Genetic knockout of pagP in avian pathogenic Escherichia coli using CRISPR-Cas9/¿-Red system and growth characterization

Authors: JIA, LINAN - Mississippi State University; FUGATE, HAILEY - Mississippi State University; YE, XIN - Mississippi State University; ARICK II, MARK - Mississippi State University; HSU, CHUAN-YU - Mississippi State University; Evans, Jeffrey; Robinson, Kelsy; ADHIKARI, PRATIMA - Mississippi State University; ZHANG, LI - Mississippi State University

Submitted to: Journal of Applied Poultry Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2026
Publication Date: 1/15/2026
Citation: Jia, L., Fugate, H., Ye, X., Arick Ii, M.A., Hsu, C., Evans, J.D., Robinson, K., Adhikari, P., Zhang, L. 2026. Genetic knockout of pagP in avian pathogenic Escherichia coli using CRISPR-Cas9/¿-Red system and growth characterization. Journal of Applied Poultry Research. 35(2026):100679. https://doi.org/10.1016/j.japr.2026.100679.
DOI: https://doi.org/10.1016/j.japr.2026.100679

Interpretive Summary: Subunit vaccines are a promising alternative to traditional live or inactivated vaccines as they can contain a unique combination of carefully selected antigens to provide wide spread protection without the need for live organisms or toxic components. This is particularly interesting for control of Avian Pathogenic Escherichia coli (APEC), as the vast genetic differences among strains has so far prevented traditional vaccines from providing adequate protection. In this study, the safety and biological relevance of the APEC gene pagP was evaluated to determine its ability to be used as a subunit vaccine. Results showed that removal of pagP did not affect growth, survivability, colonization, or development of APEC in culture or in an embryo lethality disease model. This indicates that pagP is a safe and non-toxic option for future subunit vaccine development. Further research is needed to determine its immunomodulatory potential and ability to protect against disease.

Technical Abstract: Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, leading to significant economic losses worldwide. As antibiotic use becomes increasingly restricted, subunit vaccines offer a promising alternative for APEC control. This study aimed to evaluate the biological relevance and safety of pagP, an outer membrane enzyme, as a potential subunit vaccine antigen. A CRISPR-Cas9 system was used to generate a pagP deletion mutant in APEC strain MS1657, and the mutation was confirmed by PCR, Sanger sequencing, and whole-genome sequencing. In vitro growth curve demonstrated no significant difference in doubling time between the MS1657'pagP mutant and the wild-type strain, indicating that pagP is not essential for bacterial viability under laboratory conditions. To assess the role of pagP in host interaction, we employed a chicken embryo infection model to evaluate embryo survival, bacterial colonization, and developmental outcomes. Embryos infected with either wild-type or MS1657'pagP strains showed comparable survival rates and tissue bacterial loads, suggesting that deletion of pagP does not impair virulence or replication capacity in ovo. However, the embryo-to-egg weight ratio was significantly decreased in MS1657'pagP group relative to wild-type MS1657 group (P = 0.016), indicating a modest reduction in host stress or growth disruption. No significant differences were observed in overall egg weight loss or embryo weight across groups. These findings demonstrate that pagP is non-essential for APEC growth and does not increase host damage when deleted, supporting its safety as a subunit vaccine target. Our results provide functional evidence that pagP is a biologically stable, non-toxic candidate warranting further evaluation in post-hatch immunization models.