A Trans-Acting Leader RNA from a Salmonella Virulence Gene

A trans-acting leader RNA from a Salmonella virulence gene

Eunna Choia,1, Yoontak Hana,1, Yong-Joon Chob,1, Daesil Namc, and Eun-Jin Leea,2

aDepartment of Genetic Engineering, Graduate School of Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 17104, South Korea; bUnit of Antarctic K-Route Expedition, Korea Polar Research Institute, Incheon 21990, South Korea; and cDivision of Microbiology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Suwon 16419, South Korea

Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved August 10, 2017 (received for review April 3, 2017)

Bacteria use flagella to move toward nutrients, find its host, or membrane potential (7). In addition, previous transcriptomic retract from toxic substances. Because bacterial flagellum is one of analysis revealed that mRNA levels of many genes involved in the ligands that activate the host innate immune system, its flagella machinery, including the fljB and fliC flagellin genes, synthesis should be tightly regulated during host infection, which strongly decreased when Salmonella is inside macrophages (8), is largely unknown. Here, we report that a bacterial leader mRNA implicating that flagella-based motility may not be required for from the mgtCBR virulence operon in the intracellular pathogen intramacrophage survival. However, it has been unknown how Salmonella enterica serovar Typhimurium binds to the fljB coding Salmonella achieves such down-regulation of flagellar genes region of mRNAs in the fljBA operon encoding the FljB phase 2 fla- during infection. Here, we report that a leader RNA originated gellin, a main component of bacterial flagella and the FljA repressor from the Salmonella mgtCBR virulence operon mediates down- for the FliC phase 1 flagellin, and degrades fljBA mRNAsinanRNase regulation of one of two flagellin genes inside macrophages. E-dependent fashion during infection. A nucleotide substitution of In the intracellular pathogen Salmonella enterica serovar Typhimurium, the mgtCBR operon encodes the MgtC viru- the fljB flagellin gene that prevents the mgtC leader RNA-mediated + lence protein, the MgtB Mg2 transporter, and the MgtR pep- down-regulation increases the fljB-encoded flagellin synthesis, lead- tide regulator for MgtC proteolysis (9–12). The PhoP/PhoQ ing to a hypermotile phenotype inside macrophages. Moreover, the two-component system controls transcription initiation of the fljB nucleotide substitution renders Salmonella hypervirulent, indi- mgtCBR operon from a single promoter upstream of the mgtC

+ MICROBIOLOGY cating that FljB-based motility must be compromised in the phago- gene in response to low Mg2 , mildly acidic pH, or antimicrobial somal compartment where Salmonella resides. This suggests that peptides (13–16). After transcription is initiated, the 296-nt-long this pathogen promotes pathogenicity by producing a virulence pro- leader region of the mgtCBR operon mediates further induction tein and limits locomotion by a trans-acting leader RNA from the of the mgtC gene inside macrophages in response to high ATP same virulence gene during infection. and low levels of charged tRNAPro (17–20), allowing the mgtC gene to be one of the highly expressed genes inside macrophages flagellin | mgtC | trans-acting leader RNA (8). Within the mgtCBR leader region, two short ORFs, mgtM and mgtP, enable the leader to sense and respond to respective Pro he bacterial flagellum is a long filamentous structure that is ATP- and charged tRNA signals by a mechanism similar to Tattached to the membrane and enables a bacterium to move that found in transcription attenuation (17–19). This mechanism toward nutrients or to escape from toxic chemicals (1). However, involves coupling between transcription of the mgtCBR leader during host infection, the bacterial flagellum could be dangerous region and translation of each ORF located in the leader, which for survival if it is recognized by the host innate immune system controls the formation of adjacent attenuator stem-loops preventing transcription elongation into the downstream coding as a ligand to elicit an immune response (2). Therefore, synthesis or assembly of flagella must be tightly regulated for a pathogen. The major structural component of flagella is thousands of Significance copies of flagellin monomer proteins, which are encoded by two different loci, fljB and fliC, for the intracellular pathogen Sal- The intracellular pathogen Salmonella enterica serovar Typhi- monella enterica serovar Typhimurium (1). Depending on which murium must move toward nutrients to obtain food. However, flagellin genes are expressed, Salmonella displays completely at the same time, it has to evade the host immune system. different antigenic properties that might be advantageous for this Because bacterial flagella are required for both bacterial pathogen to evade the host immune system. This so-called an- movement and immune detection, production of flagella must tigenic phase variation is mediated by an inversion event of a be tightly regulated during infection. Here, we establish that particular DNA region, which includes the hin gene encoding a Salmonella produces a leader RNA from the mgtCBR virulence recombinase and the promoter region of the fljBA operon operon and degrades mRNAs of the fljB phase 2 flagellin gene during infection. Our finding indicates a direct link between a encoding the FljB phase 2 flagellin and FljA, a repressor protein virulence determinant and motility via a trans-acting leader for the distally located FliC flagellin (3, 4) (Fig. 1A). When the RNA derived from the virulence determinant gene in a given DNA region is oriented as illustrated in Fig. 1A, the fljB and fljA host environment. genes are expressed, and, as a result, Salmonella possesses FljB-

polymerized flagella on its surface and expression of the other Author contributions: E.-J.L. designed research; E.C., Y.H., Y.-J.C., and D.N. performed fliC flagellin gene is repressed by the FljA repressor at the research; Y.-J.C. and E.-J.L. analyzed data; and E.-J.L. wrote the paper. posttranscriptional level (5) (Fig. 1A). However, when the DNA The authors declare no conflict of interest. region is switched into the opposite orientation by the Hin This article is a PNAS Direct Submission. recombinase at low frequency (6), the promoter region of the Data deposition: The data reported in this paper have been deposited in the Gene Ex- fljBA operon is flipped and the fljB and fljA genes are not pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession nos. expressed and thus Salmonella produces the FliC flagellin proteins. GSE85678 and GSE100414). Several lines of observations were reported that expression of 1E.C., Y.H., and Y.-J.C. contributed equally to this work. flagellar genes might be down-regulated during infection. In 2To whom correspondence should be addressed. Email: [email protected]. uropathogenic Escherichia coli, the virulence regulator PhoP This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. represses FliC flagellin production and motility by decreasing 1073/pnas.1705437114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1705437114 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 radiolabeled DNA probe corresponding to the mgtCBR leader region (22). This is probably due to the presence of the attenuator stem-loop structures that prevent transcription elongation within the mgtCBR leader region (17–19, 23) (Fig. S1). In this paper, we report that one of the smallest transcripts of the mgtCBR leader region functions as a trans-acting riboregulator and decreases amounts of the FljB flagellin during infection. The decrease in flagellin production by the mgtC leader RNA is required for Salmonella’s pathogenicity because the fljB substitution that prevents the mgtC leader-mediated fljB down- regulation renders Salmonella hypervirulent. Our data suggest that Salmonella’s motility powered by FljB flagella must be restricted during the course of infection. Results The mgtC Leader RNA Down-Regulates fljB and fljA mRNA Encoding the Salmonella Phase 2 Flagellin Operon. Previous Northern blot analysis using a DNA probe corresponding to the leader region of the mgtCBR operon detected multiple short transcripts in + RNA samples prepared from Salmonella grown in low Mg2 , which activates transcription from the PhoP-dependent mgtC promoter (22). An abundance and size of transcripts led us to wonder whether these short transcripts from the mgtC leader region might have a regulatory role as a trans-acting RNA else- where in the chromosome. To explore this, we constructed a Salmonella strain harboring a plasmid that expresses the smallest size of RNA fragments corresponding to nucleotides 1–113 in the mgtC leader from an arabinose-inducible promoter or the plasmid vector. [Please note that the nucleotide position at 113 is located just beyond the stem-loop B structure (Fig. 1A and Fig. Fig. 1. Regulation of the fljBA phase 2 flagellin operon by the trans-acting S1).] Then, we compared RNA profiles of the mgtC leader- and mgtCBR leader RNA. (A)InlowMg2+, the phosphorylated PhoP controls tran- the vector-expressing Salmonella after arabinose induction for scription of the mgtCBR virulence operon and generates full-length polycistronic 15 min. Surprisingly, only two mRNAs were significantly down- mRNA messages, harboring the 296-nt leader RNA with two short ORFs (mgtM regulated (greater than fourfold) in the mgtC leader-expressing and mgtP)andthemgtCBR genes encoding the MgtC virulence protein, the MgtB cells (Fig. 1B and Dataset S1); fljB mRNA encoding the phase 2+ Mg transporter, and the MgtR regulatory peptide, respectively. At the same 2 flagellin, which is a major structural component of bacterial fla- time, the phosphorylated PhoP generates multiple small RNA fragments (22), gella and fljA mRNA encoding a repressor that inhibits synthesis of including leader RNA corresponding to nucleotides 1–113. The mgtC leader RNA – the FliC phase 1 flagellin (5). Further sequence analysis revealed 1 113 binds to the fljB part of the fljBA polycistronic mRNAs encoding the FljB that fljB and fljA mRNAs are part of the same polycistronic mRNAs phase 2 flagellin and the FljA repressor for the FliC phase 1 flagellin and degrades transcribed from a promoter located in the fljBA operon (Fig. 1C). fljBA mRNAs in an RNase E-dependent fashion. Yellow boxes represent DNA regions (hixL and hixR), which are recognized by the Hin recombinase and inverted The decrease of fljB and fljA mRNA levels by overexpressing the during flagellar phase variation. (B) Scatterplot analysis of RNA sequencing in the mgtC leader RNA was confirmed by quantitative real time-PCR mgtC leader- versus the vector-expressing Salmonella. Red represents down- (Fig. 1 D and E and Dataset S2). As a control experiment, regulated genes more than fourfold in the mgtC leader-expressing Salmonella. mRNA levels of fliC gene encoding a different type of flagellin were (C) Schematic mapping of the RNA-sequencing reads against plus and minus unaffected by the mgtC leader RNA (Fig. 1 F and G)butrather strands of genes neighboring the fljBA operon in a strain expressing the mgtC slightly increased possibly due to the decrease of the FljA repressor leader RNA (Lower; pBAD-mgtC leader113) or the empty vector (Upper; vector). by the mgtC leader RNA (Fig. 1E). (D–K) Fold change in the mRNA levels of the coding regions of the fljB (D and H), fljA (E and I), and fliC (F and J) genes and the leader region of the mgtC gene (G The mgtC Leader RNA Degrades fljBA mRNAs in an Hfq- and RNase and K) in either wild type (14028s) (D–G)oranhfq deletion mutant Salmonella E-Dependent Fashion. If target mRNAs are less abundant in the (EG15349) (H–K) harboring a plasmid with nucleotides 1–113 from the mgtC presence of the trans-acting regulatory RNA, in many cases, leader (pBAD113) or the vector pBAD18. Bacteria were grown for 3 h in + target mRNAs are base-paired with the trans-acting sRNA N-minimal media containing 10 mM Mg2 , and then for an additional 15 min in 2+ guided by the Hfq RNA chaperone and degraded via the RNase the same media containing 0.5 mM Mg and 10 mM L-arabinose. Shown are the E riboendonuclease (24, 25). To test whether the mgtC leader mean and SD from three independent experiments. Expression levels of target RNA-mediated fljBA down-regulation requires the Hfq RNA genes were normalized to that of 16S ribosomal RNA rrsH gene. Fold change was fljB fljA calculated by dividing mRNA levels of cells expressing each plasmid by those of cell chaperone, we measured mRNA levels of the and genes expressing the empty vector, and thus the vector was set to 1. *P < 0.05; **P < when we expressed the mgtC leader RNA from the plasmid in an 0.01; ns, not significant; t test (relative to vector). hfq mutant background (22). As expected, the mgtC leader RNA lost the ability to decrease fljBA mRNA levels in the hfq mutant (Fig. 1 H–K). Hfq destabilized both the mgtC leader RNA and region. Therefore, any conditions that uncouple transcription of fljBA mRNA because, when transcription was blocked with ri- the mgtCBR leader and translation of each ORF, such as an fampicin treatment, the half-lives of the mgtC leader RNA and increase in ATP levels or a decrease in charged tRNAPro levels, fljBA mRNA increased in the hfq mutant compared with those in the wild type (Fig. S2 A, B, and D). Likewise, RNase E is re- are expected to promote mgtCBR transcription. Highly elevated quired for the mgtC leader RNA-promoted degradation of fljBA mRNA levels of the mgtC gene inside macrophages are corre- mRNAs because a Salmonella strain carrying the rne-3071ts allele lated with the notion that the intraphagosomal environment (26) did not decrease the mRNA levels of the fljB and fljA genes might be high in ATP levels due to phagosome acidification (21) by the mgtC leader RNA when RNase E was inactivated by and low in charged tRNAPro levels (20). Interestingly, when + transferring cells to 42 °C (Fig. S3). These data demonstrated Salmonella was grown in low Mg2 to induce PhoP-activated that the mgtC leader RNA-mediated fljBA mRNA degradation transcription, multiple short transcripts were detected with a requires both Hfq and RNase E.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1705437114 Choi et al. Downloaded by guest on October 1, 2021 The mgtC Leader RNA Binds to Near the 3′ End of the fljB Coding Region for fljBA mRNA Degradation. To investigate the binding site of the mgtC leader RNA and fljBA mRNA for the RNase E-mediated degradation, we searched for a potential pairing sequence using RNAhybrid software (27). The highest score was observed at a 14-nt base pairing between nucleotides 24–42 of the mgtC leader region and nucleotides 1362–1379 of the fljB coding region (see Fig. 3E and Fig. S4 A and B). Nucleotides 24– 42 of the mgtC leader region are required for acting on fljBA mRNA levels because a plasmid expressing mgtC leader variants with either the G27C C29G G31C substitution or the C41G G42C substitution lost the ability to decrease fljB and fljA mRNA levels (Fig. 2). By contrast, the C33G C35G or G53C G54C C55G substitution behaved just like the wild-type mgtC leader (Fig. 2). Translation of mgtM in the mgtC leader region is not required for the regulation of fljBA mRNA levels because mutation of the start codon in mgtM retained the ability to decrease fljBA mRNA levels (Fig. 2). Fig. 3. Base pairing between G42 in the mgtC leader region and C1362 in the – Next, we further examined whether the mgtC leader base-paired fljB gene is required for fljBA mRNA degradation. (A D) Fold change in the to the coding region of the fljB gene as predicted. We used in-line mRNA levels of the coding regions of the fljB (A), fljA (B), and fliC (C) genes and ′ 32 the leader region of the mgtC gene (D) produced by either wild type (14028s) probing assay examining the structure of a 5 P-labeled fljB RNA or a mutant Salmonella with the C1362G substitution in the fljB gene at its inthepresenceorabsenceofthemgtC leader RNA. The chromosomal location (EN925) harboring a plasmid with the wild-type mgtC predicted base-paired regions I and II were protected from leader from nucleotides 1–113(CL),aderivativewiththeG42Csubstitu- RNA cleavages in the presence of the mgtC leader RNA (Fig. tion (G42C), or the vector pBAD18 (vector). Bacteria were grown for 3 h in + S4C), supporting the base pairing between the mgtC leader and N-minimal media containing 10 mM Mg2 , and then transferred to the same 2+ the 3′ end of the fljB coding region. media with 0.5 mM Mg and 10 mM L-arabinose for an additional 1 h. Shown are the mean and SD from three independent experiments. Fold change was A Base Pair Between G42 in the mgtC Leader and C1362 in the fljB calculated as described in Fig. 1. *P < 0.05, **P < 0.01, t test (relative to vector).

Coding Region Determines the mgtC Leader-Mediated fljBA mRNA (E) Base pairing between the mgtC leader 113 and fljB mRNA predicted by MICROBIOLOGY Degradation. We focused interaction between the G42 position RNAhybrid (https://bibiserv2.cebitec.uni-bielefeld.de/rnahybrid). Mutated nu- in the mgtC leader RNA and C1362 position in fljB mRNA be- cleotides are colored in red with positions. cause, first, the mgtC leader derivative including the C41G G42C substitution retained mRNA levels of the wild-type leader compared with those of other mutations (Fig. 2D). Second, C1362 is located at the third position of a threonine codon in the fljB mRNA and the C1362G substitution does not alter the amino acid sequence of the FljB flagellin (Fig. 3E). To address an impact of the G42–C1362 interaction in the mgtC leader-mediated fljB destabilization, we created a chromosomal mutant with the C1362G substitution in the fljB gene. Then, we introduced a plasmid with the wild-type mgtC leader, a derivative with the G42C substitution, or the vector into the fljB C1362G chromosomal mutant or isogenic wild-type background. The G42C substitution in the mgtC leader displayed a defect in decreasing fljB and fljA mRNA levels in the wild-type background (Fig. 3 A, B,and E) and lost the ability to repress soft agar-based motility (Fig. S5C), behaving like the C41G G42C substitution (Fig. 2). However, an introduction of a compensatory mutation at the C1362 position in the fljB gene enabled the G42C-substituted mgtC leader to decrease fljB and fljA mRNA levels (Fig. 3 A, B,andE)andre- covered the ability to repress motility (Fig. S5C). These data indicate that G42–C1362 base pairing between the mgtC leader and the fljB coding region is required for destabilizing fljBA Fig. 2. The sequences corresponding to mgtM in the mgtC leader region are mRNAs. Control experiments demonstrated that neither the required for fljBA mRNA degradation. (A–D) Fold change in the mRNA levels of G42C nor C1362G substitution affected the expression behaviors the coding regions of the fljB (A), fljA (B), fliC (C), and the leader region of the of the fliC coding region and the mgtC leader region (Fig. 3 C and mgtC gene (D) produced by wild-type Salmonella (14028s) harboring a plasmid D) and the orientation of the DNA region involved in flagellar with the wild-type mgtC leader RNA corresponding to position 1–113 (wild type), phase variation (Fig. S5 A and B). derivatives with the C33G C35G (33, 35), C41G G42C (41, 42), G53C G54C C55G (53–55), G27C C29G G31C (27, 29, 31) substitutions, a derivative with mutation of STOP The C1362G Substitution Enhances FljB Flagellin Production and the mgtM start codon (mgtM ), or the vector pBAD18. Bacteria were grown 2+ + Surface Motility in Low Mg . The results presented above iden- for 3 h in N-minimal media containing 10 mM Mg2 , and then for an additional ’ 2+ tified the mgtC leader s regulatory action on fljB and fljA mRNA 15 min in the same media containing 0.5 mM Mg and 10 mM L-arabinose. Shown are the mean and SD from three independent experiments. Fold change levels when we expressed the mgtC leader RNA from a heterolo- was calculated as described in Fig. 1. *P < 0.05, **P < 0.01, t test (relative to gous promoter. We further investigated the mgtC leader-mediated vector). (E) Sequence of mgtC leader region from nucleotides 1–113. Positions of control of the fljBA operon in a physiologically relevant con- nucleotide substitutions used in the experiments (A–D) are shown above the mgtC dition. Because transcription of the mgtC leader is highly in- 2+ 2+ leader sequence. Sequences involved in fljBA degradation are colored in red, and ducedinlowMg butrepressedinhighMg (>0.05 mM) sequences adopting stem-loop structures A and B (18) are indicated as angle (16), we hypothesized that we could observe the mgtC leader- 2+ brackets. The amino acid sequences of mgtM are indicated below the nucleotide mediated destabilization of the fljBA mRNA only in low Mg 2+ 2+ sequences. The mgtM ribosome binding site is underlined. media but not high Mg .InlowMg , mRNA levels of the fljB

Choi et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 gene in wild-type Salmonella was ∼5.5-fold lower than those in + high Mg2 (Fig. 4A). This is in agreement with our expectation that mRNA levels of the fljB gene would be inversely correlated to those of the mgtC leader RNA (Fig. 4D). By contrast, the fljB derivative with the C1362G substitution was not subject to the mgtC leader-mediated control because the fljB C1362G substitution stabilized fljB mRNAs (Fig. S2A), and thus fljB mRNA + levels remained high even in low Mg2 , and also because there + was no further increase in high Mg2 (Fig. 4A). The mgtC leader RNA appeared to specifically target fljB mRNA because the fljB C1362G substitution was less effective in the fljA Fig. 5. The fljB C1362G substitution increases FljB flagellin production, 2+ mRNA levels in low Mg (Fig. 4B and Fig. S2B). As control leading to a hypermotile phenotype in low Mg2+.(A) A Coomassie-stained experiments, the fljB C1362G substitution had no effect on gel of supernatants prepared from wild type (14028s), the fljB derivative expression behaviors of the fliC gene (Fig. 4C and Fig. S2C) with the C1362G substitution (EN925), or a mutant Salmonella deleted both and the leader region of the mgtC gene (Fig. 4D). the fljB and fliC genes (EN994) grown for 5 h in N-minimal media containing + Elevated fljB mRNA levels of the fljB C1362G substitution either 0.01 or 10 mM Mg2 .(B and C) Western blot analysis of supernatants were reflected in the amounts of FljB flagellin in the super- prepared from wild type (14028s), the fljB derivative with the C1362G sub- natants from cells grown in N-minimal medium containing low stitution (EN925), a fliC deletion mutant (DN410), a fljB deletion mutant 2+ – 2+ (DN409), or the fljB fliC mutant Salmonella (EN994) grown for 5 h in Mg (Fig. 5 A D) but neither cells grown in high Mg (Fig. 5A) 2+ nor strains with the fljB deletion (Fig. 5 A–D). It is interesting to N-minimal media containing 0.01 mM Mg . The amounts of FljB and FliC note that the amounts of FljB flagellin released into the super- flagellins were determined by anti-FljB (B) or anti-flagellin (C) antibodies. (D) A Coomassie-stained gel of supernatants used in B and C.(E and F) natants from cells grown in N-minimal medium were below a Motility assay of strains listed in A. Bacteria were spotted and grown for 18 h + range of detection by Coomassie staining (Fig. 5 A and D)or on N-minimal solid media containing either 0.01 mM (E)or10mMMg2 (F). Western blot (Fig. 5 B and C) unless the fljB gene carries the C1362G substitution. Control experiments proved that the amounts of FliC flagellin remained unaffected (or slightly re- Finally, a strain deleted for both the fljB and fliC genes did duced) by the fljB C1362G substitution (Fig. 5 A and C). neither produce flagella nor migrate in all conditions tested (Fig. Each flagellin polymerizes into a flagellum that is embedded 5 and Figs. S6 and S7). in the bacterial membrane and provides motility (1). Because the fljB C1362G mutant was not subject to the mgtC leader-mediated The C1362G Substitution Increases fljB mRNA Levels and FljB- destabilization and increased fljB mRNA levels (Fig. 4) and FljB Containing Flagella Production Inside Macrophages. Because the – production (Fig. 5 A C), we expected that the fljB C1362G mgtC gene is highly expressed inside macrophages (8, 18), we substitution would accumulate FljB flagella, leading to display a 2+ hypothesized that the mgtC leader-mediated fljB destabilization hypermotile phenotype when grown in low Mg . Indeed, the fljB could be observed during course of Salmonella infection. In wild- C1362G substitution started to migrate away from the initial type Salmonella, the mRNA levels of the fljB gene decreased spotting point when grown for 18 h on N-minimal soft agar 2+ inside the macrophage-like cell line J774 A.1 over time (Fig. 6A). containing low Mg (Fig. 5E), which was not observed in wild The decrease of fljB mRNAs between 2 and 9 h appeared to be type (Fig. 5E). This is in agreement with a microscopic obser- dependent on the expression of the mgtC leader RNA because vation that the fljB C1362G mutant Salmonella was more flag- 2+ the fljB C1362G variant that prevents the mgtC leader-mediated ellated than wild type in low Mg (Fig. S6). Control experiments destabilization retained fljB mRNA levels higher than those of showed that both wild-type or the derivative Salmonella with the wild type (Fig. 6A). Likewise, immunofluorescent staining C1362G substitution did not migrate on N-minimal soft agar 2+ against the FljB flagellin detected enhanced production of FljB- containing high Mg when incubated for 18 h (Fig. 5F). How- polymerized flagella in the fljB C1362G variant at 9 h post- ever, further incubation up to 42 h allowed both strains to pro- infection (Fig. S8). The fljB C1362G substitution had a similar duce flagella and migrate to a similar extent (Figs. S6E and S7). but lesser effect on the mRNA levels of the fljA gene (Fig. 6B). However, the fljB C1362G substitution did not affect the expression behaviors of either the fliC gene or the leader region of the mgtC gene (Fig. 6 C and D). These data indicate that, during Salmonella infection, the mgtC leader RNA is highly expressed and mediates the decrease in fljB mRNA levels and FljB flagellin production.

The fljB C1362G Substitution Promotes Salmonella’s Survival Inside Macrophages and Virulence in Mice. We wondered whether enhanced production of the FljB-containing flagella in the fljB C1362G variant has physiological consequences during Salmo- nella infection. To address this, we measured replication effi- ciency of the Salmonella fljB variant inside J774 A.1 macrophages. The fljB variant with the C1362G substitution increased survival inside J774 A.1 macrophages from 9 h postinfection and reached ∼250% relative to that of wild type at 21 h after infection (Fig. 7A). This is in contrast to the fact that the ability to internalize Fig. 4. The fljB C1362G substitution fails to repress fljB mRNA expression by host cells was similar both in the wild type and the fljB C1362G the mgtC leader RNA, even when mRNA levels of the mgtC leader region are + variant Salmonella when we measured the ability to invade J774 highly induced in low Mg2 .(A–D) Relative mRNA levels of the coding regions of the fljB (A), fljA (B), and fliC (C) genes and the leader region of the A.1 cells for the first hour after infection (Fig. S9). The increase mgtC gene (D) produced by either wild type (14028s) or the fljB derivative in intramacrophage survival of the fljB variant is probably due to Salmonella with the C1362G substitution (EN925) grown for 5 h in N-minimal the hyperproduction of the FljB-polymerized flagella in the fljB + media containing either 0.01 or 10 mM Mg2 . Shown are the mean and SD variant (Fig. S8) promoting Salmonella’s survival inside macro- from three independent experiments. *P < 0.05; **P < 0.01; ns, not signifi- phages, because the fljB fliC mutant strain that is unable cant; t test (relative to wild type). to polymerize any type of flagella was completely defective for

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1705437114 Choi et al. Downloaded by guest on October 1, 2021 inhibits ATP production by interacting with the F1Fo ATP synthase (21).

Trans-Acting Riboregulators Derived from the 5′ Leader Region. As a cis-acting element, the mgtC leader RNA controls transcription elongation into the associated coding region by a transcription attenuation-like mechanism (17–19). Therefore, if ATP levels are low or charged tRNAPro levels are not limiting, transcription of the mgtC leader region and translation of mgtM or mgtP in the leader region are tightly coupled, promoting the formation of the stem-loop structures that prevent transcription elongation into the downstream region. Because the mgtC leader harbors two ORFs and responds to ATP and charged tRNAPro signals in- Fig. 6. The fljB C1362G substitution increases fljB expression inside macro- dependently (19), the presence of tandem attenuator stem-loop phages. (A–D) Relative mRNA levels of the coding regions of the fljB (A), fljA structures and different combinations of two signals could gen- (B), and fliC (C) genes and the leader region of the mgtC gene (D) produced erate transcripts of different lengths during transcription elon- by wild-type Salmonella (14028s) and the fljB derivative with the C1362G gation, which were detected in vivo (22) and in vitro (23). substitution (EN925) inside J774 A.1 macrophages at the indicated times In the sense that Salmonella uses the truncated transcripts after infection. Shown are the mean and SD from three independent ex- derived from cis-mediated transcriptional elongation control of periments. *P < 0.05, **P < 0.01, t test (relative to wild type at T2). the mgtCBR operon as a trans-acting riboregulator repressing flagellin synthesis, the mgtC leader works by a mechanism similar to the trans-encoded SAM-responsive riboswitch controlling Salmonella survival (Fig. 7B). Consistent with the previous no- the prfA virulence regulator gene in Listeria monocytogenes tion, the strains with either only the fljB gene or fliC gene deleted (29). However, the mgtC leader RNA differs from the SAM- had no significant effect on intramacrophage survival (28) riboswitch in terms of mode of action because the mgtC leader (Fig. 7B). RNA binds to 3′ end of the coding region in the fljB gene pro- Similar to enhanced survival inside macrophages, the fljB moting the RNase E-mediated degradation (Fig. S3), whereas C1362G variant displayed a hypervirulent phenotype when we the SAM-riboswitch binds to the 5′-UTR of the prfA gene for decreasing PrfA levels (29). An increasing number of small

injected the Salmonella strains listed above into mice in- MICROBIOLOGY traperitoneally (Fig. 7C). Collectively, these data indicate that transcripts originated from 5′-UTR were identified through – the mgtC leader-mediated control of FljB flagellin production is genome-wide RNA-sequencing approaches (30 32), which are critical for Salmonella virulence. likely to be produced by posttranscriptional regulation such as transcription attenuation or riboswitch-mediated control. In ad- Discussion dition to the previous finding (29), the mgtC leader’s trans- regulatory role raises a possibility that previously identified We have presented an example where a leader RNA from a ′ Salmonella virulence gene acts as a trans-encoded riboregulator 5 -UTR transcripts may participate in diverse regulatory functions, and represses a pathogen’s motility by decreasing flagellin pro- not only being by-products or intermediates produced during cis-encoded posttranscriptional regulation. duction during infection. Specifically, we established that the Salmonella mgtC leader RNA directly base pairs with mRNAs of Leader RNA-Mediated Control of Salmonella Motility in Host Environment. the fljB gene encoding flagellin (Fig. 3 and Fig. S4) and promotes The mgtC leader RNA represses Salmonella’s motility by pro- RNase E-mediated degradation (Fig. S3). The nucleotide sub- moting degradation of fljB mRNAs during infection. What are stitution in the fljB gene that disrupts the base pairing increases the benefits for decreasing FljB flagellin-based motility by the flagellin production and motility (Fig. 5), thereby promoting mgtC leader RNA? Salmonella has a unique property that survival inside macrophages and virulence in mice (Fig. 7). This switches expression of two independent flagellin loci, fljB and − − reflects that FljB flagella-based motility must be restricted in the fliC,atafrequencyof10 3 to 10 5 per generation (6, 33). This phagosomal compartment. This makes physiological sense be- regulatory switch is governed by a DNA inversion of the region cause the mgtC gene including the leader region is highly between the hin elements, which are located upstream of the expressed inside macrophages (Fig. 6) and expression of flagellin fljBA operon. Because the invertible DNA region harbors the genes is severely down-regulated during Salmonella infection (8). promoter of the fljBA operon, the orientation of the DNA Taken together, all these data demonstrate another critical role fragment determines whether Salmonella expresses either the of the mgtC virulence gene in Salmonella’s pathogenesis by FljB flagellin and the FljA repressor or the FliC flagellin (3) repressing motility via its leader RNA during infection, in ad- (Fig. 1). This antigenic phase variation enables Salmonella to dition to the previous finding that the MgtC protein directly express only one flagellin protein at a time (3), thereby having the

Fig. 7. The fljB C1362G substitution promotes Salmonella’s survival inside macrophages and mouse virulence. (A) Survival inside J774 A.1 macrophages of wild-type Salmonella (14028s) and the fljB derivative with the C1362G substitution (EN925) at indicated time after infection. (B) Survival inside J774 A.1 macrophages of wild type (14028s), the fljB deletion mutant (DN409), the fliC deletion mutant (DN410), or the fljB fliC mutant Salmonella (EN994) at 21 h after infection. Fold replication represents number of bacteria at indicated time/number of bacteria at T1. Shown are the mean and SD from three independent experiments. *P < 0.05, **P < 0.01, t test (relative to wild type). (C) Survival of C3H/HeN mice inoculated intraperitoneally with ∼103 colony-forming units of the Salmonella strains listed in A and the fljB fliC mutant Salmonella (EN994). The data are representative of two independent experiments, which gave similar results.

Choi et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 advantage to escape from recognition by the host immune re- mgtC leader RNA’s role of decreasing FljB flagellin synthe- sponse or to colonize in the gut (34). We speculated that the mgtC sis might be energetically favorable by saving the biosyn- leader RNA-mediated fljB mRNA degradation might be benefi- thetic cost because otherwise Salmonella uses a large amount cial for Salmonella to remove preexisting mRNAs of the fljB gene of ATP for synthesizing flagella, which are generally composed when the DNA inversion switches flagellin expression one from of more than 10,000 copies of each flagellin protein in a single the other. In addition to the DNA rearrangement-based phase flagellum (36). variation, this RNA-based regulatory mechanism enables a faster transition between two types of flagella and allows Salmonella to Materials and Methods express two genetically distinct flagella in a bistable manner. All procedures were performed according to approved protocols by the In- Moreover, the leader RNA-mediated flagellin down-regulation stitutional Animal Care and Use Committee from Kangwon National Uni- Salmonella is clearly required for normal virulence because the versity (protocol KW-160517-1). Bacterial strains and plasmids used in this nucleotide substitution preventing the leader-mediated down- study are listed in Table S1.AllS. enterica serovar Typhimurium strains are Salmonella regulation renders hypervirulent (Fig. 7). derived from the wild-type strain 14028s (37) and were constructed by one- During infection, MgtC reduces ATP production by inhibit- step gene inactivation method (38) and/or phage P22-mediated transduc- ing F1Fo ATP synthase inside macrophages (21). This property, ’ tions as described (39). DNA oligonucleotides are listed in Table S2. Full in turn, contributes to promoting Salmonella s pathogenicity methods are presented in SI Materials and Methods. (21) because phagosome acidification creates a large proton concentration gradient across the bacterial inner membrane to ’ ACKNOWLEDGMENTS. This work was supported, in part, by the Basic Science power ATP synthesis and MgtC s property prevents Salmonella Research Program through the National Research Foundation of Korea (NRF) from generating too much ATP in the phagosomal compart- funded by the Ministry of Science, Information and Communications Technology, ment, which could be toxic for Salmonella (35).Insucha and Future Planning (NRF-2013R1A1A2074505 and NRF-2016R1A2B2012424) condition that MgtC limits ATP production inside host, the (to E.-J.L.).

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