Transcription elongation factors GreA and GreB regulate the cellular invasion and virulence of Salmonella Enteritidis

The facultative intracellular bacterium Salmonella enterica subspecies enterica serotype Enteritidis (S. Enteritidis) is a leading cause of salmonellosis in humans and animals and has been associated with intensive poultry farming and egg production. Gre proteins (including GreA and GreB) have been identified as virulence factors in several bacteria, but their role in S. Enteritidis has not been elucidated. Here, we constructed greA and greB double gene-deletion mutants and investigated their effects on S. Enteritidis. The deletion of the Gre proteins impaired bacterial growth and motility, but enhanced the anti-oxidative stress response and biofilm formation. Thereafter, we evaluated the effects of Gre protein deletion on bacterial adhesion and invasion in vitro and in vivo. Compared to the parental strain, the ΔgreAΔgreB mutant showed significantly increased LD₅₀ values in challenged chicks. The mutant was significantly less efficient at the colonization of livers, spleens, and lungs following subcutaneous inoculation. Consistently, interference with bacterial adhesion and entry into cells was the predominant reason for weakened S. Enteritidis infectivity. Importantly, Gre factors could restore infectivity in the complemented strain. Transcriptomic analysis (RNA-Seq) showed that the loss of Gre proteins caused the differential expression of metabolism and virulence genes. Thus, our fingdings suggest that Gre factors act as transcriptional regulatory factors by influencing the transcription of metabolism-related genes, and can act as virulence elements by directly engaging with cellular invasion and/or indirectly affecting the expression of virulence genes in S. Enteritidis.