Pathogenic Roles Of Adhesion And Invasion Associated Factors Of Avian Pathogenic E. Coli DE205B

Escherichia coli typically colonize the mammalian and avian gastrointestinal tract and other mucosal surfaces. While many of these strains are commensal, certain pathogenic strains can cause severe diseases. Based on anamnestic clinical reports and virulence features, pathogenic E. coli have been categorized into intestinal pathogenic and extraintestinal pathogenic (ExPEC). Extraintestinal pathogenic E. coli (ExPEC) predominantly belong to phylogenetic group B2, which contain newborn meningitis E. coli (NMEC), uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC) based on their original host and clinical symptom. Among these typical infections caused by ExPEC in human are urinary tract infections (UTIs) and neonatal meningitis. Similarly, systemic infections caused by APEC are economically devastating to poultry industries. Previous studies showed that APEC and NMEC strains are highly similar. Moreover, virulence determinants common to APEC and NMEC have been identified, such as the type 1 and P fimbriae, the K1 capsule, aerobactin iron transport system, IbeA. And more and more proofs indicated that the APEC could be a vehicle or even a reservoir for human ExPEC strains, such as NMEC and UPEC. Thus, it is necessary to study the zoonotic potential of APEC.Microbial pathogenicity is a complex phenomenon encompassing diverse mechanisms. There are, however, several common strategies that pathogenic organisms use to sustain themselves and overcome host barriers, one of them being the firm adhesion of the microorganism to host cells. Colonization is crucial to pathogenesis of bacteria, being the earliest stage during onset of the disease and the ability to adhere to host surfaces is by far the most vital step in the successful colonization by microbial pathogens. The presence of adhesins are said to be essential to the first steps of bacterial pathogenicity. Initial colonization, after the bacteria have been acquired perinatally from the mother, is followed by transported to tissues through the bloodstream. It is necessary to cross the host barriers for most pathogenic bacteria and cause disease. Invasion associated proteins play roles in the progress of invasion.1. Pathogenic roles of Autotransporter adhesin AatA in APEC DE205BAutotransporter adhesin gene aatA was identified using Suppression Subtractive Hybridization (SSH) in our previous study, whereas the role of aatA was not clear. Thus, it is necessary to analyze the sequence and distribution of aatA in 779 different pathogenic E. coli. The mutant with in-frame deletion in the autotransporter adhesin gene aatA was generated using the lambda red recombinase method, and the complementation strain was constructed. The virulence, adhesion capacity in vivo and in vitro, aggregation and expression levels of virulence genes was compared for these strains, which help us study the role of aatA in the pathogenesis of APEC. The Open Reading Frame (ORF) of aatA gene is 3498 bp, which encoded 1165 aa protein AatA. This Autotransporter adhesin AatA comprises all structural motifs characteristic for members of the family of autotransporter proteins:a signal peptide at the N-terminus, an autotransporter repeat, a passenger domain and a translocation domain. The prevalence of aatA in the E. coli collection in China and Germany was investigated, and 186 of 779 strains were positive for aatA, accounting for 23.9%. Moreover, non pathogenic strains also were positive for aatA, which might be the horizontal transfer of aatA between different strains by transposable elements. In addition, analysis of the association of the E. coli reference (ECOR) groups and Multilocus sequence typing (MLST) with the distribution of aatA revealed that aatA was linked to group D and ST93, ST140 and ST117. The mutant strain and complementation strain showed well genetic stability, whereas the growth capacity of mutant strain was slightly reduced than that of the wild strain. The expression of aatA in fimbria-negative strain AAEC189 exhibited functions of cell aggregation and settling of cells. However, this cell aggregation is blocked by Type I fimbriae. The results of adherence inhibition assays showed that AatA and its antibody could reduce the adhesion of APEC to DF-1 cells, which indicate that AatA plays roles in adhesion of APEC. Moreover, the mutant strain showed reduced adhesion capacity in vitro and in vivo and down regulated expression levels of virulence genes, which led to defective virulence in duck and mice model compared to the wild type strain. Whereas, the adhesion capacity, virulence and expression levels of virulence genes were restored for the complementation strains, which indicated that autotransporter adhesin AatA was involved in the initial colonization of APEC to host cells.2. Pathogenic roles of invasion protein IbeA in APEC DE205B The invasion-associated gene ibeA is located on a genomic island, GimA, which is involved in the invasion and penetration Blood-Brain Barrier (BBB) of Neonatal Meningitis E. coli (NMEC). However, the roles of ibeA in the pathogenesis of APEC are unkonwn. Thus, it is necessary to analyze the sequence, distribution and pathogenic mechanism of ibeA in APEC. The ibeA gene from APEC DE205B is 1371 bp long and 100% identical to that of the other two APEC strains, BEN2908 and APEC_O1, and one NMEC strain, IHE3034, whereas the ibeA gene of another NMEC strain, RS218, has 99.3% identity to that of DE205B. This result indicated that the specificity of ibeA was not consistent along pathotypes. The prevalence of ibeA in the APEC collection in China was investigated, and 20 of 467 strains were positive for ibeA, accounting for 4.3%. In addition, analysis of the association of the E. coli reference (ECOR) groups with positive strains revealed that ibeA was linked to group B2. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and NMEC RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. A clean ibeA deletion mutant of DE205B was created using the lambda red recombinase method. The mutant strain and complementation strain showed well genetic stability, and deletion of ibeA did not affect growth kinetics of APEC. The inactivation of ibeA in APEC DE205B led to the reduced invasion capacity in vivo and in vitro, whereas the invasion ability was restored in the complementation strain. In addition, the expression of ibeA in AAEC189 exhibited enhanced invasion capacity, whereas IbeA and its antibody could block the invasion of APEC. Thus, we can assume that ibeA is involved in the invasion of APEC to host cells and BBB. The deletion of ibeA resulted in the decreased expression virulence genes associated with invasion and adhesion, which might be a reason for the decreased invasion capacity and defective virulence. The results of biofilm formation assays showed that IbeA contributed to the biofilm formation, which aids in the survival of bacteria and enhances their ability to cause disease.3. Construction and Characterization of autotransporter adhesin gene aatA and invasion-associated gene ibeA double gene mutant strains in APEC DE205BAdhesin and invasion protein play important roles in the adhesion, invasion and penetration across the blood-brain barrier for APEC. Thus, autotransporter adhesin gene aatA and invasion-associated gene ibeA double gene deletion mutant were constructed and analyzed. Thus, a clean aatA and ibeA double gene deletion mutant of DE205B was created using the lambda red recombinase method. The characterization of mutant strain and wild type strain were compared and analyzed, which showed that deletion of aatA and ibeA did not affect growth kinetics of APEC. The abilities of adhesion and invasion to DF-1 cells for double gene mutant strain significantly reduced to 59% and 42% of the parent strain. The adhesion and invasion capacities to blood, brain, lung, liver and spleen of double gene mutant strain was reduced to 37.2%、10.5%、23.6%、16.1% and 63.1% compared to wild type strain. Moreover, double gene mutant strain exhibited additive effects compared to single gene mutant strain. It is possible that interactions of these determinants with their receptors may involve different signaling pathways. In addition, double gene mutant strain showed defective virulence compared to the parent strain and single gene mutant strain, indicting that double gene mutant strain would be a candidate for the attenuated live vaccine.4. Pathogenic roles of Genetic Island GimA regulation protein IbeR in APEC DE205BibeR is located on a genomic island, GimA, which was involved in the pathogenesis of NMEC and APEC. However, the pathogenic mechanism of ibeR was unkonwn. Thus, it is necessary to analyze the sequence, distribution and compare the characterization of mutant strain, complementation strain and wild type strain, which contribute to study the role of ibeR in the pathogenesis of APEC. The ibeR gene from DE205B is 1950 bp long and 99-100% identical to that of the other E. coli strains. The prevalence of ibeR in the APEC collection in China was investigated, and 20 of 467 strains were positive for ibeR, accounting for 4.3%. Moreover, ibeR was linked to E. coli reference (ECOR) groups B2, which was same to the distribution of ibeA gene. The inactivation of ibeR in DE205B led to the reduced invasion capacity in vivo and in vitro, which might lead to the defective virulence of APEC. Thus, it could be conclude that regulatory protein IbeR was involved in the invasion of APEC.