Colibactin is a complex secondary metabolite that leads to genotoxicity that interferes with the eukaryotic cell cycle. It plays an important role in many diseases, including neonatal mouse sepsis and meningitis. Avian pathogenic Escherichia coli (APEC) is responsible for several diseases in the poultry industry and may threaten human health due to its potential zoonosis. In this study, we confirmed that clbG was necessary for the APEC XM strain to produce colibactin. The deletion of clbG on APEC XM contributed to lowered γH2AX expression, no megalocytosis, and no cell cycle arrest in vitro. None of the 4-week Institute of Cancer Research mice infected with the APEC XM ΔclbG contracted meningitis or displayed weakened clinical symptoms. Fewer histopathological lesions were observed in the APEC XM ΔclbG group. The bacterial colonization of tissues and the relative expression of cytokines (IL-1β, IL-6, and TNF-α) in the brains decreased significantly in the APEC XM ΔclbG group compared to those in the APEC XM group. The tight junction proteins (claudin-5, occludin, and ZO-1) were not significantly destroyed in APEC XM ΔclbG group in vivo and in vitro. In conclusion, clbG is necessary for the synthesis of the genotoxin colibactin and affects the development of APEC meningitis in mice.

Avian pathogenic Escherichia coli (APEC) is an important member of extraintestinal pathogenic Escherichia coli (ExPEC). It shares similar pathogenic strategies with neonatal meningitis E. coli (NMEC) and may threaten human health due to its potential zoonosis. RyhB is a small non-coding RNA that regulates iron homeostasis in E. coli. However, it is unclear whether RyhB regulates meningitis occurrence. To investigate the function of RyhB in the development of meningitis, we constructed the deletion mutant APEC XM∆ryhB and the complemented mutant APEC XM∆ryhB/pryhB, established a mouse meningitis model and evaluated the role of RyhB in virulence of APEC. The results showed that the deletion of ryhB decreased biofilm formation, adhesion to the brain microvascular endothelial cell line bEnd.3 and serum resistance. RNA-seq data showed that the expression of multiple virulence-related genes changed in the ryhB deletion mutant in the presence of duck serum. Deletion of ryhB reduced the clinical symptoms of mice, such as opisthotonus, diarrhea and neurological signs, when challenged with APEC. Compared with the mice infected with the wild-type APEC, fewer histopathological lesions were observed in the brain of mice infected with the ryhB deletion mutant APEC XM∆ryhB. The bacterial loads in the tissues and the relative expression of cytokines (IL-1β, IL-6, and TNF-α) in the brain significantly decreased when challenged with the APEC XM∆ryhB. The expressions of tight junction proteins (claudin-5, occludin and ZO-1) were not reduced in the brain of mice infected with APEC XM∆ryhB; that is, the blood-brain barrier permeability of mice was not significantly damaged. In conclusion, RyhB contributes to the pathogenicity of APEC XM in the meningitis-causing process by promoting biofilm formation, adhesion to endothelial cells, serum resistance and virulence-related genes expression.