sparse than that of XJ10;The LD₅₀0 of XJ10-?cyaA was 1.8times higher than that of XJ10,the difference was significant?P<0.05?,and the LD₅₀0 of cyaA⁺/?cyaA was1.3 times lower than that of XJ10-?cyaA.?3?The number of adhesion to TC-1 of XJ10-?cyaA was significantly lower than that of XJ10,and the difference was significant?P<0.05?.The adhesion bacteria number of cyaA⁺/?cyaA was less than that of XJ10 at each time point,but more than XJ10-?cyaA and the difference was significant?P<0.05?.Compared with XJ10,the number of invasive bacteria of XJ10-?cyaA decreased at each time point,and the difference was significant?P<0.05?.Although the invasion number of cyaA⁺/?cyaA was less than that of XJ10 at each time point,it was more than XJ10-?cyaA,the difference was not significant?P>0.05?.At 48 h,the mRNA expression levels of occluding,zo-1 and mmp-9 of the 3strains peaked.Compared with XJ10,the expression of occluding and zo-1 mRNA of XJ10-?cyaA decreased,and the difference was significant?P<0.05?,the mRNA expression of mmp-9 increased,the difference was significant?P<0.05?.Conclusion:?1?The cyaA gene deletion decreased the growth rate of XJ10 and the ability to utilize lactose.?2?The cyaA deletion led to down-regulation of the genes transcription level of XJ10 related to carbon source utilization and adhesion-invasive,and BF formation ability and virulence were obvious decline.?3?The cyaA gene deletion reduced the adhesion and invasion of XJ10 to TC-1,and also reduced genes transcriptional level related to the tight junctions and basement membrane of the lungs from mice infected with XJ10.