| Pathogenic bacteria utilize secretion systems to deliver effector proteins into the host cells,which facilitate bacterial survival and infections.Type Ⅲ secretion systems(T3SSs)play crucial roles in bacterial virulence,which are present in the many bacteria,such as pathogenic Escherichia coli,Salmonella.A second T3 SS,designated the E.coli type Ⅲ secretion system 2(ETT2),was identified by sequencing the genome of an EHEC O157:H7.ETT2 is known to be presented,in whole or in part,in the majority of E.coli strains.ETT2 plays an important role in adhesion/invasion,intracellular survival,biofilm formation of pathogenic E.coli.Avian pathogenic E.coli(APEC)belonged to Extraintestinal pathogenic Escherichia coli(ExPEC),could cause avian colibacillosis,which are economically devastating to poultry industries.APEC share common virulence factors and pathogenic mechanism with human ExPEC,indicating that the APEC could be a vehicle or even a reservoir for human ExPEC.Thus,this study investigated the prevalence and characteristics of ETT2 in APEC isolates to elucidate the potential zoonotic risk of APEC.Moreover,we determined the effects of the ETT2 ATPase EivC on the phenotype and virulence of APEC,which provide knowledgement of APEC ETT2 pathogenicity.1.Epidemiological investigation of ETT2 isoforms in APECEight pairs of overlapping PCR primers were designed according to the ETT2 sequence of EHEC O157:H7,which were used to detect the distribution of ETT2 in APEC clinical isolates.Then,the PCR products were sequenced,analyzed and submitted to GenBank.ETT2 were sequenced and analyzed for ETT2 isoforms.On the other hand,the serotype,phylogenetic groups of each APEC isolate was identified by an allele-specific PCR assay.In total,57.6%(141/245)APEC isolates were positive for ETT2 gene sequences.PCR amplification and sequencing identified five different ETT2 isoforms among the APEC isolates,including intact ETT2 which was designated the as type A isoform and,mutant ETT2 isoforms which was designated the type B,C,D,E isoform.The ETT2 locus was present in the predominant APEC serotypes O1,O2 and O78.Moreover,the intact ETT2 island(type A)was found only in serotypes O1 and O2 and notably the O78 isolates always contained deleted isoforms of ETT2.Interestingly,a putative second type Ⅲ secretion-associated locus(eip locus)was present only in the isolates with an intact ETT2.A comparison of the distribution of the ETT2 cluster among phylogenetic groups revealed revealed that 66.7%(32/48),64.8%(35/54),59.6%(31/52)and 44.9%(35/78)of the group B1,D,A and B2 isolates,respectively.However,there was no distinct correlation between ETT2 and other virulence factors in APEC.This study showed that ETT2 was more widely distributed in APEC isolates and exhibited more isoforms compared to ETT2 in human ExPEC,suggesting that APEC might be a potential risk to human health.2.Cloning,expression and enzyme activity analysis of ETT2 ATPase EivCTo determine the key catalytic sites of ETT2 ATPase EivC in APEC,we selected the key points of APEC ETT2 EivC by comparing the sequence of ATPase of other pathogenic bacteria.The eivC gene were amplified and subcloned into pET28a(+)vector.The fusion protein His-EivC was expressed by isopropyl-beta-D-thiogalactopyranoside(IPTG)induction.Then,the ATPase activity of His-EivC fusion protein was determined using an ATPase/GTPase activity assay kit.The amino acid sequence analysis indicated that EivC contains a glycine-rich region(Walker box A)and a nucleotide-binding protein region(Walker box B),which was the best conserved motif of the F0F1 ATPase.These data suggested that the eiv C gene likely encodes a F0F1 ATPase.The purified His-EivC fusion protein showed ATPase activity,with phosphate release rate of 0.28 ± 0.08 μmol/min/mg.According to the key site of the ATPase in pathogenic bacteria,the site-directed mutations at 175,199,201,and 233 amino acids of EivC were successful constructed.However,there was no significant difference in the ATPase activity between the recombinant EivC protein of mutations and wild-type EivC protein.The study indicated that 175,199,201 and 233 amino acids did not affect ATPase activity of EivC.3.Construction and characterization of eivC gene mutant strain and complementary strain in avian pathogenic Escherichia coliIn order to determine the effect of ETT2 ATPase EivC on APEC phenotype and virulence,the eivC gene mutant strain was constructed using Red homologous recombination system.The complementary strain was constructed using low-copy plasmid pSTV28.Then the growth curve,motility,resistance to serum bactericidal capacity,adhesion and invasion capacity to DF-1 cells,intracellular survival capacity in HD-11 cells and virulence to duck of wild-type,mutant and complementary strains were compared and analyzed.Expression of bacterial virulence genes in wild-type,mutant and complementary strains were investigated by quantitative real-time reverse transcription PCR.In addition,expression of interleukin(IL)-1β and IL-8 genes in HD-11 cells infected with the wild-type strain,mutant strain and complementary strain were also investigated.Morphological changes of the APCE94,APCE94ΔeivC and APCE94CΔeivC strains were determined by transmission electronmicroscopy(TEM).Our results showed that inactivation of eivC led to impaired flagella production and augmented fimbriae on the bacterial surface,consequently,reduced bacterial motility.In addition,the eivC mutant strain exhibited attenuated virulence in ducks,diminished serum resistance,reduced survival in macrophage cells and in ducks,upregulated fimbrial gene expression,and downregulated flagellar and virulence gene expression.The expression of the inflammatory cytokines interleukin IL-1β and IL-8 were increased in HD-11 macrophages infected with the eiv C mutant strain,compared with the wild-type strain.These virulence-related phenotypes were restored by genetic complementation.These findings demonstrated that ETT2 ATPase EivC was involved in the motility and pathogenicity of APEC. |
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