RyhB-1 and RyhB-2 are small non-coding RNAs in Salmonella
that act as regulators of iron homeostasis by sensing the environmental iron concentration. Expressions of RyhB paralogs from Salmonella
Typhimurium are increased within microphages. RyhB paralogs restrain the growth of S.
Typhimurium in RAW264.7 macrophages by modulating the expression of Salmonella
pathogenicity island 1 (SPI-1) genes sicA
. However, little is known about the regulatory role of RyhBs and their virulence-associated targets in Salmonella
Enteritidis. We studied candidate targets of RyhB paralogs via RNA-Seq in conditions of iron limitation and hypoxia. RyhB paralogs were expressed when the S.
Enteritidis strain CMCC(B)50336 (SE50336) interacted with the chicken macrophage line HD11. We analyzed gene expression associated with Salmonella
survival and replication in macrophages in wild-type strain SE50336 and the RyhB deletion mutants after co-incubation with HD11 and screened out targets regulated by RyhBs. The expressions of both RyhB-1 and RyhB-2 were increased after co-incubation with HD11 for 8 h and several survival-associated genes within macrophages, such as ssaI
, and hns
, were upregulated in the ryhB
deletion mutant. Specifically, ssaI
, the type-three secretion system 2 (T3SS-2) effector encoded by SPI-2, which promoted the survival of Salmonella
in macrophages, was upregulated more than 3-fold in the ryhB
deletion mutant. We confirmed that both RyhB-1 and RyhB-2 downregulated the expression of ssaI
to repress its mRNA translation by directly interacting with its coding sequence (CDS) region via an incomplete complementary base-pairing mechanism. The SPI-2 gene sseA
was indirectly modulated by RyhB-1. The survival assays in macrophages showed that the ability of intracellular survival of ryhB
deletion mutants in HD11 was higher than that of the wild-type strain. These results indicate that RyhB paralogs downregulate survival-related virulence factors and attenuate the survival of S
. Enteritidis inside chicken macrophage HD11.