Abstract

   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 and rtsB. 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,
   sseA, pagC, sodC, mgtC, yaeB, pocR, and hns, were upregulated in the
   ryhB-1 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-1 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-1 and/or
   ryhB-2 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.

   Keywords: Salmonella Enteritidis, RyhB paralogs, ssaI, survival,
   macrophages

1. Introduction

   Salmonella enterica serovar Enteritidis is a facultative intracellular
   pathogen that causes nontyphoidal salmonellosis in hosts, such as
   humans and poultry [[38]1]. As a major foodborne pathogen, S.
   Enteritidis is mainly transmitted through contaminated poultry products
   and eggs and causes enteritis or food poisoning [[39]2]. When infecting
   the intestine, S. Enteritidis penetrates the mucus layer, invades and
   internalizes into the intestinal epithelium, and multiplies within
   non-phagocytic and phagocytic cells. During this process, S.
   Enteritidis must respond to, and cope with, a series of stress
   conditions in the host internal environment, including iron deficiency
   and hypoxia [[40]3]. Small non-coding RNA (sRNA) quickly responds to
   stressful conditions and modulates target gene expression at the
   post-transcriptional level to resist host defenses [[41]4,[42]5].

   Survival and replication of Salmonella within macrophages are essential
   for its pathogenicity in hosts. Many sRNAs contribute to its survival
   and replication in macrophages [[43]6]. In Salmonella Typhimurium,
   transcriptome analysis showed that 88% of the 280 sRNAs are expressed
   and 34 sRNAs are upregulated within macrophages, compared to bacterial
   cultures at the early stationary phase. Among these upregulated sRNAs,
   RyhB-1 and RyhB-2, two RyhB paralogs, are the most highly upregulated
   sRNAs [[44]7]. RyhB paralogs regulate a variety of physiological
   processes in Salmonella, including iron homeostasis, nitrate
   homeostasis, and adaptive response to oxidative stress
   [[45]8,[46]9,[47]10]. RyhB-1 and RyhB-2 in Salmonella Typhi (named as
   RfrA and RfrB) are essential for the replication of S. Typhi inside
   macrophages [[48]11]. RyhB-1 and RyhB-2 restrict the growth of S.
   Typhimurium within RAW264.7 macrophages by modulating Salmonella
   pathogenicity island 1 (SPI-1) gene expression and directly regulating
   the mRNAs of the invasion chaperone SicA and the regulatory protein
   RtsB [[49]6]. Although some targets related to virulence in macrophages
   have been identified in S. Typhimurium, the targets and regulation
   mechanism of RyhB paralogs in S. Enteritidis are poorly understood and
   require study.

   To investigate the regulatory function of S. Enteritidis RyhB paralogs
   during infection of chicken HD11 macrophages, we screened out novel
   target genes of RyhB paralogs via RNA-Seq and quantitative real-time
   PCR (qRT-PCR) in vitro and analyzed the regulation mechanism of RyhB
   paralogs to targets. Furthermore, we analyzed the survival of S.
   Enteritidis mutants lacking RyhB-1 or/and RyhB-2. Our study enriches
   the regulatory network of RyhB paralogs and provides ideas for reducing
   S. Enteritidis infection.

2. Materials and Methods

2.1. Bacteria, Plasmids, and Cell-Growth Conditions

   The bacterial strains and plasmids used in this study are listed in
   [50]Table 1. All bacteria were grown in Luria-Bertani (LB) broth or on
   LB plates at 37 °C with shaking at 180 rpm. Complemented mutants
   harboring antibiotic-resistance genes were cultured in LB containing
   ampicillin (Amp, 100 μg/mL) or chloramphenicol (Cm, 34 μg/mL) when
   appropriate. Anaerobic growth was achieved via static culture at 37 °C
   in the anaerobic workstation (DG250, Don Whitley Scientific, Bingley,
   UK) with mixed gas (10% H[2], 10% CO[2], and 80% N[2]). For RNA-Seq
   analysis, all strains were cultured under iron-limited, hypoxic, and
   nutrient-limited conditions. This stressful culture was achieved by
   culturing the S. Enteritidis wild-type (WT) strain and all the deletion
   mutants in a medium containing 0.05 mol/L KH[2]PO[4] and 10 g/L trypsin
   with 0.2 mM 2, 2′-dipyridyl at 37 °C in an anaerobic workstation
   [[51]12]. Chicken macrophage HD11 cells (accession number OTWO,
   HTX2259) were cultivated in Dulbecco’s Minimal Essential Medium (DMEM)
   (HyClone, Logan, UT, USA) containing 10% heat-inactivated fetal bovine
   serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% chicken serum (VivaCell,
   Shanghai, China). Cells were maintained in an atmosphere of 5% CO[2] at
   37 °C.

Table 1.

   Bacteria and plasmids used in this study.
   Strain/Plasmids Characteristics References