Roles Of Related Iron Acquisition Systems And Regulatory Protein RfaH And Capsule Virulence Genes In Pathogenesis Of APEC And UPEC

Escherichia coli commonly colonize the mammalian and avian gastrointestinal tract or other mucosal surfaces. While most of these strains are commensal, certain few pathogenic strains can cause intestinal or extraintestinal infection. E.coli strains isolated from infections outside of the intestinal tract, e.g., uropathogenic E.coli (UPEC), neonatal meningitis-associated E.coli (NMEC) and sepsis-causing E.coli (SEPEC) have been grouped as extraintestinal pathogenic E.coli (ExPEC) by Johnson and Russo. In addition, strains which cause systemic infection in poultry, avian pathogenic E.coli (APEC), resemble certain human-pathogenic ExPEC variants. Virulence determinants common to APEC and UPEC have been identified, such as the type1and P fimbriae, the K1capsule, iron acquisition system, IbeA. It is proposed that APEC could be a vehicle or even a reservoir for human ExPEC strains, such as NMEC and UPEC.In this study, the role of capsule, iron acquisition system and regulatory protein RfaH in virulence of APEC and UPEC were focused on. The similarity of virulence factors and pathogenic mechanism between these two type strains were compared, so as to deepen our understanding of virulence genes formation, evolution, and mechanism of pathogenesis. It is becoming more and more apparent that the common presence of a set of virulence associated genes among APEC and UPEC strains as well as similar disease patterns indicate a genetic relationship between APEC and UPEC isolates. The hypothesis of APEC could be a vehicle or even a reservoir for human ExPEC strains should be fully considered.1. Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coliAPEC and UPEC are the two main subsets of ExPEC. Both types have multiple iron acquisition systems, including heme and siderophores. Although iron transport systems involved in the pathogenesis of APEC or UPEC have been documented individually in corresponding animal models, the contribution of these systems during simultaneous APEC and UPEC infection is not well described. To determine the contribution of each individual iron acquisition system to the virulence of APEC and UPEC, isogenic mutants affecting iron uptake in APEC E058and UPEC U17were constructed and compared in a chicken challenge model. Salmochelin-defective mutants E058△iroD and U17AiroD showed significantly decreased pathogenicity compared to the wild-type strains. Aerobactin defective mutants E058△iucD and U17△iucD demonstrated reduced colonization in several internal organs, whereas the heme defective mutants E058AchuT and U17△AchuT colonized internal organs to the same extent as their wild-type strains. The triple mutant AchuT△iroDAiucD in both E058and U17showed decreased pathogenicity compared to each of the single mutants. The histopathological lesions in visceral organs of birds challenged with the wild-type strains were more severe than those from birds challenged with AiroD, AiucD or the triple mutants. Conversely, chickens inoculated with the AchuT mutants had lesions comparable to those in chickens inoculated with the wild-type strains. However, no significant differences were observed between the mutants and the wild-type strains in resistance to serum, cellular ingestion and intracellular survival in HD-11, and growth in iron-rich or iron-restricted medium. Results indicated that APEC and UPEC utilize similar iron acquisition mechanisms in chickens. Both salmochelin and aerobactin systems appeared to be important in APEC and UPEC virulence, while salmochelin contributed more to the virulence. Heme bounded by ChuT in the periplasm appeared to be redundant in this model, indicating that other periplasmic binding proteins likely contributed to the observed no phenotype for the heme uptake mutant.2. Regulatory protein RfaH regulates the expression of related genes and the role in virulence of extraintestinal pathogenic Escherichia coliIn E. coli and many other bacterial species, a regulatory protein RfaH acts as a transcriptional antiterminator that reduces the polarity of long operons encoding cell components. RfaH was shown to be essential for the expression of cell components encoded on long operons in E. coli, including the expression of F plasmid, different capsules, and hemin uptake receptor, as well as the toxins alpha-hemolysin and cytotoxic necrotizing factor1. The direct effect of RfaH on virulence of APEC has not been investigated so far. Our results showed that the inactivation of rfaH significantly decreased the virulence of APEC E058and UPEC U17. The attenuation was assessed by in vivo and in vitro assays, including chicken infection assays, ingestion and intracellular survival assay, and bactericidal assay with serum complement.1-day-old bird lethal test showed that loss of rfaH resulted in abolishment of virulence of E058and U17in birds. Our results demonstrated that the resistance to serum and phagocytosis was impaired through the inactivation of the rfaH. Meanwhile, the results of the qRT-PCR analysis demonstrated that the inactivation of rfaH decreased the transcription level of virulence genes involved in the capsule, hemin uptake, serum resistance and colicin V synthesis, which may contribute to the reduced colonization and proliferation capacities of APEC E058and UPEC U17.3. Construction of capsule transport double mutant of APEC E058and UPEC U17and evaluation of their pathogenesis in chickensCapsule plays an important role in serum complement resistance and cell phagocytosis resistance for E.coli. Thus, capsule transport associated genes kpsE and kpsD were chosen for double gene deletion. The clean kpsE and kpsD double gene deletion mutant of APEC E058and UPEC U17were created using the lambda red recombinase method. The characterization of mutant strains and wild type strains were compared and analyzed. The results showed that the deletion of kpsED significantly decreased the virulence of APEC E058and UPEC U17. The attenuation was assessed by in vivo and in vitro assays, including bactericidal assay with serum complement, ingestion and intracellular survival assay, and chicken infection assays. The growth curves in LB showed that the deletion of kpsED did not affect growth kinetics of APEC E058and UPEC U17. The abilities of resistance to serum and killing by chicken macrophages were significantly impaired. LD50results showed that the double mutants completely abolished the virulence, and the colonization and coinfection model demonstrated that the deletion of kpsED leads to attenuation of virulence, since the double mutant showed significantly decreased colonization compared with the wild type strains in all organs tested in birds, indicting that double gene mutant strain would be a candidate for the attenuated live vaccine.