LeuO fine-tunes the virulence attributable to Salmonella pathogenicity island-2 via repressing ssrA transcription in Salmonella Typhimurium

LeuO, initially identified as a leucine regulator in Escherichia coli, has since been identified as a global regulator required for bacterial pathogenicity in a broad range of bacteria, including Salmonella, Shigella, and Vibrio. This study aimed to determine the regulatory role of LeuO in Salmonella pathogenicity island (SPI)-2, essential for the intracellular proliferation of Salmonella enterica serovar Typhimurium (S. Typhimurium). Overexpression of LeuO repressed the transcription of SPI-2 genes and accordingly decreased its protein levels. Chromatin immunoprecipitation sequencing revealed the genome-wide binding sites of LeuO in S. Typhimurium 14,028 and identified a distinctive 23-nucleotide motif with high similarity to that previously discovered in E. coli. Notably, multiple LeuO-binding sites were predicted within SPI-2, primarily adjacent to the ssrA and ssrB loci. In vitro binding assays verified the high binding affinity between LeuO and three specific motifs located at positions −35 to −12 (ssrA1),+231 to + 254 (ssrA2) near ssrA, and at positions −622 to −599 (ssrB3) near ssrB, relative to their transcription start sites. Furthermore, LeuO overexpression abolished the transcription of lacZ fused to the ssrA promoter containing ssrA1 and ssrA2, suggesting the direct repression of ssrA via LeuO-binding. The absence of LeuO increased the intracellular survival of S. Typhimurium within macrophages, whereas its overexpression attenuated bacterial replication, which was presumably associated with the downregulation of SPI-2 by LeuO. This study reveals the versatile regulatory mechanisms of LeuO and underscores its pivotal role in modulating SPI-2 expression, thereby providing key insights into the fine-tuning of virulence by Salmonella during systemic infection.