YgeP, a global regulator within the ETT2 pathogenicity island, coordinates motility-biofilm balance and negatively controls serum resistance in avian pathogenic Escherichia coli.

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

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.

Molecular mechanism underlying YibT-mediated RpoS regulation of virulence variation in Salmonella typhimurium.

Salmonella typhimurium, a Gram-negative pathogen widely distributed in the environment, poses a serious threat to public health by contaminating food and causing foodborne diseases. Our preliminary study found that under thymol stress, the expression of YibT, a poorly characterized factor in salmonella, was significantly reduced, and deletion of the yibT gene markedly impaired biofilm formation. However, the biological role of yibT in salmonella pathogenesis remains unclear. In this study, the λ-Red homologous recombination system and pET28a vector were used to construct the single deletion mutant STΔrpoS, the double deletion mutant STΔrpoSΔyibT, complementation strains, and an overexpression strain. Promoter reporter vectors were constructed using pKP302. Growth characteristics, biofilm formation, adhesion, invasion, virulence, and pathogenicity were evaluated. The regulatory relationship between rpoS and yibT was examined by β-galactosidase assays. Results showed that deletion of rpoS and yibT led to slow growth, reduced biofilm formation, and decreased flagella and surface appendages under transmission electron microscopy. qRT-PCRshowed that there is a negative feedback loop between yibT and rpoS, where RpoS positively regulates yibT transcription and YibT negatively regulates RpoS expression. Preliminary detection of β-galactosidase confirms that yibT may be located downstream of rpoS. Cellular infection models demonstrated that deletion of yibT and rpoS significantly reduced salmonella adhesion, invasion, and intracellular survival. In mouse infection experiments, bacterial loads in the liver, spleen, and ileum were markedly decreased in mutant-infected mice, with further reduction under quercetin intervention. Flow cytometry analysis of T-cell subsets suggested attenuated immune modulation by the mutant strains, indicating reduced pathogenicity. Collectively, yibT contributes to regulate adhesion and invasion of S. typhimurium, and its deletion attenuates bacterial virulence and host pathogenicity. YibT may participate in the virulence regulatory network by interacting with the transcriptional regulator RpoS. This study provides a theoretical basis for elucidating the molecular mechanism by which YibT modulates salmonella pathogenicity.

Effects of the ecpA gene on the biological characteristics and pathogenicity of avian pathogenic Escherichia coli strain FJLY68.

Avian pathogenic Escherichia coli (APEC) is a major cause of colibacillosis in poultry, yet the role of the ecpA gene, which encodes the major structural subunit of the Escherichia coli common pilus (ECP), remains incompletely defined in APEC pathogenesis. To investigate the role of ecpA in the biological characteristics and pathogenicity of Avian Pathogenic Escherichia coli (APEC) strain FJLY68, an ecpA deletion mutant (ΔecpA) and its corresponding complemented strain (CΔecpA) were constructed using the CRISPR/Cas9 system and verified by PCR and Sanger sequencing. Phenotypic analyses revealed that the ΔecpA mutation significantly impaired bacterial motility, biofilm formation, adherence to chicken embryonic fibroblast (DF-1) cells, and fimbriae assembly. Transcriptomic analysis identified 1720 differentially expressed genes in the ΔecpA mutant, significantly enriched in pathways associated with flagellar assembly, chemotaxis, and metabolism, consistent with the observed phenotypic changes. Although in vitro growth was unaffected, the ΔecpA mutant exhibited markedly attenuated virulence in a chick infection model, as indicated by an increased LD50, attenuated clinical signs and pathological lesions, and reduced bacterial colonisation in tissues. Full genetic complementation restored all observed defects to wild-type levels. This study identifies ecpA as a critical determinant of APEC pathogenesis, directly linking its function to bacterial motility, biofilm formation, adhesion, and in vivo virulence, and provides a theoretical basis for developing novel control strategies targeting this virulence factor.

System CpxRA Negatively Regulates the Locus of Enterocyte Effacement of Enterohemorrhagic Escherichia coli Involving 32 σ and Lon protease RETRACTED: The Two-Component

PrfA, the master virulence regulator of Listeria monocytogenes: structural–functional insights, regulatory architecture, and antivirulence strategies

Pathogen-enriched transcriptional regulators modulate biofilm formation and virulence in Escherichia coli O157:H7.

Pathogenic Escherichia coli O157:H7 encodes numerous lineage-enriched transcriptional regulatory proteins that are absent or poorly conserved in non-pathogenic strains, yet their functions in transcriptional programming and biofilm-associated virulence remain largely uncharacterized. Here, we identified eight previously unreported biofilm-associated regulatory proteins involved in biofilm formation in enterohemorrhagic E. coli (EHEC) O157:H7 via comparative genomic analysis. Conserved domain analysis revealed that these regulators belong to diverse DNA-binding protein families, including LysR-, GntR-, LuxR-, AraC/XylS-, Cro/CI-, and Ogr/Delta-like families. Among these, two regulators (ECs_2620 and ECs_4457) were confirmed as direct DNA-binding transcriptional regulators through in vitro and in vivo DNA-binding assays. Functional analyses showed that overexpression of these regulators enhanced biofilm formation, whereas gene deletion impaired biofilm development and reduced bacterial virulence in both THP-1 cell and mouse infection models. Transcriptomic analysis revealed that these regulators primarily modulate genes involved in bacterial chemotaxis and flagellar assembly, representing a regulatory framework distinct from the classical c-di-GMP-centered regulatory paradigm described in non-pathogenic E. coli. Furthermore, we identified that the specific DNA-binding motifs of two regulators with direct in vitro DNA-binding activity. Evolutionary analysis showed that these regulators are highly conserved among pathogenic E. coli lineages, including EHEC, enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC), but are absent or truncated in non-pathogenic E. coli K-12 strains. Collectively, this study characterizes a set of pathogen-enriched transcriptional regulatory proteins and provides new insights into the macromolecular regulatory mechanisms underlying biofilm formation and virulence in pathogenic E. coli.