Abstract
Growing evidence indicates that small noncoding RNAs (sRNAs) play important regulatory roles during bacterial infection. In Salmonella Typhimurium, several sRNAs are strongly up-regulated within macrophages, but little is known about their role during the infection process. Among these sRNAs, the well-characterized paralogs RyhB-1 and RyhB-2 are two regulators of gene expression mainly related with the response to iron availability. To investigate the role of the sRNAs RyhB-1 and RyhB-2 from S. Typhimurium in the infection of RAW264.7 macrophages, we analyzed several phenotypic traits from intracellular mutant strains lacking one and both sRNAs. Deletion of RyhB-1 and/or RyhB-2 resulted in increased intracellular survival and faster replication within macrophages. The bacterial metabolic status inside macrophages was also analyzed, revealing that all the mutant strains exhibited higher intracellular levels of ATP and lower NAD+/NADH ratios than the wild type. Expression analyses from bacteria infecting macrophages showed that RyhB-1 and RyhB-2 affect the intra-macrophage expression of bacterial genes associated with the Salmonella pathogenicity island 1 (SPI-1) and the type III secretion system (T3SS). With a two-plasmid system and compensatory mutations, we confirmed that RyhB-1 and RyhB-2 directly interact with the mRNAs of the invasion chaperone SicA and the regulatory protein RtsB. Altogether, these results indicate that the RyhB homologs contribute to the S. Typhimurium virulence modulation inside macrophages by reducing the intracellular growth and down-regulating the SPI-1 gene expression.
Highlights
Typhimurium strain overexpressing the island-encoded sRNA RyhB-2, we identified putative regulatory targets related with iron homeostasis and nitrosative stress and with genes belonging to the Salmonella pathogenicity island 1 (SPI-1), the type III secretion system (T3SS), and some regulatory factors associated with the SPI-1 T3SS [13]
Since the expression of both RyhB-1 and RyhB-2 is highly induced inside murine macrophages [7,8], suggesting that they could potentially play a regulatory role during infection, we first examined the effect of the sRNAs on the S
We confirmed that the expression of both RyhB-1 and RyhB-2 is highly induced inside RAW264.7 macrophages at 8 h post-infection (Figure S1)
Summary
Salmonella Typhimurium must cope with several stress conditions during the passage from the stomach to the intestine. The pathogen can enter and proliferate within non-phagocytic and phagocytic cells, such as epithelial cells and macrophages, respectively. Typhimurium secretes effector proteins that generate a specialized intracellular compartment, the Salmonella-containing-vacuole (SCV). The SCV allows bacteria to evade macrophage killing, and these phagocytic cells become the vehicle for the systemic bacterial spread and the niche of persister cells [1,2].
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