Effects Of Escherichia Coli Type ? Secretion System 2 (ETT2) Effector And Two-component System Cpx R On The Virulence Of Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)can cause severe respiratory infections and systemic diseases in poultry,which are economically devastating to poultry industries worldwide.APEC belongs to Extraintestinal pathogenic E.coli(ExPEC),which share a broad range of virulence factors and similar pathogenic mechanism with human Ex PEC.It has been indicated that APEC could be a reservoir of virulence genes and drug resistance genes,which are potential zoonotic risks for public health.Secretion systems and two-component systems(TCSs)are involved in the process of APEC infection.The secretion systems are used to deliver effector proteins into eukaryotic hosts or other bacteria,which facilitate bacterial infections..Bacteria utilize TCSs to detect environmental cues or stresses and precisely modulate gene expression.Our previous studies indicated that E.coli Type III secretion system 2(ETT2)and Type VI secretion system 2(T6SS2)contributed to intracellular survival,interbacterial competition activity and virulence of APEC.However,the roles of ETT2 effectors and the regulatory mechanism of T6SS2 are remain unclear.Therefore,this study was proformed to screen and identify the ETT2 effectors and determine the influence of ETT2 effector EspE3 on the pathogenicity of APEC.Moreover,the molecular mechanism of TCS CpxR facilitating the pathogenity of APEC by regulating T6SS2 were analyzed.Secretion systems and regulatory system have become the vaccine and drug target due to its roles in bacterial virulence.Under the circumstances of the continuous emergence of drug-resistant pathogens and the challenge of antibiotic treatment,it is improtant to determine the molecular pathogenesis of ETT2 effectors and TCSs,which can provide new strategies and drug targets for the design of new antibacterial drugs.1.Screening and identification of ETT2 effectors in APECIn order to screen the ETT2 effectors of APEC,the ETT2 core gene eiv C mutant strain was constructed using Red recombination system.The expression levels of ETT2 in different growth conditions were determined by qRT-PCR.For screening of ETT2 effectors,the secretory proteins of FJ1 B and mutant strain ?eiv C were analyzed and compared by comparative proteomics and bioinformatics.Finally,the functions of ETT2 effectors were analyzed by Western blot,expression and enzyme activity assays.The results showed that eivC mutant strain was constructed successfully.All the ETT2 operons showed up-regulated expression during the late logarithmic growth phase.A total of 9845 peptides,1322 protein groups and 725 unique proteins were identified by the label free quantification of secretory proteins.Bioinformatics analysis showed that Esp E3 contained a leucine repeat sequence(LRR),which might encode a putative E3 ubiquitin ligase.Western blot and in vitro ubiquitination assays showed that ETT2 effector Esp E3 is an E3 ubiquitin ligase,which catalyzed the polyubiquitination of K63 site.The screening and identification of ETT2 effectors provides a basis for elucidating the pathogenic mechanism of ETT2 in APEC.2.Effects of ETT2 effector Esp E3 on the virulence of APECTo determine the roles of ETT2 effector Esp E3 on the pathogenicity of APEC,we constructed the espE3 gene mutant and complemented strains via the Red recombination system and complementary plasmid.Then,the effects of EspE3 on the growth characteristics,motility,adhesion and invasion capacity and virulence of APEC were determined.In addition,we analyzed the influence of Esp E3 on the inflammatory cytokines IL-1?,IL-8 expressions in host cells by APEC infection and EspE3 transfection.Moreover,the host cellular proteins interacting with EspE3 were screened and identified by GST pull-down.The results showed that the deletion of esp E3 gene did not affect the growth rate,motility and invasion capacity of APEC.Whereas,EspE3 could promote the adhesion capacity,colonization and pathogenicity of APEC.In addition,the effector Esp E3 could inhibit the expressions of host inflammatory cytokines IL-1? and IL-8.A total of 77 host cellular proteins were identified,which should be further studied in future.These data indicated that EspE3 was involved in the pathogenicity of APEC,which would help us to understand the pathogenic mechanism of ETT2 in APEC.3.Molecular mechanism of two-component system CpxR regulating T6SS2 and promoting virulence of avian pathogenic E.coliTo determine the molecular regulatory mechanism of the two-component system Cpx R in APEC,the cpxR gene mutant and complemented strains were constructed using the Red recombination system and complementary plasmid.Then,we analyzed the effects of CpxR on the growth characteristics,motility,biofilm formation ability,serum bactericidal resistance,drug sensitivity,interbacterial competition activity,adhesion capacity and virulence of APEC.The regulation roles and mechanism of CpxR on the T6SS2 were determined by qRT-PCR,Western blot,?-galactosidase assays and electrophoretic mobility shift assay(EMSA).The results showed that cpxR gene deletion did not affect the growth rate,motility,biofilm formation ability and adhesion capacity of APEC.Inactivation of cpx R leads to significant defects in the drug resistance,serum bactericidal resistance,interbacterial competition activity,invasion and survival of APEC.Moreover,activation of CpxR positive regulates the expression of the APEC T6SS2 hcp2 B operon genes in APEC.EMSA revealed that CpxR could directly bind to the T6SS2 hcp2 B promoter region.This study broadens the understanding of the regulatory effect of Cpx TCS on T6SS2,thus elucidating the mechanisms of the adaptability and virulence of APEC.