The (p)ppGpp Synthetase RelA Contributes to Virulence, Competition Capability and Antibiotic Resistance of Avian Pathogenic Escherichia coli

Abstract

Avian pathogenic Escherichia coli (APEC) induces avian colibacillosis and brings huge economic losses to global poultry production. The small alarmone (p)ppGpp mediates the bacterial stringent response, a vital pathway modulating microbial stress adaptation and pathogenic capacity. The functions of the (p)ppGpp synthase gene relA in APEC pathogenesis remain poorly characterized. In this study, we constructed a relA deletion mutant (ΔrelA) and its complemented strain (CΔrelA). The phenotypic and pathogenic characteristics of these strains were investigated. The results showed that deletion of relA did not significantly affect bacterial growth or motility. However, the ΔrelA strain showed increased susceptibility to aminoglycoside antibiotics. Furthermore, the enhanced interbacterial competition of the mutant was associated with the upregulation of core genes in the type VI secretion system (T6SS). Importantly, relA was essential for APEC adhesion to and invasion of avian DF-1 cells, as well as for colonization and virulence in ducklings, where ΔrelA exhibited significantly attenuated infectivity and reduced bacterial loads in the liver and spleen. Furthermore, transcriptomic analysis revealed that RelA deletion downregulated genes involved in integral components of the membrane, and further assays confirmed compromised membrane integrity in the mutant strain. These findings suggest that RelA maintains membrane integrity, which underpins its contributions to antibiotic resistance and virulence. These findings indicate that relA plays a key role in APEC virulence, antibiotic resistance, and membrane homeostasis, and could provide a theoretical basis for targeting the stringent response as a potential strategy to control avian colibacillosis.