ABSTRACT: 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 gram-negative pathogens, such as Salmonella, Shigella, and Vibrio; and gram-positive bacteria, such as Streptococcus pneumoniae. However, the regulatory roles and targets of LeuO vary among species. In the Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), LeuO represses the transcription of Salmonella pathogenicity island (SPI)-1, thus diminishing the ability of the bacteria to invade host cells. However, its regulatory effect on SPI-2, essential for survival within macrophages, remains poorly understood. This study aimed to determine the regulatory role of LeuO in the intracellular persistence of 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 14028 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 persistence, 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.
