Non-typhoidal Salmonella (NTS), including Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), are significant global causes of bacterial gastroenteritis, posing a major public health challenge due to their adaptability to diverse environments and hosts. NTS infection involves gut colonization, intestinal invasion, and crucially, immune permissiveness. S. Typhimurium's disease-causing ability is tied to virulence factors in Salmonella Pathogenicity Island’s (SPI). In the realm of SPI, SPI-2 stands out as the pivotal player responsible for the maintenance and replication of the bacterium within macrophages, a crucial aspect facilitating systematic dissemination. Although numerous research endeavors have employed transcriptomic analyses to unravel the intricate regulatory networks governing SPI-2 in cellular models, the arduous task of RNA isolation has presented a significant obstacle. In this context, a simplified differential centrifugation method has been rigorously validated for the isolation of intracellular Salmonella RNA, rendering it amenable to sequencing and reinforcing its reliability for comprehensive analysis. This study was initiated by leveraging transcriptomic data in the quest for a regulatory gene capable of orchestrating Salmonella's survival within macrophages. Validation of RNA-Seq data, along with the screening of thirteen candidate genes using a gentamicin protection assay against macrophages, led to the identification of a final candidate gene, leuO which highlighted the absence of leuO led to higher survival rates of Salmonella inside macrophages. Subsequent analyses demonstrated that the overexpression of leuO markedly attenuated the transcriptional activity of SPI-2 genes, leading to the consequential cessation of Type III Secretion System 2 (T3SS-2) protein expression. These observations robustly reinforce the contributory role of leuO within the regulatory framework controlling SPI-2. Moreover, the extensive inhibition of SPI-2 gene expression, without concurrently affecting the expression of upstream regulatory genes, indicates that the master regulators, ssrA and ssrB, are probable targets for leuO-mediated repression of SPI-2 transcription._x000D_
<br> LeuO is recognized as a global regulator, making it suitable for exploring its binding profiles within Salmonella Typhimurium. To delve deeper into this, ChIP-Seq analysis was conducted to identify LeuO's DNA binding profile, which led to the discovery of LeuO's binding motif. This allowed for in silico binding predictions across the Salmonella genome, including SPI-2 genes such as ssrA and ssrB, each harboring two potential LeuO binding motifs. These motifs underwent extensive in vitro assays, ultimately revealing that LeuO represses ssrA but not ssrB. However, efforts to validate the repressive effect of LeuO within macrophages were inconclusive at 9 hours post-infection (p.i). Monitoring the translocation of SseJ-CyaA, a ssrAB-regulated effector, revealed an elevated translocation period occurring around 4 to 5 hours p.i. in the absence of leuO. However, contrary to expectations, there was no observed concurrent increase in SPI-2 genes. Chloroquine was employed to remove intravacuolar bacteria and keep cytosolic Salmonella intact. This resulted in a significant upregulation of SPI-2 genes, notably sseJ, in ΔleuO at 5 hours p.i, accompanied by a marked surge in the cytosolic bacterial population at this time point. This supports the hypothesis that cytosolic Salmonella are indeed targeted by LeuO-mediated suppression of SPI-2 genes early in the infection process. Further investigation revealed that the increased cytosolic population was not due to an escape mechanism, but rather seemed susceptible to autophagy-like elimination. However, with prolonged infection, the ΔleuO mutant exhibited a simultaneous increase in extracellular bacterial load and macrophage cytotoxicity, along with decreased macrophage viability. This pattern resembled a pyroptosis phenotype in long-term macrophage infections of Salmonella lacking leuO. This pattern underscores the potential consequences of an early, unregulated surge in SPI-2 activity at the infection's onset. Such a surge might be a crucial factor in the detrimental long-term effects on macrophage function. This study identifies the concealed regulation that connects the causal relationships governing these complex dynamics during extended infection periods._x000D_
<br> Finally, deepening the understanding of the intricate mechanisms employed by Salmonella during infection could yield valuable tools and insights, potentially having far-reaching implications for public health, microbiology, and the development of novel therapeutic strategies.
