Expression of avian pathogenic Escherichia coli (APEC) virulence factors, Iss and HlyF, as potential sub-unit vaccine candidates
Van der Westhuizen, Wouter Andre
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Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis in poultry and leads to economic losses in the poultry industry. Due to rising concerns of antibiotic resistance and antibiotic carry-over into food, bans have been implemented on antibiotic use in animal production. The process of discovering new antibiotics and having them registered and approved can take up to eight years, and their application will most likely be limited to human-use. Alternative therapies for the control of bacterial diseases in animals, including poultry, are therefore becoming increasingly important. Alternative treatment options could include the use of bacteriophages, bacteriophage enzymes, essential oils and vaccines. Bacterial vaccines are generally based on whole bacterial cells, and in the case of commensal bacteria such as E. coli, this can lead to poor gut health. Therefore, the need for highly specific bacterial vaccines are required, such as sub-unit vaccines containing only antigens found in pathogenic strains of E. coli. Various virulence genes have been found in APEC that contribute to the pathogenicity of the strains. Five of these genes have been found to be highly prevalent in most clinical cases of avian colibacillosis. In this study, two of these genes were selected as potential candidates for sub-unit vaccine development, namely increased serum survival (iss) and haemolysin F (hlyF). The increased serum survival gene, an outer-membrane protein of APEC, was successfully expressed in E. coli BL21 (DE3) using the pET28b(+) vector system, although the protein was mostly water-insoluble due to hydrophobic N -and C-terminals. The sequence of iss was then modified to create a truncated version of the gene encoding the hydrophilic region of the gene, also the potential epitope of the Iss antigen, to improve solubility during over-expression. Water-soluble truncated Iss was produced in conjunction with the full Iss protein, solubilised using a zwitterionic detergent, purified using the hexahistidine regions flanking the inserted gene in the pET28b(+) vector system. The purified proteins were mixed with adjuvant and injected into chickens raise antibodies against the two expressed proteins. The antibodies obtained from the chickens were used to perform western blotting and ELISA and both proteins were confirmed to be immunogenic. Furthermore, the obtained serum was cross-reacted with the full and truncated forms of the protein, indicating potentially similar epitopes, showing promise of using the highly water-soluble truncated Iss protein for potential future vaccine development. Yarrowia lipolytica heterologous protein expression was also attempted with the full iss gene. However, no expressed protein was detected using SDS-PAGE, so alternative methods of expression, purification and isolation were attempted. An enterokinase proteolytic cleavage site was introduced between the iss gene and the GPI-anchor sequence, the secretion vector pINA1317 and the C-terminus hexahistidine region bearing pINA1317 secretion vector were used as alternatives methods to express the full iss gene sequence. These methods failed to produce the desired results. Western blotting using antiserum raised against E. coli expressed protein could also not detect any expressed Iss protein. It was then concluded that a yet unknown issue is preventing adequate, if not any, expression of the iss gene in Y. lipolytica and further research is required. During the study, published literature indicated that the putative avian haemolysin F, was not a haemolysin but an enzyme involved in outer-membrane vesicle (OMV) biogenesis. This was validated by our failure to detect haemolytic activity after hlyF-overexpression and the presence of OMVs observed by TEM. As this was very recent research into the involvement of this gene in OMV biogenesis, the impact of the expression of the hlyF gene was investigated regarding the regulation of outer-membrane proteins which are scavenged during OMV release. Relative quantitative PCR was used to compare hlyF-expressing and non-expressing cells, and it was shown that ompA expression is increased during hlyF expression, while ompF expression remained nearly the same, which could lead to osmotic-stress susceptible cells during hlyF induction. It was concluded that a decrease in ompA expression is not involved in the mechanism of hlyF-induced OMV biogenesis, contrary to one of the biogenesis mechanisms described in literature. Sub-unit bacterial vaccines could be the future method of preventing bacterial diseases in the poultry industry. Alternative methods such as bacteriophage therapy might not be possible due to the non-linear pharmacokinetics observed, which will hinder registration of these products. Sub-unit vaccines should elicit highly specific immune responses to virulence-related antigens and not target commensal bacteria. Various antigens accessible to the immune system of the host are outer-membrane or cell wall-associated proteins, protein expression of these antigens could pose problems such as high costs for production and problems during the development of expression systems for these proteins, even complete failure of expression. Knowledge and correct characterisation of virulence-related proteins is also essential, as seen with the HlyF protein expressed in this study which is not directly suitable for vaccine production as previously thought. Future work is required in the optimisation of protein expression parameters, and chicken challenge studies with APEC will need to be conducted to determine if protective immunity is gained by vaccination with the E. coli-expressed Iss proteins. It will also be highly advantageous if the problems encountered during protein expression in Y. lipolytica could solved and the yeast-expressed Iss protein tested during challenge studies with APEC.