3′UTR-derived small RNA couples acid resistance to metabolic reprogramming of Salmonella within macrophages

Abstract

Acid resistance is crucial for enterobacteria to withstand host acidic environments during infection, including the gastrointestinal tract and macrophage phagosomes. A key acid resistance mechanism of the facultative intracellular pathogen Salmonella is the expression of the arginine decarboxylase AdiA. While AdiA confers acid resistance via an H⁺-consuming reaction, we discover that the 3′-untranslated region (UTR) of adiA mRNA is processed by RNase E into a regulatory small RNA, AdiZ. Through RNA–RNA interactome profiling and transcriptomic analysis, followed by in vitro structural probing and in vivo validations, we demonstrate that AdiZ directly base-pairs with and negatively regulates ptsG, pykF, and dmsA mRNAs involved in glucose uptake, glycolysis, and anaerobic respiration, respectively. Intriguingly, AdiZ is induced and facilitates Salmonella survival within macrophages, where acidic and hypoxic stresses prevail. Thus, simultaneous expression of AdiA and AdiZ from a single mRNA ties arginine-dependent acid resistance to metabolic reprogramming of Salmonella in the host intracellular niches.

Graphical Abstract

Graphical Abstract.

Introduction

Salmonella enterica (hereafter referred to as Salmonella) is a globally important pathogen responsible for over 100 million infections annually [1]. This bacterium is primarily transmitted through the fecal-oral route, with a higher prevalence in regions without proper sanitation or access to clean drinking water [2, 3]. During infection, Salmonella experiences different acidic environments; extracellularly, throughout the gastrointestinal tract [4], and intracellularly, during its proliferation inside the mildly acidic Salmonella-containing vacuole (SCV) within host macrophages [5]. For Salmonella to adapt to acidic environments, key enzymes for its survival in acidic environments are inducible amino acid decarboxylases. The Salmonella genome encodes three acid-inducible decarboxylases, AdiA, CadA, and SpeF, which catalyze the H⁺-consuming conversion from arginine, lysine, and ornithine to agmatine, cadaverine, and putrescine, respectively. The cognate antiporters, AdiC, CadB, and PotE, transport the polyamines out of the cell in exchange for their respective amino acids (Fig. 1A) [6, 7]. The CadA/CadB and SpeF/PotE systems are required to counteract growth inhibition by short-chain fatty acids produced by the gut microbiota in the large intestine [8].

Figure 1.

Conservation, secondary structure, and expression profile of AdiZ sRNA. (A) Schematic representation of the adiAYC cluster (top) and the AdiA/C acid resistance system (bottom). AdiA: arginine decarboxylase; AdiY: transcriptional activator of adiA and adiC; AdiC: arginine:agmatine antiporter. AdiA consumes intracellular H⁺ through arginine decarboxylation. adiZ is located 12 bp downstream of the adiA stop codon in Salmonella. (B) Phylogenetic tree of adiZ constructed using MAFFT [93]. (C) Multiple sequence alignment of adiZ was performed using MultAlin [94]. Fully, partially, and poorly conserved nucleotides are indicated in red, blue, and black, respectively. The single-stranded region in Salmonella AdiZ and the conserved intrinsic terminator are indicated. (D) In vitro structure probing of 5′end-labeled Salmonella AdiZ. “T1” and “OH” ladders represent digestion under denaturing conditions with RNase T1 (lane 1, cleaving unpaired G residues) and alkali treatment (lane 2, cleaving at all positions), respectively. Lane 3 (“control”) shows untreated AdiZ, while lanes 4 and 5 depict cleavages induced by Pb(II)-acetate (cleaving single-stranded regions) and RNase T1 under native conditions, respectively. (E) Secondary structure of Salmonella AdiZ predicted from in vitro structure probing data and computational analysis using mfold [52]. Unpaired G residues identified in (D) are marked with arrowheads. Three seed regions, as defined in Fig. 4A, are indicated. The region shared by seed 2 and seed 3 is shown in black. (F) AdiZ expression is highest under acidic (pH 5.5) and anaerobic conditions. Salmonella cultures were grown at pH 7.0 under aerobic or anaerobic conditions until the late exponential phase. The culture pH was shifted to acidic (pH 5.5), alkaline (pH 8.0), or maintained at neutral pH (pH 7.0). Total RNA was extracted 30 min after the pH shift and analyzed by northern blot. AdiZ was detected using probe MMO-1469. 5S rRNA detected by MMO-1056 served as a loading control.

Among these acid resistance systems, the arginine-dependent system is the most effective in protecting Salmonella under anoxic conditions against an extreme acidic stress (pH 2.3) [9]. The transcription of adiA is activated under acidic and anaerobic conditions by an AraC/XylS family transcriptional regulator, AdiY [10], which is encoded between adiA and adiC genes on the same strand (Fig. 1A). The adiA gene contains an intrinsic terminator, which attenuates the transcription of downstream genes [11] and whose transcript binds the RNA chaperone Hfq [12]. The 3′-untranslated region (UTR) of the monocistronic adiA mRNA accumulates as a 95-nt small RNA (sRNA), annotated as STnc1180 [13–15]. However, the mechanism of STnc1180 biogenesis and the potential regulatory function of this sRNA were unclear.

Hfq-dependent sRNAs typically repress translation of target mRNAs by base-pairing near the ribosome-binding site, thereby blocking ribosome access, which often accompanies the degradation of the target mRNA [16–18]. sRNAs derived from mRNA 3′UTRs similarly function as canonical Hfq-dependent sRNAs [19–22]. Given that the nucleotide sequences in the 3′UTRs are highly conserved among bacterial species, their complementarity to the other mRNAs often in the 5′UTRs implicates profound links in various biological processes such as central metabolism [23–27], anaerobic respiration [28], and membrane integrity [29]. Recent advances in RNA-seq methodologies have expanded our knowledge of bacterial RNA-RNA interactions [30–32], but most of them await further investigation.

In this study, we extend the physiological roles of the adi locus beyond its well-established function in acid resistance. We demonstrate that STnc1180, which we rename to AdiZ, is released by RNase E from the 3′UTR of adiA mRNA, whose expression is induced under acidic and anaerobic conditions. By analyzing both transcriptome and RNA-RNA interactome of Salmonella grown under specific conditions, we identify pykF, ptsG, and dmsA as direct targets of AdiZ. These genes are critical for glycolysis [33], glucose uptake [34], and anaerobic respiration [35], respectively, thereby linking acid resistance with key metabolic processes of Salmonella within macrophages. AdiZ specifically interacts with these mRNAs using three distinct seed sequences. These post-transcriptional regulations support Salmonella proliferation within the acidic and anoxic environment inside macrophages.

Materials and methods

Bacterial strains and growth conditions

The derivatives of Salmonella enterica serovar Typhimurium strain SL1344 and Escherichia coli used in this study are listed in Supplementary Table S1. Bacterial cells were grown at 37°C, or 30°C for temperature-sensitive mutants, in LB Miller medium either aerobically with reciprocal shaking at 180 rpm or anaerobically under 76% N₂, 20% CO₂, 4% H₂ atmosphere in a Coy anaerobic chamber (COY). Where required, the culture pH was adjusted to 8.0 or 5.5 with 100 mM 3-morpholinopropanesulfonic acid (MOPS, pH 8.6) or 100 mM 2-morpholinoethanesulfonic acid (MES, pH 5.4), respectively. Media were supplemented with antibiotics when necessary at the following concentrations: 50 µg/ml ampicillin (Amp), 50 µg/ml kanamycin (Km), 12.5 µg/ml chloramphenicol (Cm), and 20 mg/ml tetracycline (Tet).

Animal studies

Animal experiment procedures were approved by the University of Tsukuba animal experiment committee. 8-week-old female C57BL/6J mice were purchased from CLEA Japan and housed in groups of 5 or 6 in ventilated cages. Mice were orally administered 20 mg of streptomycin 24 h before Salmonella inoculation.

Salmonella cells grown aerobically in LB (pH 7.0) for 21 h were harvested by centrifugation (10 min at 3,200 g, 20°C), washed twice with PBS, and diluted in PBS to a concentration of 10⁷ colony-forming unit (CFU)/ml. Bacterial suspensions of the two strains were then mixed, and 100 µl (5 × 10⁵ CFU of each strain) was orally administered to the mice. The strain combinations used are listed in Supplementary Table S2. Fresh fecal samples were collected at 24 h post-infection (h.p.i.). Mice were euthanized 48 h.p.i. and colonic luminal contents, livers, and spleens were harvested. The samples were homogenized, serially diluted in PBS, and plated onto LB agar containing Cm. The plates were incubated at 37°C overnight. Fluorescence signals from superfolder GFP (sfGFP) or mScarlet-I (mSc) within the colonies were detected using a trans illuminator (BIO CRAFT) to determine the CFU of each strain.

Plasmid construction

The plasmids and oligonucleotides used in this study are listed in Supplementary Tables S3 and S4, respectively. For cloning of the adiA-adiZ and adiZ regions, DNA fragments were amplified using primer pairs MMO-1496/MMO-0544 and MMO-0543/MMO-0544, respectively. The amplified fragments were digested with XbaI and ligated into the vectors pKP8-35 (for arabinose-inducible expression [36]) or pJV300 (for constitutive expression [37]). To generate the Δ8U mutant, the plasmid-borne adiZ was mutated by inverse PCR using primer pair MMO-1813/MMO-1814, followed by DpnI digestion and self-ligation of the PCR product. The G10C, G32C, and C46G mutants were generated using overlapping primer pairs MMO-1933/MMO-1934, MMO-2095/MMO-2096, and MMO-1807/MMO-1808, respectively, followed by DpnI digestion. Translational fusion plasmids based on pXG10-sf and pXG30-sf were constructed as described previously [38, 39]. Single-nucleotide mutations were introduced by inverse PCR using overlapping primer pairs: MMO-1935/MMO-1936 for pykF G10C, MMO-2093/MMO-2094 for ptsG G32C, and MMO-1815/MMO-1816 for dmsA C46G, followed by DpnI digestion. pYC963 was generated by amplifying the backbone from pJV300 using primer pair YCO-3283/YCO-3284, and the mCherry insert from pCWU6-mCherry using YCO-3285/YCO-3286. The two fragments were assembled using the Ready-to-Use Seamless Cloning Kit (Sangon Biotech). To construct pYC964, the plasmid backbone was amplified from pYC963 using YCO-3367/YCO-3368, and the adiA promoter region was amplified using YCO-3369/YCO-3370. The resulting fragments were similarly fused using the Ready-to-Use Seamless Cloning Kit. pXG-10-tnaC_eco5′UTR-sfGFP was constructed by inverse PCR with MMO-1937/MMO-1987 using pXG-10sf-tnaC_eco as a template, followed by self‐ligation of the PCR products. For pXG-10-tnaC_eco5′UTR-mSc, the sfGFP sequence in pXG-10sf-tnaC_eco was removed by inverse PCR with MMO-1982/MMO-2000. The mSc sequence [40], codon-optimized for efficient expression in bacteria, was synthesized and amplified by PCR with MMO-1998/MMO-1999. Both fragments were digested with NheI and XbaI, then ligated.

Strain construction

Deletion mutants were constructed using pKD46 for the λ Red recombination [41] and pKD13 as a template for amplifying the Km resistance cassette. The resulting Km‐resistant strains were confirmed by PCR, and the mutant loci were transduced into appropriate genetic backgrounds using P22 and P1 phages for Salmonella and E. coli, respectively. The ΔadiZ mutants of both Salmonella and E. coli were transformed with the temperature‐sensitive plasmid pCP20 expressing FLP recombinase to excise the resistance gene from the chromosome. The rne-1 allele was transduced from strain TK40 [42] into the E. coli ΔadiZ::FRT strain using P1 phage.

Chromosomal point mutations in adiZ (G10C, G32C, and C46G) and its target genes (pykF-C-22G, ptsG-C-19G, and dmsA-G-22C) were introduced by scarless mutagenesis using the two-step λ Red system [43, 44]. A DNA fragment containing a Cm resistance marker and an I-SceI recognition site was amplified from the template plasmid pWRG100 with primer pair MMO-2126/MMO-2127 and integrated into the adiZ locus of the chromosome by λ Red recombinase expressed from pKD46. Similarly, Km resistance cassettes containing an I-SceI recognition site were amplified from pWRG717 using primer pairs AWO-2438/AWO-2439, AWO-2440/AWO-2441, or AWO-2442/AWO-2443, and inserted into the pykF, ptsG, or dmsA loci, respectively. The resultant mutants were then transformed with pWRG99. The adiZ mutant alleles were amplified from pP_L-AdiZ derivatives (Supplementary Table S3) using MMO-2129/MMO-2130 for G10C, or MMO-2128/MMO-2130 for G32C and C46G. The pykF-C-22G, ptsG-C-19G, and dmsA-G-22C alleles were amplified from pXG10-sf derivatives (Supplementary Table S3) using AWO-2444/AWO-2445, AWO-2446/AWO-2447, and AWO-2448/AWO-2449, respectively. These alleles were integrated into the chromosome by λ Red recombinase expressed from pWRG99. Recombinants were selected on LB agar plates supplemented with Amp and 5 µg/ml anhydrotetracycline to induce I-SceI endonuclease. Successful recombinants were confirmed by Cm or Km sensitivity, PCR, and sequencing.

For western blot detection of PykF, PtsG, and DmsA, a 3xFLAG epitope tag was fused to the C-terminus of the corresponding proteins. Primer pairs MMO-2146/MMO-2147 and MMO-2148/MMO-2149 were used for PykF and DmsA tagging, respectively, with the template plasmid pSUB11 [45] for PCR amplification. The PCR products were integrated into the chromosome by λ Red recombinase expressed from pKD46. The resulting Km-resistant strains were confirmed by PCR, and the mutant loci were transduced into appropriate genetic backgrounds using P22 phage. For tagging PtsG, a ptsG-3xFLAG-Km^R allele was transduced from strain JVS-4141 [46] using P22 phage.

For macrophage and mouse infection assays, the hisG mutation causing histidine auxotrophy in the Salmonella SL1344 strain was restored to functionality by introducing a C206T (Pro → Leu) reverse mutation via two-step PCR. In the first PCR step, primer pairs MMO-1943/MMO-1946 and MMO-1944/MMO-1945 were used with genomic DNA of wild-type Salmonella as the template. The purified products were used as templates for the second PCR step with primer pair MMO-1943/MMO-1945. The final PCR product was transformed into a Salmonella strain harboring pKD46 and plated onto M9 minimal medium supplemented with 0.4% glucose. The resulting strains were confirmed by sequencing, and the mutant locus was transduced into the appropriate genetic backgrounds using P22 phage.

To introduce fluorescence markers into the Salmonella chromosome, a DNA fragment, P_LtetO-tnaC_eco5′UTR-sfGFP-Cm^R or P_LtetO-tnaC_eco5′UTR-mSc-Cm^R, was amplified by PCR using primer pair MMO-1988/MMO-1989 and the template plasmid pXG-10-tnaC_eco5′UTR-sfGFP or pXG-10-tnaC_eco5′UTR-mSc (Supplementary Table S4), respectively. The amplified fragment was integrated into the chromosomal putA-putP intergenic region by λ Red recombinase expressed from pKD46. The resulting Cm-resistant strains were confirmed by fluorescence and PCR, and the mutant loci were transduced into appropriate genetic backgrounds using P22 phage.

Structure probing

DNA templates for in vitro transcription were amplified from wild-type Salmonella genomic DNA using the following primer pairs carrying a T7 promoter on the sense oligo: AWO-1787/AWO-1788 for adiZ, AWO-1801/AWO-1802 for pykF, AWO-1803/AWO-1804 for ptsG, and AWO-1805/AWO-1806 for dmsA. For each reaction, 0.4 pmol of labeled AdiZ RNA was denatured and incubated at 37°C for 15 min in the presence of RNA Structure Buffer (Thermo Fisher Scientific) and 1 µg of yeast RNA (Thermo Fisher Scientific) in a final volume of 10 µL. Pb(II)-induced cleavage was initiated by adding 2 µL of 25 nM Pb(II)-acetate, followed by incubation at 37°C for 90 s. Reactions were terminated by adding 12 µL of Gel Loading Buffer II (Thermo Fisher Scientific). A control reaction was performed by denaturing 1 pmol of labeled RNA at 95°C in 10 µL of water, followed by immediate cooling on ice with 10 µL of GL II RNA loading dye. To generate an alkaline hydrolysis ladder, 1 pmol of labeled RNA was incubated in Alkaline Hydrolysis Buffer (Thermo Fisher Scientific) at 95°C for 5 min. For RNase T1 digestion, 1 pmol of labeled RNA was first denatured in water at 95°C for 1 min, then incubated with RNase T1 at 37°C for 3 min. All reactions were stopped using the same procedure described above. Around 10 µL of each reaction was heated at 95°C for 3 min, then loaded onto a 10% polyacrylamide gel containing 7 M urea and subjected to electrophoresis at 45 W for 3 h.

In-line probing assay

To identify nucleotide positions involved in RNA-RNA interactions, in-line probing was carried out using radiolabeled in vitro transcribed AdiZ RNA (0.2 pmol, 5′end-[³²P]). The RNA was incubated at room temperature for 40 h in a reaction buffer composed of 100 mM KCl, 20 mM MgCl₂, and 50 mM Tris-HCl (pH 8.3), in the presence of 0, 0.2, or 2 pmol of unlabeled target RNAs. Reactions were stopped by adding 10 µL of denaturing gel-loading buffer containing 10 M urea and 1.5 mM EDTA (pH 8.0).

To generate an RNase T1 digestion ladder, 0.4 pmol of labeled RNA was first heat-denatured at 95°C for 1 min in sequencing buffer (Ambion), followed by treatment with 0.1 U of RNase T1 at 37°C for 5 min. For the alkaline hydrolysis ladder, 0.4 pmol of labeled RNA was incubated in 9 µL of alkaline hydrolysis buffer (Ambion) at 95°C for 5 min. Both reactions were stopped with 12 µL of loading buffer II and kept on ice. RNA fragments were resolved on a 10% polyacrylamide gel containing 7 M urea, followed by electrophoresis at 45 W for 2–3 h. Gels were then dried and visualized by phosphorimager (FLA‐3000 Series, Fuji).

Northern blot

For analysis of endogenous AdiZ, Salmonella cells were cultured in LB (pH 7.0) either aerobically or anaerobically until the late exponential phase (OD₆₆₀ = 0.5). The culture pH was then shifted to 8.0, 5.5, or maintained at 7.0 by adding 100 mM MOPS (pH 8.6), 100 mM MES (pH 5.4), or H₂O, respectively. After the pH shift, cells were cultured for an additional 30 min. For Salmonella ΔadiZ strains harboring pBAD-AdiAZ or pBAD-AdiZ, 0.2% l-arabinose was added to the cultures, and cells were further incubated for 10 min. For E. coli ΔadiZ Δhfq strains harboring pBAD-AdiAZ or pBAD-AdiZ, cells were cultured aerobically at 37°C until OD₆₆₀ reached 1.0, then 0.2% l-arabinose was added, followed by a 10-min incubation. For an E. coli ΔadiZ rne-1 strain harboring pBAD-AdiAZ, cells were cultured aerobically at 30°C until OD₆₆₀ reached 0.7. The temperature was then shifted to 42°C or maintained at 30°C, and cells were cultured for an additional 30 min. Subsequently, 0.2% l-arabinose was added, followed by a 10-min incubation. Cell cultures were immediately mixed with 20% (v/v) of stop solution (95% ethanol, 5% phenol), and total RNA was isolated using the TRIzol reagent (Invitrogen).

Total RNA (5 µg) was separated on 6% polyacrylamide/7 M urea gels in 1 × TBE buffer at 300 V for 2.5 h using a Biometra Eco-Maxi system (Analytik-Jena). DynaMarker RNA Low II ssRNA fragment (BioDynamics Laboratory) and/or Low Range ssRNA Ladder (NEB) served as size markers. RNA was transferred onto a Hybond-N+ membrane (Cytiva) by electroblotting at 50 V for 1 h using the same device. The membrane was UV-crosslinked at 120 mJ/cm², prehybridized in Rapid-Hyb buffer (Cytiva) at 42°C for 1 h, and hybridized overnight at 42°C with a [³²P]-labeled probe (Supplementary Table S5). The membrane was washed in three 15-min steps with 5 × SSC/0.1% SDS, 1 × SSC/0.1% SDS, and 0.5 × SSC/0.1% SDS buffers at 42°C. Signals were visualized on a Typhoon FLA7000 scanner (GE Healthcare).

qRT-PCR

cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). qRT-PCR was performed using TB Green Premix Ex Taq™ II (Takara Bio) on QuantStudio 5 Real-Time PCR System (Thermo Scientific). Each target-gene mRNA level was normalized to a reference gene transcript (rpoB mRNA) from the same RNA sample. Fold changes were determined using the 2^−ΔΔC_T method [47]. The sequences of the primers used are shown in Supplementary Table S4.

RNA-seq

Biological duplicates of Salmonella ΔadiZ::FRT strains harboring pKP8-35 or pBAD-AdiZ were cultured in LB (pH 7.0) anaerobically until OD₆₆₀ reached 0.5. The culture pH was shifted to 5.5 by adding 100 mM MES (pH 5.4), and cells were cultured for an additional 30 min. Subsequently, 0.2% l-arabinose was added, and cells were further incubated for 10 min. To stabilize cellular RNA, two volumes of RNA Protect Bacterial Reagent (Qiagen) were added to one volume of the cultures. Total RNA was extracted using NucleoSpin RNA (Macherey-Nagel) according to the manufacturer’s instructions and treated with TURBO DNase (Invitrogen) at room temperature for 30 min. DNase I was then denatured and removed via phenol‒chloroform extraction, followed by ethanol precipitation. RNA quality was assessed using Bioanalyzer 2100 with the Agilent RNA 6000 Nano Kit (Agilent Technologies).

For cDNA library preparation, ribosomal RNA was depleted using the Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina). Strand-specific cDNA libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit (Illumina) after transcript fragmentation. Libraries were sequenced with 150-bp paired-end reads on a NovaSeq 6000 system (Illumina). Raw reads were quality-checked using FastQC (ver. 0.11.7), and low-quality bases (< 20) and adapter sequences were trimmed using Trimmomatic (ver. 0.38) with the following parameters: ILLUMINACLIP:path/to/adapter.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36. Trimmed reads were aligned to the S. enterica subsp. enterica serovar Typhimurium strain SL1344 reference genome (ASM21085v2) using HISAT2 (ver. 2.1.0). Details of read counts and mapping rates are shown in Supplementary Table S5. The resulting .sam files were converted to .bam files using Samtools (ver. 1.9), and uniquely mapped reads were quantified with featureCounts (ver. 1.6.3). Differential expression analysis was performed with DESeq2 (ver. 1.24.0), using relative log normalization (RLE). Genes were considered differentially expressed if |Log2(Fold-Change)| > 1 and the adjusted P-value (Benjamini-Hochberg method) was < 0.05.

iRIL-seq procedure

iRIL-seq was performed as previously described [31], with minor modifications. Biological duplicates of Salmonella hfq::3xFLAG strain harboring the plasmid pBAD-t4rnl1 (pYC582) were cultured in LB (pH 7.0) either aerobically or anaerobically until OD₆₆₀ reached 0.5. The culture pH was then shifted to 5.5 by adding 100 mM MES (pH 5.4) or maintained at 7.0 with H₂O, and cells were cultured for an additional 30 min. Subsequently, 0.2% l-arabinose was added to induce expression of T4 RNA ligase, and cells were further incubated for 30 min. Cells corresponding to 50 OD₆₆₀ units (e.g. 100 ml at OD₆₆₀ = 0.5) were harvested by centrifugation at 12,000 g for 5 min at 4°C. Pellets were washed twice with 10 ml pre-chilled PBS and stored at −80°C until further use.

Hfq co-IP was carried out as described in [48]. Bacterial pellets were resuspended in 600 µl ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM KCl, 1 mM MgCl₂, 1 mM DTT, 0.05% Tween-20). Cells were lysed by bead-beating with 500 µl glass beads using a Shake Master NEO (Biomedical Science) at 4°C for 18 min. Lysates were cleared by centrifugation at 20,000 g for 30 min at 4°C and transferred to fresh tubes. For co-IP, lysates were incubated with Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) for 1 h at 4°C with gentle rotation. Beads were washed five times with 500 µl ice-cold lysis buffer and resuspended in 100 µl of the same buffer. Hfq-associated RNA was purified using RNA Clean & Concentrator columns (Sangon Biotech) and eluted in nuclease-free water.

iRIL-seq libraries were prepared using a modified RNAtag-Seq protocol through the following steps [49]. Briefly, RNA was fragmented, treated with DNase I, and purified using 2.5 × Agencourt RNAClean XP beads (Beckman Coulter) and 1.5 × isopropanol. RNA was ligated to a 3′-barcoded adaptor and purified with 2.5 × RNAClean XP beads and 1.5 × isopropanol. Ribosomal RNA was removed using the Ribo-off rRNA Depletion Kit (Vazyme), followed by purification with 2 × RNAclean XP beads and 1 × isopropanol. First-strand cDNA was synthesized with HiScript II First Strand cDNA Synthesis Kit (Vazyme). Residual RNA was hydrolyzed with 1 M NaOH, and cDNA was purified using 2 × RNAClean XP beads and 1 × isopropanol. The cDNA was ligated with a second adaptor and purified twice with 2 × RNAclean XP beads and 1 × isopropanol. Libraries were PCR amplified with Illumina P5 and P7 primers using Q5 High-Fidelity DNA Polymerase (NEB), and purified with 1.3 × RNAClean XP beads. Final libraries were sequenced on an Illumina NovaSeq X Plus platform with 150-bp paired-end reads.

iRIL-seq data were analyzed using ChimericFragments [50]. Paired-end reads that mapped within a distance of 1,000 nt or within the same transcript were considered as singletons, whereas reads mapped to two different loci were defined as chimeras. Fisher’s exact test (two-sided) was applied to assign an odds ratio and a P-value to each chimera, and those with a false discovery rate ≤ 0.05 and an odds ratio ≥ 1 were defined as significant chimeras (S-chimeras). Only S-chimeras supported by ≥ 10 chimeric reads were included in further analysis.

GFP fluorescence quantification

The E. coli MG1655 ΔadiZ::FRT strains harboring a combination of the sfGFP translational fusions and pJV300, pP_L-AdiZ, or its derivatives were grown in 100 µl of LB (pH 7.0) in 96-well optical bottom black microtiter plates (Thermo Scientific). The plates were incubated at 37°C with rotary shaking at 180 rpm and a 3-mm amplitude in Humidity Cassette using Spark plate reader (Tecan). Both OD₆₀₀ and fluorescence (excitation at 485 nm and emission at 535 nm with a dichroic mirror of 510 nm, fixed gain value of 50) were continuously measured every 10 min. At OD₆₀₀ of 0.6, the relative fluorescence unit (RFU) was calculated by subtracting the autofluorescence of the same strains without the sfGFP-reporter plasmids, then normalized to the OD₆₀₀ value.

Western blot

Salmonella cells were cultured anaerobically in pH 5.5 LB supplemented with 100 mM MES (pH 5.4) in the absence or presence of 0.4% glucose. When OD₆₆₀ reached 0.4, the cultures were collected by centrifugation at 3,200 g for 10 min at 4°C. The pellets were dissolved in 1 × Laemmli Sample Buffer (Bio-Rad) to a final concentration of 0.01 OD/µl, then heated at 95°C for 5 min. For PykF-3xFLAG analysis, 0.0007 OD of whole-cell samples was separated on 10% TGX gels (Bio-Rad). For PtsG-3xFLAG and DmsA-3xFLAG analysis, 0.01 OD of each sample was separated on 10% and 7.5% gels, respectively. Proteins were transferred onto Hybond P 0.2 PVDF membranes (Cytiva) at 10 V for 1 h using a semi-dry blotter (Anatech) with Trans-Blot Turbo transfer buffer (Bio-Rad). The membranes were blocked in Bullet Blocking One (Nacalai Tesque) at room temperature for 10 min and then incubated with mouse monoclonal α-FLAG (1:5,000, Sigma-Aldrich) or rabbit polyclonal α-GroEL (1:10,000, Sigma-Aldrich) antibodies diluted in Bullet Blocking One for 1 h at room temperature or overnight at 4°C. After washing three times for 15 min each with 1 × TBST buffer at room temperature, the membranes were incubated with secondary HRP-linked α-mouse or α-rabbit antibodies (1:10,000, Cell Signaling Technology) in Bullet Blocking One for 1 h at room temperature. The membranes were then washed again in three 15-min steps with 1 × TBST buffer. Chemiluminescent signals were developed using Amersham ECL Prime reagents (Cytiva), visualized on LAS4000 (GE Healthcare), and quantified using ImageJ software.

Arginine decarboxylase activity assay

Salmonella cells were cultured anaerobically in LB (pH 7.0) until the late exponential phase (OD₆₆₀ = 0.5). The culture pH was then shifted to 5.5 by adding 100 mM MES (pH 5.4). Cells were further cultured for 30 min, harvested by centrifugation (10 min at 3,200 g, 20°C), and washed with 0.85% NaCl to normalize cell concentrations. Aliquots of each cell suspension (1.25, 2.5, 5.0, 10, or 20 × 10⁷ cells) were transferred to 200 μl of the arginine decarboxylase assay reagent (1 g l-arginine, 0.05 g bromocresol green, 90 g NaCl, and 3 ml Triton X-100 per liter of distilled water, with the pH adjusted to 3.4 with HCl, [11]). The reaction mixtures were incubated at 37°C for 90 min and qualitatively assessed for decarboxylase activity based on a color change from yellow to blue.

Growth assay

For growth assessment under acidic conditions, Salmonella cells grown aerobically in LB (pH 7.0) for 21 h were diluted 1:200 in 200 µl of pH 5.5 LB supplemented with 100 mM MES (pH 5.4) in the absence or presence of 1% DMSO, 0.4% glucose, or both, in 96-well flat-bottom clear microtiter plates (Iwaki) in a Coy anaerobic chamber (COY). For general growth assessment of strains used in macrophage and mouse competitive infection assays, cells were diluted 1:200 in pH 7.0 LB either in ambient air or in a Coy anaerobic chamber. To maintain anaerobic conditions, the plates were sealed with SealPlate (Excel Scientific). Cells were incubated at 37°C with reciprocal shaking at 1,096 rpm and a 1-mm amplitude in a Synergy H1 microplate reader (BioTek). Growth was continuously monitored by measuring OD₆₀₀ every 10 min for 24 h.

Transcriptional reporter assay

Salmonella strains carrying either plasmid pYC964 or its vector control pJV300 were cultured aerobically in LB medium adjusted to pH 7.0, 6.0, or 4.0 with HCl. Cultures were grown to the exponential phase, and fluorescence was measured using an Agilent BioTek Synergy H1 plate reader (excitation: 587 nm; emission: 610 nm). RFU was normalized to the corresponding OD₆₀₀ value.

Single-cell fluorescence imaging and analysis

Around 5 × 10⁴ mouse leukemic monocyte/macrophage cells (RAW264.7; ATCC TIB-71) were seeded in 500 µl of RPMI1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; Biochrom) in a 24-well plate (Corning) and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 2 days. A Salmonella GFP-reporter strain (hisG⁺  putAP::P_Llac-O-tnaC_eco5′UTR-sfGFP-Cm^R) carrying either pYC964 or its control vector pJV300 was grown overnight in LB medium. The cultures were diluted 1:100 into fresh LB and grown to OD₆₀₀ = 2.0. Bacteria were harvested, washed twice with PBS, resuspended in RPMI medium, and added to the cells at a multiplicity of infection (MOI) of 10. Plates were centrifuged at 250 g for 10 min and incubated at 37°C for 30 min. To eliminate extracellular bacteria, the medium was replaced with RPMI containing 50 µg/ml gentamicin for 30 min, followed by RPMI containing 20 µg/ml gentamicin for the remainder of the experiment. At 0, 1, 3, 5, or 20 h.p.i., cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Nuclei were stained with Hoechst 33258 (Thermo Fisher Scientific). Images were acquired using an Olympus SpinSR10 Ixplore spinning disk confocal microscope equipped with a UPlanApo 60×/1.5 NA oil immersion objective. Image analysis was performed using ImageJ software.

Macrophage infection assay

Around 2 × 10⁵ RAW264.7 cells were seeded in 2 ml of RPMI1640 medium (Thermo Fisher Scientific) supplemented with 10% FCS (Biochrom), 2 mM L-glutamine (Gibco), and 1 mM sodium pyruvate (Gibco) in a 6-well plate (OmniAb) and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 2 days.

Salmonella cells were grown aerobically in LB (pH 7.0) for 16 h and harvested by centrifugation (2 min at 10,000 g, room temperature). The pellet corresponding to 2 OD₆₆₀ was resuspended in 2 ml of RPMI supplemented with 10% mouse serum (Sigma-Aldrich). For competition assays between two strains, equal volumes of the two suspensions were mixed at a 1:1 ratio. The strain combinations used are listed in Supplementary Table S2. The final mixture was incubated at room temperature for 20 min prior to infection. To check CFU, the mixture was serially diluted in PBS and plated onto LB agar plates. The plates were incubated at 37°C overnight.

RAW264.7 cells were infected by adding 100 µl of the bacterial suspension to each well at an MOI of 25. Following bacterial addition, the plates were centrifuged at 250 g for 10 min at room temperature and then incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 30 min. The medium was then replaced with RPMI containing 50 µg/ml gentamicin to eliminate extracellular bacteria, followed by a 30-min incubation. Subsequently, the medium was replaced again with fresh RPMI containing 10 µg/ml gentamicin, and the infected cells were incubated under the same conditions. Time point 0 was defined as the time of the first gentamicin addition.

At 2 or 20 h.p.i., the supernatant was collected, and the infected cells were solubilized using PBS containing 0.1% Triton X-100 (Gibco). Both the supernatant and lysate samples were serially diluted in PBS and plated onto LB agar plates. The plates were incubated at 37°C overnight. Fluorescence signals from sfGFP or mSc within the colonies were detected using a trans illuminator (Nippon Genetics) to determine the CFU for each strain.

Results

AdiZ is expressed under acidic and anaerobic conditions

To investigate the genetic distribution of AdiZ, we compared the sequences of the adiA 3′UTR across members of the Enterobacteriaceae family, where adiA is predominantly conserved [51]. The adiZ homologs were grouped into three distinct clusters, Escherichia-Shigella, Citrobacter, and Salmonella (Fig. 1B). In all clusters, the Hfq-binding module, composed of the intrinsic terminator preceded by a U-rich sequence [12], was nearly perfectly conserved (Fig. 1C). In contrast, the other sequences of AdiZ are relatively variable and exhibit cluster-specific characteristics. In vitro structure probing using Pb(II) and RNase T1 combined with in silico prediction by mfold [52] revealed that Salmonella AdiZ contains a stem-loop with a 24-nt single-stranded region, which would be available for base pairing with target mRNAs (Fig. 1D and 1E).

To profile the expression of AdiZ, we performed northern blot analysis. Salmonella was cultured in LB (pH 7.0) under either aerobic or anaerobic conditions until the late exponential phase. The culture was then acidified (pH 5.5), alkalinized (pH 8.0), or kept neutral (pH 7.0). Total RNA was extracted 30 min after the pH shift and analyzed by northern blot, which revealed a single ∼90-nt transcript (Fig. 1F). The signal intensity was highest at pH 5.5 under anaerobic conditions, and the expression pattern closely mirrored that of the parental adiA mRNA, which was quantified in parallel by qRT-PCR (Supplementary Fig. S1A).

AdiZ is derived from the 3′UTR of adiA mRNA via processing by RNase E

3′UTR-derived sRNAs are either expressed as primary transcripts driven from internal promoters or endonucleolytically processed from their parental mRNAs [19]. To distinguish between these biogenesis pathways for AdiZ, we first assessed the role of AdiY, a transcriptional activator required for adiA expression [10]. Total RNA was extracted from a Salmonella ΔadiY mutant and the wild-type strain anaerobically grown at pH 5.5. The levels of AdiZ and its parental adiA mRNA were analyzed by northern blot and qRT-PCR, respectively. In the ΔadiY mutant, the adiA mRNA levels were reduced to ∼1/20 of those in the wild type (Fig. 2A). Correspondingly, AdiZ expression was abolished in the ΔadiY mutant, indicating that AdiY is indispensable for the transcription of both adiA and adiZ. Notably, expression of adiY was induced under anaerobic conditions but not by acidic treatment (Supplementary Fig. S1B). These results suggest that, under anaerobic and acidic conditions, oxygen depletion triggers adiY expression through an as-yet-unknown mechanism. The activity of AdiY is likely further enhanced in response to acid stress via a conformational change [53], thereby driving transcription of adiA and adiZ.

Figure 2.

Biogenesis of Salmonella AdiZ from the 3′UTR of adiA mRNA. (A) Transcriptional activator AdiY is essential for the expression of adiA and adiZ. Wild-type Salmonella and the ΔadiY strain were cultured in LB at pH 5.5 under anaerobic conditions. Total RNA was analyzed by qRT-PCR to quantify adiA mRNA and by northern blot to detect AdiZ sRNA. qRT-PCR results were normalized to rpoB mRNA levels and are presented as mean ± standard error from three independent experiments (n = 3). Statistical significance was assessed using a two-tailed Student’s t-test (***P < 0.001). (B) AdiZ transcription depends on the promoter of adiA. The ΔadiZ strain harboring either pBAD-AdiZ, pBAD-AdiAZ, or pBAD-AdiAZ Δ8U was cultured in LB at pH 5.5 under anaerobic conditions. At the late exponential phase, 0.2% l-arabinose was added to half of the cultures. After 10 min, total RNA was extracted and analyzed by northern blot. The arrowhead indicates the primary transcript. (C) Hfq and RNase E are required for AdiZ expression. Wild-type and Δhfq strains, both with the ΔadiZ background harboring either pBAD-AdiAZ or pBAD-AdiZ, were cultured in LB at 37°C. At the late exponential phase, 0.2% l-arabinose was added, and the cells were incubated for 10 min. For wild-type and rne-1 (temperature-sensitive RNase E mutant) strains, both in the ΔadiZ background harboring pBAD-AdiAZ, cells were cultured at 30°C until the late exponential phase, then either shifted to 42°C or kept at 30°C for 30 min. 0.2% l-arabinose was added, and cells were further incubated for 10 min. Total RNA was analyzed by northern blot. As anticipated, precursors of 5S rRNA accumulated in the rne-1 mutant at 42°C. (D) Model for AdiZ biogenesis. AdiZ was detected using the probe MMO-1469, and 5S rRNA detected by MMO-1056 served as a loading control in (A), (B), and (C).

Next, we constructed two plasmids: pBAD-AdiAZ, encompassing the adiA transcription start site to the adiZ terminator, and pBAD-AdiZ, spanning the adiA stop codon to the same downstream region, both under the control of the arabinose-inducible P_BAD promoter. A Salmonella ΔadiZ mutant harboring either plasmid was anaerobically grown at pH 5.5. In the presence of arabinose, AdiZ was strongly upregulated in both constructs (Fig. 2B). However, in the absence of arabinose, AdiZ expression was negligible, indicating the absence of an internal promoter and supporting the hypothesis that AdiZ is derived from the parental adiA mRNA.

The RNA chaperone Hfq is critical for the stability and functionality of numerous sRNAs in Salmonella [16], and endoribonuclease RNase E plays a central role in sRNA processing and maturation [54]. AdiZ has been previously identified in Hfq co-immunoprecipitation (coIP) assays [14, 55], and TIER-seq (transient inactivation of RNase E followed by sequencing) revealed RNase E cleavage sites near its 5′end [54]. To investigate the involvement of these proteins in AdiZ expression, we pulse-expressed either the adiA-adiZ region or adiZ alone in the Δhfq mutant and the temperature-sensitive RNase E mutant (rne-1), respectively. In the Δhfq mutant, AdiZ levels from both constructs were significantly reduced compared to the wild type (Fig. 2C). In the rne-1 mutant, AdiZ expression was comparable to wild-type levels at a permissive temperature (30°C) but was completely abolished by heat inactivation of RNase E at 42°C. We identified a consensus U-rich sequence, a hallmark of RNase E recognition [54], immediately downstream of the predicted processing site [14] (Fig. 1C). Deletion of the eight consecutive U residues (Δ8U) in pBAD-AdiAZ significantly reduced AdiZ accumulation without affecting the partially degraded adiA mRNA fragments (Fig. 2B).

Collectively, these results show that AdiZ is cleaved from the adiA transcript by RNase E at the consecutive U-rich sequence and is stabilized through its interaction with Hfq (Fig. 2D).

RNA-seq-based approaches reveal putative target mRNAs of AdiZ

To uncover potential target RNAs post-transcriptionally regulated by AdiZ, we first performed RNA-seq analysis following AdiZ pulse expression. A Salmonella ΔadiZ mutant harboring pBAD-AdiZ or its vector control was anaerobically cultured at pH 5.5, where the endogenous AdiZ expression was maximal (Fig. 1F). Ectopic AdiZ was pulse-expressed by adding arabinose, and total RNA was extracted after 10 min for transcriptomic analysis. RNA-seq revealed significant changes in transcript levels, with 62 mRNAs showing decreased expression and 12 mRNAs showing increased expression in the AdiZ-expressing strain compared to the control strain (|Log₂(Fold-Change)| > 1, adjusted P-value < 0.05; Fig. 3A and B; Supplementary Tables S6 and S7).

Figure 3.

Target RNA candidates of AdiZ revealed by RNA-seq-based approaches. (A) Transcriptomic changes upon AdiZ pulse expression. Salmonella ΔadiZ strains harboring pBAD-AdiZ or its vector control (pKP8-35) were grown anaerobically in LB (pH 5.5). At the late exponential phase, 0.2% l-arabinose was added to the cultures, followed by a 10-min incubation. Differentially expressed genes (DEGs; adjusted P-value < 0.05, n = 2) are shown: blue for downregulated (Log₂(Fold-Change) < -1) and red for upregulated (Log₂(Fold-Change) > 1). Note that AdiZ is not represented in this dataset due to the use of a column-based mRNA enrichment protocol during RNA extraction and an annotation pipeline that excluded sRNAs. (B) Genome browser screenshots showing decreased mRNA levels of pykF and dmsA upon AdiZ pulse expression. (C) iRIL-seq detected AdiZ under anaerobic conditions. Salmonella hfq::3xFLAG strain harboring pBAD-t4rnl1 was grown either aerobically or anaerobically in LB at different pH values (7.0 or 5.5). At the late exponential phase, 0.2% l-arabinose was added to the cultures, followed by a 30-min incubation. Genome browser screenshots showing the genomic locations of adiZ covered by singleton or S-chimera (P < 0.05, two-sided Fisher’s exact test). (D) Relative abundance of chimeric RNAs with AdiZ detected by iRIL-seq. Percentages were calculated as the number of fragments corresponding to each RNA divided by the total number of chimeric fragments with AdiZ. (E) Genome browser screenshots showing pykF, ptsG, and dmsA singletons, and AdiZ chimeras mapped to ptsG, detected by iRIL-seq under anaerobic conditions at pH 5.5. (F) Venn diagram showing the overlap between potential AdiZ targets identified by RNA-seq following pulse expression and iRIL-seq.

Next, to identify transcripts that directly interact with AdiZ, we employed iRIL-seq analysis (intracellular RNA interaction by ligation and sequencing) to profile the RNA–RNA interactome [31]. By pulse-expressing T4 RNA ligase 1 from a P_BAD promoter, iRIL-seq enables in vivo proximity ligation of sRNAs with their interaction partners in living bacterial cells. Hfq-bound ligation products (RNA chimeras) are subsequently enriched via Hfq-coIP, followed by RNA-seq analysis. Here, Salmonella cells were cultured either aerobically or anaerobically at different pH values (7.0 or 5.5). As expected, AdiZ reads were most enriched as both chimeras and non-chimeric singletons under the anaerobic condition at pH 5.5 (Fig. 3C). By integrating iRIL-seq data across all four conditions, we identified a total of 12 RNAs interacting with AdiZ (Fig. 3D, Supplementary Tables S8–S12). 25.4% of the chimeric reads were mapped to the ptsG 5′UTR (Fig. 3E). Only one gene (SL1344_RS16625, encoding methyl-accepting chemotaxis protein CheM) was identified as a common candidate target by both RNA-seq-based approaches (Fig. 3F).

AdiZ represses pykF, ptsG, and dmsA via direct base-pairing

To assess whether AdiZ interacts with and regulates the candidate target RNAs identified by the two RNA-seq-based approaches as described above, we conducted in silico base-pairing predictions using IntaRNA [56], followed by translational reporter assays using the established two-plasmid system [39]. For the IntaRNA analysis, candidate sequences were selected from transcription start sites retrieved from SalCom v1.0 (https://bioinf.gen.tcd.ie/cgi-bin/salcom.pl?_HL [14]), or -200 nt for intraoperonic candidates, to + 200 nt relative to the start codons. Sequences with strong base-pairing potential (hybridization energy < -10 kcal/mol) to the main body of AdiZ were fused to sfGFP derived from pXG-10sf and pXG-30sf plasmids, which are suitable for analyzing standalone and intraoperonic targeted mRNAs, respectively [39]. An E. coli ΔadiZ mutant harboring a combination of the sfGFP-reporter plasmid and either an AdiZ-expressing plasmid or its vector control was cultured, and sfGFP fluorescence was measured. RFU normalized to OD₆₀₀ revealed that pykF, ptsG, and dmsA were significantly downregulated by AdiZ (≤ 50% relative to the vector control), but the other 25 candidates were not (Fig. 4A and Supplementary Fig. S2).

Figure 4.

AdiZ represses pykF, ptsG, and dmsA expression via direct base-pairing. (A) (Left) Base-pairing interactions predicted by the IntaRNA program [56]. Numbers above and below the nucleotide sequences indicate the position relative to the start codon of the mRNA and the cleavage site (i.e. 5′end) of AdiZ, respectively. (Right) sfGFP-reporter assay in E. coli ΔadiZ strains harboring a combination of the sfGFP-reporter plasmid and either pJV300 (vector control), pP_L-AdiZ, or its derivatives expressing AdiZ point mutants (G10C, G32C, or C46G). RFU was calculated by subtracting the autofluorescence of the same strains without sfGFP-reporter plasmids and normalized to OD₆₀₀ value. Data are presented as mean ± standard error from three independent experiments (n = 3). Statistical significance was assessed using one-way ANOVA followed by two-tailed Student’s t-test with Bonferroni correction (***P < 0.001). (B) In-line probing assay for 0.2 pmol radiolabeled AdiZ incubated with increasing concentrations of cold target mRNAs: pykF, ptsG, or dmsA (“+”: 0.2 pmol, “++”: 2 pmol). The “ctrl” lane represents untreated labeled RNAs, the “T1” lane represents RNase T1-digested RNAs for a G-ladder, and the “OH” lane represents alkaline-digested RNAs. Vertical labeled lines indicate protected regions corresponding to the base-pairing interactions validated in (A).

Notably, nearly half of the genes in the Salmonella pathogenicity island (SPI)-2 locus were significantly downregulated by AdiZ in the RNA-seq dataset (Supplementary Table S7). However, no strong regulation by AdiZ was detected for any of these genes in the sfGFP-reporter assay, including the genes encoding the key regulators SpiR and SsrB (Supplementary Fig. S2B), indicating that the downregulation of SPI-2 genes upon AdiZ pulse expression occurs through an indirect mechanism. This effect is likely a secondary consequence of Hfq titration by AdiZ, consistent with previous observations that transcript levels of SPI-2 genes are markedly reduced in a Δhfq mutant [57].

pykF encodes pyruvate kinase I, a key enzyme catalyzing the last step of glycolysis [33], while ptsG encodes the enzyme IIBC component of the glucose-specific phosphotransferase system, which mediates glucose uptake [34]. dmsA encodes the dimethyl sulfoxide (DMSO) reductase subunit A, an essential component for DMSO respiration under anaerobic conditions [58]. While pykF and dmsA were predicted to be AdiZ targets by the pulse expression approach, ptsG was identified as a target candidate via iRIL-seq (Fig. 3F), illustrating the added value of orthogonal sRNA target screens.

To validate direct base-pairing between AdiZ and its target mRNAs, point mutations were introduced into both the AdiZ-expressing plasmid and the sfGFP-reporter plasmids. The G10C, G32C, and C46G mutations in AdiZ abrogated repression of pykF, ptsG, and dmsA, respectively, whereas mutations outside the base-pairing regions did not affect the regulation (e.g. AdiZ mutants G32C and C46G repressed PykF levels comparable to wild-type AdiZ) (Fig. 4A). Conversely, complementary mutations in the target mRNAs (C-22G in pykF, C-19G in ptsG, and G-22C in dmsA) rather reversed the response to the wild-type AdiZ, but restored repression when combined with the corresponding AdiZ mutants. We hypothesize that AdiZ utilizes three distinct seed sequences to post-transcriptionally repress pykF, ptsG, and dmsA through direct base-pairing with their 5′UTRs.

To further identify the exact base-pairing regions, we probed the structure of AdiZ in association with in vitro-transcribed mRNA fragments (Fig. 4B and Supplementary Fig. S3). A ten-fold excess of pykF, ptsG, and dmsA mRNAs suppressed cleavage at the AdiZ regions G10-A19, C28-U36, and G37-U60, respectively, consistent with the in silico predictions (Fig. 4A). Binding to the dmsA mRNA also protected the A11-C18 region, which is complementary to the G55-U64 and forms the stem-loop in AdiZ (Fig. 1E). Notably, even equimolar amounts of dmsA mRNA produced a similar protection pattern to that observed with a ten-fold excess. These results validate the predicted base-pair interactions and suggest that the dmsA mRNA may bind to AdiZ with higher affinity than pykF and ptsG.

Physiological effects of AdiZ on the expression of its target proteins

To examine the regulatory effects of AdiZ on the endogenous protein levels of its targets in Salmonella, a 3xFLAG tag was introduced at their C-termini, and protein levels were analyzed by western blot. Under the anaerobic condition in LB medium supplemented with MES (pH 5.5), plasmid-driven overexpression of AdiZ in the ΔadiZ background strongly repressed all targets to < 20% of the levels observed with the vector control (Fig. 5A). The G10C, G32C, and C46G mutations in AdiZ abrogated the repression of pykF, ptsG, and dmsA, respectively, while mutations outside the base-pairing regions had no effect (Fig. 5A), consistent with the results of the sfGFP-reporter assay (Fig. 4A). DmsA contains a 45-amino-acid twin-arginine leader peptide, which functions as a signal for membrane targeting and is proposed to be proteolytically cleaved immediately after this sequence [58, 59]. Two bands corresponding to DmsA-3xFLAG were observed only in the vector control and the adiZ-C46G mutant: the upper band represents the precursor form, while the lower band corresponds to the mature form (Fig. 5A). Since AdiZ overexpression decreased the levels of both forms (Supplementary Fig. S4A and S4D), AdiZ likely represses dmsA translation rather than its post-translational processing, a conclusion supported by its interaction with the 5′UTR of dmsA mRNA (Fig. 4A).

Figure 5.

AdiZ represses endogenous protein levels of PykF, PtsG, and DmsA. (A) Salmonella ΔadiZ mutants with C-terminal 3xFLAG fusions to PykF, PtsG, or DmsA were transformed with pJV300 (vector control), pP_L-AdiZ, or its derivatives expressing AdiZ point mutants (G10C, G32C, or C46G). Cells were cultured anaerobically at pH 5.5 until the late exponential phase. (B, C) Wild-type Salmonella, the ΔadiZ mutant, and the chromosomal point mutants (adiZ-G10C, G32C, or C46G) with C-terminal 3xFLAG fusions to PykF, PtsG, or DmsA were cultured anaerobically in LB (pH 5.5) in (B), and in LB (pH 5.5) supplemented with 0.4% glucose in (C), until the late exponential phase. RNA extracted under the same conditions was analyzed by northern blot. AdiZ was detected with MMO-2143, and 5S rRNA detected with MMO-1056 served as a loading control. A representative image from three independent experiments is shown. Protein levels of PykF, PtsG, and DmsA were examined by western blot, and representative images from three independent experiments are shown. Band intensities were normalized to GroEL and are presented as mean ± standard error (n = 3). For DmsA, the total band intensity of the precursor (upper band) and mature (lower band) forms was measured. Statistical significance was assessed using one-way ANOVA followed by two-tailed Student’s t-test with Bonferroni correction (*P < 0.05, **P < 0.01, ***P < 0.001).

We next examined the effect of endogenous AdiZ on target expression using the ΔadiZ mutant and the chromosomal point mutants. Under the same conditions, PykF levels in the ΔadiZ mutant and the G10C mutant were significantly increased to 1.3-fold compared to those in the wild type (Fig. 5B, upper panels). For DmsA, in addition to the increased levels of the mature form, precursor forms were observed exclusively in the ΔadiZ mutant and the C46G mutant (Supplementary Fig. S4B and E). Since DmsA predominantly exists in its mature form in the wild type, we suggest that AdiZ tightly regulates the translation of DmsA to keep pace with its processing. Northern blot analysis revealed that the levels of the AdiZ G10C mutant were lower, while those of the C46G mutant were higher compared to the wild type (Fig. 5B, lower panels). Nonetheless, these differences in AdiZ abundance did not affect the extent of regulation since AdiZ-G10C still repressed DmsA expression. In contrast to PykF and DmsA, the PtsG levels were not significantly affected by any of the adiZ mutations under this condition.

The expression of ptsG is tightly regulated in response to glucose availability. Glucose represses ptsG through two mechanisms: by reducing intracellular cAMP levels, which abolishes cAMP-CRP complex-dependent activation [60], and by inducing SgrS sRNA, which downregulates ptsG via translation inhibition and mRNA degradation [61, 62]. Consistent with this, when cells were anaerobically cultured in LB containing 0.4% glucose at pH 5.5, PtsG levels dropped markedly to ∼20% of those observed in the absence of glucose (Fig. 5C). Under this condition, although the level of AdiZ remains constant, PtsG levels in the ΔadiZ mutant and the G32C point mutant increased to ∼1.5-fold compared to those in the wild type. In contrast, PykF levels remained unaffected in all the adiZ mutants under this condition (Fig. 5C). Nonetheless, the levels of both the precursor and mature forms of DmsA increased in the ΔadiZ and C46G mutants regardless of glucose supplementation (Supplementary Fig. S4C and F), reflecting the higher affinity of the dmsA mRNA to AdiZ (Fig. 4B).

AdiZ attenuates DMSO respiration coupled with glycolysis

To uncover the physiological significance of the post-transcriptional regulation by AdiZ, we screened for phenotypes of a Salmonella ΔadiZ mutant, in which the adiA 3′UTR upstream of the intrinsic terminator is replaced with the FRT sequence. We first examined whether the deletion of adiZ affects the expression or activity of AdiA protein, which catalyzes arginine decarboxylation coupled with H⁺ consumption. Under the anaerobic condition in LB medium supplemented with MES (pH 5.5), the deletion of adiZ alone did not affect the arginine-dependent decarboxylation, while it was markedly reduced in both ΔadiA and ΔadiAZ mutants (Fig. 6A). This result indicates that the expression of AdiA protein is not altered by deleting adiZ.

Figure 6.

AdiZ negatively affects Salmonella growth under acidic and anaerobic conditions in the presence of DMSO and glucose. (A) Deletion of adiZ does not affect AdiA expression. Left: schematic representation of the adiA-adiZ locus and individual deletion mutants, with deleted regions indicated by dashed lines. The intrinsic terminator remains intact in all strains. Right: Salmonella cells were cultured anaerobically in acidic LB (pH 5.5), washed with 0.85% NaCl, and aliquots (1.25, 2.5, 5.0, 10, or 20 × 10⁷ cells) were transferred to the arginine decarboxylase assay reagent (pH 3.4) containing bromocresol green as a pH indicator. Arginine decarboxylase raises the reagent pH, resulting in a color change from yellow to blue. (B) Biological triplicates of wild-type Salmonella and the ΔadiZ mutant were cultured anaerobically in LB (pH 5.5) in the absence or presence of 1% DMSO, 0.4% glucose, or both. Growth was continuously monitored by measuring OD₆₀₀ every 10 min for 24 h. (C) Salmonella ΔadiZ mutants harboring either pJV300 (vector control) or pP_L-AdiZ were cultured as in (B). Data are presented as mean ± standard error (n = 3), and OD₆₀₀ values at 24 h were statistically analyzed using the two-tailed Student’s t-test (*P < 0.05) in (B) and (C).

However, transcript levels of all three genes in the adiAYC locus were increased by > 2-fold in the ΔadiZ mutant compared to the wild type under the same conditions (Supplementary Fig. S5A). Conversely, overexpression of AdiZ from the pP_L-AdiZ plasmid repressed total arginine decarboxylase activity relative to the vector control (Supplementary Fig. S5B). Given that AdiZ does not affect the translation of adiY, encoding the essential regulator of this operon, as shown by the sfGFP-reporter assay (Supplementary Fig. S2A), we conclude that the observed inverse correlation between AdiZ and adiAYC expression levels is likely indirect. One possible mechanism is Hfq sequestration by AdiZ, which could influence adiY mRNA levels, consistent with previous reports that hfq deletion reduces adiY transcript levels in Salmonella [57].

Given that PykF and PtsG play key roles in glucose metabolism [33, 34] and DmsA is required for anaerobic respiration using DMSO as the terminal electron acceptor [58], we hypothesized that AdiZ might influence DMSO respiration-dependent growth of Salmonella. To test this, wild-type Salmonella and the ΔadiZ mutant were cultured anaerobically in LB supplemented with MES (pH 5.5) in the presence or absence of 1% DMSO, 0.4% glucose, or both for 24 h. The final cell density (OD₆₀₀ value) of the ΔadiZ mutant was 1.3-fold higher than that of the wild type in the presence of both DMSO and glucose (Fig. 6B). In addition, AdiZ overexpression in the ΔadiZ mutant reduced the final OD₆₀₀ to ∼80% of that observed in the vector control under the same conditions (Fig. 6C). No significant differences were observed under conditions lacking either DMSO or glucose. These results suggest that AdiZ attenuates a series of DMSO respiration reactions, where electrons are transferred from NADH generated through glucose metabolism, thereby modulating metabolic flux under anaerobic, glucose-rich conditions.

AdiZ contributes to Salmonella infection within macrophages

In addition to glycolytic flux [63], recent studies indicate that DmsA-dependent respiration is also crucial for Salmonella survival within macrophages, where the bacterium faces a hypoxic (∼1% O₂) and acidic (pH < 5) environment [5, 35]. To counteract the dissipation of ΔpH caused by reactive oxygen species (ROS) from phagocyte NADPH oxidase, Salmonella shifts its metabolic flux from oxidative phosphorylation to glycolysis [64]. This metabolic shift of Salmonella within macrophages is also supported by transcriptional profiling using a comprehensive reporter library [65]. Additionally, phagocyte NADPH oxidase generates methionine sulfoxide (MetSO), which serves as a biologically relevant terminal electron acceptor, and DmsA-mediated respiration using MetSO further supports redox balance and contributes to cytoplasmic alkalinization in Salmonella [35]. Based on these recent findings, we hypothesize that AdiZ, by coordinating the necessary metabolic adaptations, could play a pivotal role in Salmonella survival within macrophages.

To test this hypothesis, we first examined whether the adiAZ locus is expressed within macrophages. A transcriptional reporter plasmid was constructed by fusing the adiA promoter to the mCherry reporter gene. Expression of mCherry was induced under acidic conditions (Supplementary Fig. S6), consistent with the adiA mRNA expression pattern observed in Supplementary Fig. S1A. To assess adiA promoter activity within host cells, sfGFP-expressing Salmonella harboring either the P_adiA-mCherry reporter or the empty vector control were added to cultures of mouse macrophage-like RAW264.7 cells, and the fluorescence was analyzed by microscopy up to 20 h.p.i.. As expected, no mCherry signal was detected at any time point for the empty vector control (Supplementary Fig. S7). In the case of the P_adiA reporter, a weak mCherry signal was detectable at 1 h.p.i., and it became stronger at 3 and 5 h.p.i. (Fig. 7A and B). By 20 h.p.i., most intracellular Salmonella exhibited mCherry fluorescence, with a median of 89.4%. These results indicate that adiAZ expression is activated following Salmonella entry into macrophages.

Figure 7.

AdiZ promotes Salmonella infection in vitro and in vivo. (A) Activation of the adiA promoter following Salmonella entry into macrophages. An sfGFP-expressing Salmonella strain harboring the P_adiA-mCherry reporter plasmid (pZE12-P_adiA-mCherry) was added to cultures of mouse macrophage-like RAW264.7 cells. At the indicated time points, cells were fixed, stained with Hoechst, and analyzed by fluorescence microscopy. (B) Time-dependent proportion of Salmonella cells expressing mCherry within macrophages. At 0, 1, 3, 5, and 20 h.p.i., the percentage was calculated by dividing the number of mCherry-positive cells by the total number of intracellular Salmonella. In each of two independent experiments (n = 2), at least fifteen host cells were analyzed per time point. Due to variation in bacterial load, the number of Salmonella cells analyzed varied: ∼80–160 until 5 h.p.i., and ∼500 at 20 h.p.i. Red bars indicate the median with interquartile ranges. (C) Competitive infection assay between wild-type Salmonella and adiZ mutants within macrophages. Salmonella cells opsonized with mouse serum were added to cultures of mouse macrophage-like RAW264.7 cells. Fitness was assessed 2 and 20 h.p.i. by calculating the CI between strains carrying different fluorescence markers (sfGFP adiZ-WT versus mSc adiZ-WT, deletion, G10C, G32C, or C46G) within host cells. The dashed line represents CI = 1. Red bars represent the mean CI with SEM from biological replicates (n = 3 or 5). (D) Competitive infection assay between wild-type Salmonella and the ΔadiZ mutant in a mouse. Salmonella cells were orally administered to streptomycin-pretreated 8–9-week-old female C57BL/6J mice. Fecal samples were collected 1 d.p.i., and colonic luminal contents, livers, and spleens were harvested 2 d.p.i. Bacterial fitness was assessed by calculating the CI between strains carrying different fluorescence markers (sfGFP adiZ-WT vs. mSc adiZ-WT or ΔadiZ). The dashed line represents CI = 1. Red bars represent the median CI with interquartile ranges from biological replicates (n = 5 or 6 mice). Statistical significance was determined using the two-tailed Student’s t-test in (C) and Mann–Whitney U test in (D). *P < 0.05, “ns” indicates no significant difference.

Next, we evaluated the fitness of the ΔadiZ mutant within macrophages by conducting competitive infection assays in an in vitro infection model. We constructed Salmonella strains that chromosomally carry a functional hisG (hisG⁺), a fluorescence marker (sfGFP or mSc), and one of the adiZ variants (wild type, deletion, G10C, G32C, or C46G). All strains exhibited nearly identical growth in LB under both aerobic and anaerobic conditions, confirming that neither the fluorescence marker insertions nor the adiZ mutations affect the general growth of Salmonella (Supplementary Fig. S8). Mixed suspensions of two differently labeled strains were added to cultures of RAW264.7 cells, following opsonization with mouse serum. The competitive index (CI) was determined at 2 and 20 h.p.i. based on CFUs. As an important control, competition between the wild-type strains (WT sfGFP vs. WT mSc) showed no significant fitness differences at either time point (Fig. 7C). Although ΔadiZ CFUs did not significantly differ from wild-type CFUs at 2 h.p.i. inside the macrophages, the intramacrophage CFU counts were significantly reduced at 20 h.p.i., with a mean CI value of 0.56 (Fig. 7C). No significant differences were observed in the supernatants at either time point (Supplementary Fig. S9). Furthermore, fluorescence marker swapping (WT mSc vs. ΔadiZ sfGFP) consistently reproduced the lower fitness of the ΔadiZ mutant (CI = 0.50, Supplementary Fig. S10). These results reveal the positive effect of AdiZ-mediated regulations in promoting Salmonella survival within macrophages.

To pinpoint which AdiZ-targeted gene(s) is (are) responsible for the above phenotype, we conducted competition assays using the adiZ G10C, G32C, and C46G mutant strains against the wild-type strain. Each of the three mutants exhibited a modest but significant fitness defect, with mean CI values of 0.71, 0.81, and 0.76, respectively (Fig. 7C), suggesting additive effects of the disabled regulation of all three target genes. Furthermore, competition between each single point mutant and the ΔadiZ mutant revealed no significant differences at either time point in the cells or supernatant (Supplementary Fig. S11). These findings suggest that AdiZ-mediated regulations of pykF, ptsG, and dmsA additively contribute to Salmonella survival within macrophages.

To verify the contribution of each target regulation individually, we constructed Salmonella strains carrying compensatory point mutations that restore AdiZ regulation: namely, adiZ-G10C pykF-C-22G, adiZ-G32C ptsG-C-19G, and adiZ-C46G dmsA-G-22C. The ptsG- and dmsA-regulation-restored mutants showed growth comparable to the wild type in LB under both aerobic and anaerobic conditions (Supplementary Fig. S12). Importantly, their fitness within macrophages at 20 h.p.i. was also similar to that of the wild type, with mean CI values of 1.78 and 1.37 for the ptsG- and dmsA-complementary strains, respectively (Supplementary Fig. S13), thereby compensating for the reduced fitness observed in the corresponding adiZ point mutants (Fig. 7C). In contrast, the pykF-regulation-restored strain exhibited prolonged lag times in LB under both aerobic and anaerobic conditions, together with a markedly reduced maximum growth rate under anaerobic conditions, where Salmonella relies more heavily on glycolysis for growth (Supplementary Fig. S12). This is likely due to the pykF-C-22G mutation, which reduces translation efficiency as demonstrated by the sfGFP-reporter assay (Fig. 4A). In macrophage infection assays, the pykF-regulation-restored strain was outcompeted by the wild type within the cells at both 2 and 20 h.p.i, likely reflecting its general growth defect (Supplementary Fig. S13). Collectively, these complementation experiments confirm that AdiZ promotes Salmonella survival within macrophages through the specific regulation of its targets, at least ptsG and dmsA.

Lastly, we assessed the fitness of the ΔadiZ mutant in mice. Mixed suspensions of two strains were orally administered to 8–9-week-old female mice. Fecal samples were collected at 1 day post-infection (d.p.i.), and colonic luminal contents, liver, and spleen samples were harvested at 2 d.p.i., homogenized, and plated to determine CI values based on CFU counts. As observed in vitro, competition between the wild-type strains (WT sfGFP versus WT mSc) showed no significant fitness differences in any samples (Fig. 7D). In contrast, the ΔadiZ mutant exhibited a slightly but significantly reduced fitness in the spleen compared to the wild-type, with a median CI of 0.65 (Fig. 7D). Together, these findings suggest that AdiZ promotes Salmonella survival within macrophages by fine-tuning metabolic reprogramming, thereby facilitating its establishment in the host environment (Fig. 8).

Figure 8.

Model illustrating AdiZ-mediated fine-tuning of metabolic reprogramming in Salmonella within macrophages. Upon entry into macrophages, Salmonella encounters an acidic and hypoxic environment within the SCV, which induces the expression of AdiZ sRNA (the biogenesis mechanism is depicted in Fig. 2D). AdiZ fine-tunes the reprogramming of two key metabolic pathways that support intracellular adaptation: glycolysis and MetSO-dependent anaerobic respiration, by directly targeting the pykF, ptsG, and dmsA mRNAs via three distinct seed regions, shown in blue, orange, and green, respectively. The flow of electrons (e⁻), generated through glycolysis and the TCA cycle and transferred to the DmsABC complex, is indicated by a dotted arrow. This coordinated modulation of metabolic gene expression enhances Salmonella adaptation to, and survival within, the intracellular niche. SCV, Salmonella-containing vacuole; OM, outer membrane; IM, inner membrane; MetSO, methionine sulfoxide; G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate; TCA, tricarboxylic acid cycle; MQ, menaquinone.

Discussion

Bacterial acid resistance has been extensively studied since the 1990s due to its potential relevance to pathogenicity by enabling survival in severe acidic environments within the host. In the facultative intracellular pathogen Salmonella, the arginine decarboxylase encoded by the adi locus has been identified as the most effective system for resisting extremely acidic conditions, such as those found in the stomach [9]. In this study, we extend the physiological significance of the adi locus beyond its classic role in acid resistance by characterizing the adiA 3′UTR-derived sRNA AdiZ. AdiZ is transcribed as part of the adiA mRNA under acidic and anaerobic conditions, cleaved by RNase E, and stabilized by Hfq (Fig. 2D). Moreover, the adiZ deletion mutant displayed reduced fitness in both macrophages and mice (Fig. 7C and D), revealing a previously unappreciated contribution of the adi locus to intracellular adaptation. Based on our comprehensive target identification, we propose that AdiZ facilitates Salmonella infection by fine-tuning two key metabolic pathways: glycolysis and DmsA-dependent anaerobic respiration. Glucose transport via PtsG and subsequent catabolism are crucial to mitigate ROS-induced stress within macrophages [64]. DmsA, the primary AdiZ target, is essential for anaerobic respiration using MetSO as a terminal electron acceptor, contributing to redox balance and cytoplasmic alkalinization [35]. Notably, point mutations in three distinct AdiZ seed regions (G10C, G32C, and C46G), each impairing regulation of the specific target gene, individually reduced intracellular fitness (Fig. 7C), suggesting that coordinated regulation of all three targets collectively enhances adaptation to the hostile intracellular milieu.

AdiZ connects anaerobic respiration and sugar metabolism with acid resistance

One major challenge in the characterization of AdiZ is its absence under standard culture conditions, such as aerobiosis and neutral pH. Recent studies exploring the global RNA-RNA interactome in Salmonella and E. coli under normoxic conditions failed to capture significant reads of AdiZ [31, 66–71]. However, to fully uncover the infectious strategies of pathogenic bacteria, it is crucial to investigate sRNAs expressed under specific conditions, especially relevant to host niches, as these regulatory RNAs could enable rapid adaptation to the dynamic environments within the host [14, 72, 73].

Our two RNA-seq-based approaches, both conducted under anaerobic conditions, successfully provided a comprehensive list of potential AdiZ target RNAs (Fig. 3). Following in silico prediction, reporter assay, structure probing, and western blot collectively identified pykF, ptsG, and dmsA mRNAs as direct AdiZ targets (Fig. 4 and 5). Among the three validated targets, dmsA binds AdiZ with the highest affinity (Fig. 4B), supporting its designation as the primary target of AdiZ. Moreover, the deletion or C46G mutation of chromosomal adiZ strikingly increased the levels of the premature form of DmsA (Supplementary Fig. S4B and S4C), suggesting that AdiZ inhibits the translation initiation of DmsA to coordinate the processing of the leader peptide and the translocation of the mature form into the membrane [58, 59]. It is noteworthy that DmsA is involved in cytoplasmic alkalinization of intracellular Salmonella by anaerobically respirating MetSO [35]. Similar to other 3′UTR-derived sRNAs [19, 20], the adiAZ mRNA produces both the arginine decarboxylase and AdiZ sRNA, and the post-transcriptional regulation of dmsA balances the expression of two distinct acid resistance systems in the intracellular Salmonella.

Both pykF and ptsG are involved in sugar metabolism, implying their physiological roles under anaerobic and acidic conditions. Notably, the AdiZ G10C and G32C mutants, which have lost their ability to regulate pykF and ptsG, respectively, exhibited significantly reduced fitness within macrophages compared to the wild type (Fig. 7C). This emphasizes the importance of sRNA-mediated fine-tuning of sugar metabolism for the intracellular survival of Salmonella. Interestingly, both pykF and dmsA were not contained in chimeras with AdiZ in the iRIL-seq dataset, whereas ptsG was not downregulated upon AdiZ pulse expression (Fig. 3B, E, and F). Transcripts that are rapidly degraded upon AdiZ interaction are more likely to be detected by the pulse expression RNA-seq but not by iRIL-seq, and this may be the case for pykF and dmsA. Conversely, ptsG may be regulated by AdiZ primarily at the translational level without affecting mRNA abundance, explaining its presence in the iRIL-seq data but not in the pulse expression RNA-seq data. ptsG is also a well-known target of the SgrS sRNA, which promotes its rapid degradation during glucose-phosphate stress [61]. Intriguingly, Salmonella and Citrobacter AdiZ harbor and utilize the complete six-nucleotide seed sequence of SgrS that forms the critical core interaction with ptsG mRNA [74], whereas this sequence is not conserved in Escherichia or Shigella (Supplementary Fig. S14). Despite targeting the same site, AdiZ and SgrS appear to exert mechanistically distinct modes of regulation: SgrS triggers mRNA degradation, while AdiZ modulates translation without altering transcript stability. AdiZ may act on the residual pool of ptsG transcripts that escape SgrS-mediated degradation, potentially explaining why AdiZ-dependent modulation of PtsG protein levels becomes detectable only under the glucose-replete condition (Fig. 5C).

sRNAs orchestrate Salmonella virulence and sugar metabolism

Recent findings provide further evidence for the regulatory interplay between Salmonella virulence and sugar metabolism [75]. The Salmonella-specific sRNA PinT, for example, is a key regulator of virulence, controlling both SPI-1 and -2 by regulating key transcription factors HilA, RtsA, and SsrB, as well as the global carbon metabolism regulator CRP [76, 77]. CRP suppression by PinT influences SPI-2 expression during intracellular survival [76]. Furthermore, the acid-inducible sRNA RyeC represses translation of PtsI, a key component of the phosphoenolpyruvate:sugar phosphotransferase system, and promotes intracellular survival within macrophages [78]. More recently, the 3′UTR-derived sRNA ManS was shown to contribute to Salmonella colonization in the host by coordinating sialic acid metabolism [22].

SgrS represses the expression of multiple sugar transporters, including PtsG, in response to sugar-phosphate stress, and specifically in Salmonella, also directly regulates the SPI-1 effector SopD [79], suggesting that Salmonella senses subcellular host environments via carbon source availability and fine-tunes effector expression. Intriguingly, SgrS also directly base-pairs with adiY mRNA, leading to its translational inhibition and degradation in E. coli [80]. Our findings that AdiY strongly induces AdiZ expression and AdiZ represses ptsG thus suggest the existence of a coordinated sRNA-based regulatory circuit that ensures precise and robust control of ptsG expression in response to environmental cues.

The adiAZYC locus functions in the SCV environment

We argue that AdiZ-mediated post-transcriptional regulation of pykF, ptsG, and dmsA takes place within macrophages, where Salmonella faces an acidic and hypoxic environment. Our mCherry-based reporter detected activation of the adiAZ promoter following macrophage entry (Fig. 7A and 7B). However, a recent transcriptional profiling using a comprehensive GFP-reporter library failed to detect such activation [65]. One possible explanation for this discrepancy is that GFP—but not mCherry—is highly sensitive to acidic pH [81]; thus, genes induced in response to SCV acidification may be missed when using GFP-based reporter systems. While RNA-seq-based studies did not identify increased expression of the adi locus inside murine or porcine macrophages [73, 76], early transcriptome profiling using microarray analysis did detect an upregulation of adiY within murine macrophages [82]. Transcriptional activation of the adi locus may therefore depend on the timing of infection, possibly requiring both hypoxia and acidification, which are gradually established inside the SCV during maturation.

Noteworthy, transposon-directed insertion-site sequencing identified the adi locus as crucial for systemic infection in mice and intestinal colonization in chickens, pigs, and cattle [83]. Disruption of the adiAZ terminator significantly reduced fitness in pigs and cattle [83], which may further underscore the importance of the AdiZ-mediated regulations during infection. In addition to these observations, several studies have demonstrated that arginine metabolism itself plays a crucial role in Salmonella survival within macrophages. Arginine is one of the seven essential metabolites that support Salmonella colonization in mice [84] and is critical for intracellular Salmonella to withstand ROS and nitric oxide (NO) produced by phagocyte NADPH oxidase and inducible NO synthase (iNOS), respectively [85]. In addition to de novo synthesis, Salmonella acquires host-derived arginine through two ABC transporter systems and a putative l-Arg/Orn antiporter, which limit host NO production by sequestering arginine away from the iNOS pathway [85–87]. Furthermore, Salmonella utilizes polyamines generated through host arginine catabolism to facilitate type-III secretion system (T3SS) assembly [88]. Further studies will be required to elucidate when AdiA functions within macrophages and how it influences host arginine and polyamine metabolism.

Inside macrophages, Salmonella senses SCV acidity through a cascade of EnvZ/OmpR, PhoQ/PhoP, and SpiR/SsrB two-component systems to induce SPI-2 [89]. OmpR and PhoP activate the transcription of spiR and ssrB, respectively [90], and SsrB directly responds to acidic pH through a conserved histidine residue to stimulate the transcription of SPI-2 genes encoding T3SS and its effector proteins required for intracellular proliferation [91]. Interestingly, OmpR also represses the CadA/CadB acid resistance system to prevent cytoplasmic neutralization [92]. How other regulators control the transcription of adi locus in conjunction with AdiY requires further investigation.

Notes

Present address: Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), 97080 Würzburg, Germany; Department of Microbiology, Biocenter, University of Würzburg, 97074 Würzburg, Germany

Supplementary data

Supplementary data is available at NAR online.

Conflict of interest

None declared.

Funding

This study was supported by JSPS KAKENHI grant numbers JP22K14809 and JP22KJ0376 to T.K, and JP19H03464, 19KK0406, and JP24K01661 to M.M; and by international collaboration grants from JSPS-CAS Bilateral Joint Research Project (JPJSBP120237201, 176002GJHZ2022022MI) and National Key R&D Program of China (2022YFE0111800) to Y.C and M.M. T.K. was supported by JSPS Postdoctoral Fellowship, IFO scholarship for young researchers (Y-2022-2-028), and Kato Memorial Bioscience Foundation (2023B-101), and is currently supported by JSPS Overseas Research Fellowship. Research in the Miyakoshi lab is supported by Mishima Kaiun Memorial Foundation, Asahi Group Foundation, and Takeda Science Foundation. Research in the Chao lab is supported by Natural Science Foundation of China (92478118, 32270064) and Shanghai Municipal Science and Technology Commission (24ZR1493200). Research in the Westermann lab is supported by the European Research Council (ERC Starting Grant #101040214). Funding to pay the Open Access publication charges for this article was provided by the Japan Society for the Promotion of Science.

Data availability

Raw RNA-seq data from AdiZ pulse expression and iRIL-seq experiments have been deposited in the DDBJ Sequence Read Archive (DRA) database under BioProjects PRJDB35407 and PRJDB38015, respectively.