| The extraintestinal pathogenic E. coli (ExPEC) contains the classical phenotype avian pathogenic E. coli (APEC), which is a facultative intracellular bacterial pathogen. The colonization of avian respiratory tract is earliest infection for APEC, accompanying with inflammation and histopathological changes at air sacs and lung. To infect the host extraintestinally, highly virulent APEC can spread in bloodstream and cause fatal multisystemic infection. In recent years, due to significant mortality, drug resistance, and economic losses, avian colibacillosis caused by APEC attracts more and more attention. In this study, high virulent APEC strains isolated were whole-genome sequenced and comparative genomics analysis was performed to reveal the genetic information of APEC O2:K1 strains. The pathogenicity tests of APEC strains (isolated in China) were conducted through four animal models. Characterization of the novel autotransporter adhesions and global regulators were conducted by a serial of experimental tests. These results enrich knowledge of pathogenic mechanisms of avian pathogenic E. coli.1. Complete genome sequencing and comparative genomic analysis of APEC O2:K1 isolate IMT5155Avian pathogenic E. coli and human extraintestinal pathogenic E. coli serotypes O1,O2 and O18 strains isolated from different hosts are generally located in phylogroup B2 and ST complex 95, and they share similar genetic characteristics and pathogenicity, with no or minimal host specificity. They are popular objects for the study of ExPEC genetic characteristics and pathogenesis in recent years. In this study, we investigated the evolution and genetic blueprint of APEC pathotype by performing phylogenetic and comparative genome analysis of avian pathogenic E. coli strain IMT5155 with other E. coli pathotypes.Phylogeny analyses indicated that IMT5155 has closest evolutionary relationship with APEC O1, IHE3034, and UTI89. Comparative genomic analysis showed that IMT5155 and APEC O1 shared significant genetic overlap/similarities with human ExPEC dominant O18:K1 strains (IHE3034 and UTI89). Furthermore, the unique PAI I5155 (GI-12) was identified and found to be conserved in APEC O2 serotype isolates. GI-7 and GI- 6 encoding two typical T6SSs in IMT5155 might be useful markers for the identification of ExPEC dominant serotypes (O1, O2, and O18) strains. IMT5155 contained a ColV plasmid p1ColV5155, which defined the APEC pathotype. The distribution analysis of 10 sequenced ExPEC pan-genome virulence factors among 47 sequenced E. coli strains provided meaningful information for B2 APEC/ExPEC-specific virulence factors, including several adhesins, invasins, toxins, iron acquisition systems, and so on. The pathogenicity tests of IMT5155 and other APEC O1:K1 and O2:K1 serotypes strains (isolated in China) through four animal models showed that they were highly virulent for avian colisepticemia and able to cause septicemia and meningitis in neonatal rats, suggesting zoonotic potential of APEC O1:K1 and O2:K1 isolates.2. Characterization of a novel autotransporter adhesin AatB in avian pathogenic Escherichia coliA novel AT adhesin gene aatB was identified in APEC DE205B. The ORF of aatB was 1,017 bp, encoding a 36.3-kDa protein. The AatB included the classic structural motifs for AT proteins: a signal peptide,a passenger domain,and a translocator domain. The predicted 3D structure of AatB contained two distinct domains,the C-terminal「β-barrel translocator’domain and an N-terminal passenger domain. The aatB gene was present in 26.4% (72/273)APEC strains, and the aatB was strongly associated with ECOR D and B2. Moreover, AatB could elicit antibodies in infected ducks, indicating that AatB is involved in APEC pathogenicity. Thus, the aatB mutant strain and complemented strains with the aatB gene were constructed. Deletion of gene aatB in DE205B resulted in a reduced capacity to adhere to DF-1 cells, defective virulence capacity in vivo, and decreased colonization capacity in lung during systemic infection. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatB makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, biofilm formation assays with strain AAEC189 expressing AatB indicated that AatB mediates biofilm formation.3. Characterization of a novel autotransporter adhesin UpaB in avian pathogenic Escherichia coliHere,a conventional autotransporter UpaB in APEC DE205B genome was characterized. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells,and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655△fim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50% lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205B△upaB/aatA/aatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL,yadN,and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.4. AutA and AutR,two novel global transcriptional regulators,facilitate avian pathogenic Escherichia coli infectionIn this study, two novel regulators in APEC were identified and designated as AutA and AutR. AutA and AutR co-regulated the expression of adhesin UpaB in APEC strain DE205B. AutA and AutR could directly bind the upaB promoter DNA. AutA could activate the upaB transcription, while AutR inhibited the upaB transcription due to directly suppressing the activating effect of AutA on UpaB expression. Transcriptome analysis showed that AutA and AutR coherently affected the expression of hundreds of genes that are involved in adhesion, capsule synthesis, acid resistance, and etc. Our study confirmed that AutA and AutR co-regulated the expression of DE205B K1 capsule and acid resistance systems in E. coli acid fitness island (AFI). Moreover, phenotypic heterogeneity in expression of K1 capsule and acid resistance systems in AFI during host-pathogen interaction was associated with the regulation of AutA and AutR. Collectively speaking,this was the first case study and outstanding identification that AutA and AutR facilitated APEC infection and played roles for reciprocal phenotype regulation under host-induced stimuli by coordinately regulating the expression of adhesins, K1 capsule, and acid resistance systems. |
