| Diarrhea in newborn and weaned piglets is the most common gastrointestinal disease affecting the pig industry worldwide.It is mainly related to the adhesion,colonization and infection of procine enterotoxigenic Escherichia coil(ETEC)F4,F5,F6,F18,F41 in the intestine of piglets,and these pathogens can cause a variety of diseases such as yellow scour of new born piglets,white scour,edema disease and weaned piglet diarrhea,which resulting in significant economic losses in swine production.Actually,F4ac+ETEC is the main pathogenic bacteria,causes porcine post-weaning diarrhea(PWD)and even causes piglets to die.Considering CpxRA two-component system as a global control system,it has been reported that in order to couple with a wide range of external conditions,bacteria use CpxRA two-component system to sense and respond quickly to external signals to adapt to special situations,to control the expression of some virulence factors,and to provide adaptive advantages for the survival of bacteria in the host.Although the CpxRA two-component system has important regulatory effects involved in bacterial virulence factors such as fimbrial adhesins,biofilm formation ability and antibiotic resistance,the roles of CpxRA two-component system in the regulation of virulence factors of F4ac+ ETEC remain to be studied.In this study,we explore the role of CpxRA two-component system in the regulation of virulence factors from F4ac+ETEC reference C83902(O8:H19:F4ac+LT+STa+STb+),in order to better understand the pathogenesis mechanism of ETEC.According to the ?-Red recombination system,we constrcted the CS3902 ? cpxA?C83902 ? cpxR?C83902? cpxRA mutants successfully.On the base of mutant strains,we electroporated recombinant plasmid pBR322 expressing CpxA?CpxR or CpxRA protein into the second recombination mutants to construct the complemental strains C83902 ? cpxA/pcpxA,C83902 ? cpxR/pcpxR and C83902 ? cpxRA/pcpxRA.Bacterial growth curve test showed that there were no significant differences in the OD600 values among wild type,? cpxR,? cpxRA mutants and complemental strains,while compared to wild type,the ? cpxA mutants had a weakening effect on the growth.The result of showed growth curve test indicated that complete CpxRA two-component system is essential for bacterial growth.Motility test showed that ? cpxA,? cpxR,? cpxRA mutants decreased their motility on semisolid medium compared to the wild and complemental strains,in which?cpxA had the weakest motility;Meanwhile,biofilm formation tests showed that compared with WT strain biofilm formation ability in ? cpxA mutant decreased 78.3%(p<0.01),while? cpxR,? cpxRA mutant separately increased 30.8%,35.1%(p<0.05),further confirming the important role of the complete CpxRA two-component system in biofilm formation ability of C83902.In vitro,the adhesion test of IPEC-J2 cells showed that the adhesion ability of ? cpxA,?cpxR,?cpxRA mutants decreased by 55%(p<0.01),27%(p<0.05),20%(p<0.05)respectively.The result of adhesion test showed that the complete CpxRA two-component system plays an essential in C83902 motility,biofilm formation ability,and fimbrial adhesion.Sequentially we used Real-time PCR to detect the transcriptional changes of fliC(the main subunit of flagellum),fimA(the main subunit of type ? fimbria),faeG(the F4 fimbria main subunit)in mutants and complemental strains.The results showed that the transcriptional level of fliC,fimA and faeG in ?cpxA mutant decreased by 73%,70%,83%,89%,respectively(p<0.01).The transcriptional level of fliC,fimA and faeG in ? cpxR mutant decreased by 48%,40%,34%(p<0.05).The transcriptional level of fliC,fimA and faeG in ? cpxRA mutant decreased by 32%,27%,42%(p<0.05).Meanwhile,we used Real-time PCR to detect the transcriptional changes of the other virulence factors in mutants and complemental strains.The results indicated that the transcriptional level of luxS and pfs genes in type II quorum sensing system and heat-sensitive enterotoxin eltB were significantly decreased.The results suggested that the CpxRA two-component system can regulate the expression of F4ac+ETEC virulence factors.In addition,resistance tests of aminoglycoside antibiotics showed that? cpxA mutant could significantly increase the resistance of C83902 to aminoglycomine antibiotics,such as caminomycin,carnamycin,st reptomycin,and the absence of the cpxR and cpxRA genes didn't affect the resistance to aminoglycoside antibiotics of C83902.Sequentially we used Real-time PCR to detect the transcriptional changes of cpxR response regulator in mutants and complemental strains.The results shows that the transcriptional level of cpxR in? cpxA mutant increased by 35%.The high levels of CpxR response regulator are responsible for the resistance to aminoglycoside antibiotics of the ? cpxA mutant.but,the underlying mechanism of the resistance to aminoglycoside antibiotics remians to be studied.In summary,the complete CpxRA two-component system of F4ac+ETEC is essential for the expression of F4ac+ETEC virulence factors,and it also affects the resistance of bacteria to aminoglycoside antibiotics,which plays an important role in the response of bacteria to antibiotic stress.The study provided some helpful data and good information for further elucidation of the molecular pathogenic mechanism and the mechanism of drug resistance of F4ac+ ETEC. |
PDF Full Text Request