RNAIII of the Staphylococcus Aureus Agr System Activates Global Regulator Mgra by Stabilizing Mrna

Correction

MICROBIOLOGY Correction for “RNAIII of the Staphylococcus aureus agr system (112:14036–14041; first published October 26, 2015; 10.1073/ activates global regulator MgrA by stabilizing mRNA,” by Ravi pnas.1509251112). Kr. Gupta, Thanh T. Luong, and Chia Y. Lee, which appeared The authors note that Fig. 2 appeared incorrectly. The cor- in issue 45, November 10, 2015, of Proc Natl Acad Sci USA rected figure and its legend appear below.

A B 6 6 * * * 5 ** ** * 5 * 4 4 3 3 2 2 1 1 Half-life (min) 0 Half-life (min) 0

agr agr agr

(11346) Newman P1 UTR P2 UTR P1 UTR P1 UTR P2 UTR P2 UTR (1844) (1845) +RNAIII +pML100 +RNAIII 5-nt mutant(12916) (A22) +pML100 (12501) (12502) P1 UTR P2 UTR 5-nt revertant

Fig. 2. Stability of mgrA mRNA. (A) Stability in various chromosomal mutants. (B) RNAIII complementation of deletion mutations in mgrA UTR. mRNA stability expressed as half-life in minutes. The numbers in parentheses represent strain number. *P < 0.05, **P < 0.01 (unpaired two-tailed Student t test between Newman and each mutant, n = 3).

www.pnas.org/cgi/doi/10.1073/pnas.1523895113

E7306 | PNAS | December 29, 2015 | vol. 112 | no. 52 www.pnas.org Downloaded by guest on September 26, 2021 RNAIII of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA

Ravi Kr. Gupta, Thanh T. Luong, and Chia Y. Lee1

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205

Edited by Richard P. Novick, New York University School of Medicine, New York, NY, and approved September 9, 2015 (received for review May 12, 2015)

RNAIII, the effector of the agr quorum-sensing system, plays a key (13) to postulate that RNAIII must interact with one or more role in virulence gene regulation in Staphylococcus aureus, but how pleiotropic regulators. Here, we report that RNAIII can also affect RNAIII transcriptionally regulates its downstream genes is not com- its downstream genes through another important S. aureus global pletely understood. Here, we show that RNAIII stabilizes mgrA mRNA, transcriptional regulator, MgrA, which has been shown to affect thereby increasing the production of MgrA, a global transcriptional more than 350 genes involved in virulence, antibiotic resistance, regulator that affects the expression of many genes. The mgrA gene autolysis, and biofilm formation (21–25). Our results suggest that is transcribed from two promoters, P1 and P2, to produce two mRNA part of the MgrA regulon is affected by RNAIII. ′ transcriptswithlong5 UTR. Two adjacent regions of the mgrA mRNA Results UTR transcribed from the upstream P2 promoter, but not the P1 promoter, form a stable complex with two regions of RNAIII near the mgrA Gene Is Transcribed by Two Promoters. We have previously 5′ and 3′ ends. We further demonstrate that the interaction has sev- identified the mgrA gene (22). To map the mgrA promoter, we eral biological effects. We propose that MgrA can serve as an inter- performed primer extension experiments. We found three tran- mediary regulator through which agr exerts its regulatory function. scriptional start sites: at nucleotides −123, −301, and −303 upstream from the ATG start codon (Fig. S1), which closely agree − − Staphylococcus aureus | RNAIII | MgrA | virulence regulation with the nucleotide 124 and 302 sites previously reported (21). Most likely, nucleotides −301 and −303 represent start sites from the same promoter with the former being the preferred start site. mall regulatory RNAs (sRNAs) play an important role in gene We then designated the promoters as P1 and P2, as shown in Fig. Sregulation in both eukaryotes and prokaryotes. In bacteria, 1. To further define the promoters, we constructed promoter fu- sRNAs typically act by base pairing with target mRNAs with lim- sions to the xylE reporter gene in plasmid pLL38 with successive ited or extended complementarity (antisense mechanism), but they deletions of DNA extending from 368 bp upstream of the mgrA can also modulate protein activity (1, 2). sRNAs commonly act on ATG start codon. Our results showed that deletion of P2 reduced their target mRNA near the translation initiation site by base reporter activity by about 33–43%, whereas deletion of both pro- — — pairing to block and in some cases promote translation. Some moters resulted in about 92% reduction (Fig. 1), indicating that sRNAs cause alterations in the secondary structure of the target both promoters were functional and that the predicted promoters mRNA, thereby enhancing or reducing mRNA degradation (3, 4). are most likely correct. It should be noted here that the P2 pro- Staphylococcus aureus is an important human pathogen that moter is embedded within the coding region of a divergently produces many virulence factors that are regulated by an equally transcribed upstream gene encoding a putative ABC transporter. impressive number of regulators (5, 6). A key regulatory system involved in virulence regulation in S. aureus is the agr quorum- P2 5′ UTR Affects the Stability of mgrA mRNA. The 123-nt and 301/ sensing system. A 514-nt RNA, RNAIII, is the effector through 303-nt UTRs transcribed from the P1 and P2 promoters, re- which most genes in the agr regulon are regulated (7, 8). RNAIII is spectively, are unusually long for bacterial mRNAs. For conve- one of the largest known sRNAs that regulates gene expression via nience, herein we refer to the region between the P2 and P1 antisense mechanism by base pairing with target mRNAs (5, 9). transcriptional start sites as the P2 UTR and the region between RNAIII is capable of forming a stable structure that is characterized by 14 stem-loop motifs and three long-distance interacting helices (9). Several examples of regulation by direct interaction between Significance RNAIII and its target mRNAs have been reported (10–14). A common denominator in these regulatory interactions is the tar- In Staphylococcus aureus,theagr quorum-sensing system is geting of translation initiation sequences, through base pairing of critical for regulation of many virulence genes, primarily through the CU-rich loop domains of RNAIII with regions containing the RNAIII, a small RNA. RNAIII functions by interacting with trans- Shine and Dalgarno (SD) sequence or AUG start codon of the lational elements of its downstream genes typically by inhibiting target mRNA, resulting in negative control of translation (15, 16). translation initiation. Only a few direct targets of RNAIII have A notable exception is the activation of Hla by RNAIII, in which been identified, one of which is global regulator Rot. However, the interaction unmasks the SD sequence to facilitate translation of Rot can only partially account for RNAIII regulation. We show hla mRNA (10). Although a recent report shows that RNAIII here that RNAIII can affect gene expression through another activates map expression, the mechanism of activation is un- global regulator MgrA by enhancing MgrA production via mRNA known (17). More recently, RNAIII has also been shown to neg- atively control sbi translation through direct interaction with the stabilization, a novel mechanism of RNAIII regulation. Our find- translation initiation sequences of the sbi mRNA. However, in this ings provide evidence linking two major master regulators, case, three distant RNAIII domains that do not include the CU- thereby advancing our understanding of virulence regulation in rich loop domains have been shown to directly interact with three this important human and animal pathogen. distinct sites in the sbi mRNA to repress sbi translation (18). Rot is a pleiotropic regulator that affects many toxins and sur- Author contributions: R.K.G. and C.Y.L. designed research; R.K.G. and T.T.L. performed face proteins (19). The finding that RNAIII represses rot mRNA research; R.K.G., T.T.L., and C.Y.L. analyzed data; and R.K.G. and C.Y.L. wrote the paper. translation has advanced the understanding of agr regulation The authors declare no conflict of interest. (12, 13). However, RNAIII and Rot transcriptomes only partially This article is a PNAS Direct Submission. overlap (19, 20). Thus, RNAIII repression of Rot cannot com- 1To whom correspondence should be addressed. Email: [email protected]. pletely account for how RNAIII regulates the majority of its This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. downstream genes. This led Geisinger et al. (12) and Boisset et al. 1073/pnas.1509251112/-/DCSupplemental.

14036–14041 | PNAS | November 10, 2015 | vol. 112 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1509251112 -368 P2 P1 100bp +82 Promoter mRNA level of the genes involved (27). Thus, it is possible that Plasmid activity (%) -35 -10 -35 -10 mgrA pTL3234 100±16.3 RNAIII could affect NWMN_0656 expression, thereby affecting Δ59bp pTL3219 57.8±2.6 mgrA mRNA levels. However, we showed that RNAIII had no effect Δ103bp pTL3235 65.8±5.0 Δ152bp pTL3236 67.7±4.9 on NWMN_0656 transcription (Fig. S4), indicating that RNAIII Δ216bp pTL3221 8.3±2.6 does not regulate mgrA mRNA levels through NWMN_0656. Δ95bp pTL3245 66.8±7.8 -492 pTL3289 100±22.3 Δ138bp pTL3271 53.4±10.2 mgrA UTR Interacts with RNAIII at Two Separate Domains. To determine whether the complementary regions identified above are involved in Fig. 1. Analysis of mgrA promoter region. DNA fragments with various de- the interaction between RNAIII and mgrA P2 UTR, we used RNA- letions, as indicated, were fused to the xylE reporter in pLL38. All deletions RNA EMSA. RNA corresponding to the mgrA P2 UTR was syn- were derived from pTL3234 with a 450-bp insert containing both promoters and 82 bp of the mgrA coding sequence, except pTL3271, which was derived thesized in vitro and labeled with biotin. Various regions of the from pTL3289 with an additional 124 bp of upstream DNA (dashed line; not to RNAIII were also synthesized in vitro. Incubation of the labeled scale). XylE activities were assayed in Newman. The promoter activities are mgrA P2 UTR probe with full-length RNAIII resulted in a shifted expressed as percentages of the activity of the parent plasmid. band (Fig. 3A), which could be effectively competed away with 10- to 30-fold excess of cold fragment (lanes 2 and 3 from the right in Fig. 3A). An irrelevant RNA fragment derived from the 5′-end of the P1 transcriptional start site and the AUG start codon as the P1 clpC (negative control) did not result in a band shift. By using partial UTR. To determine whether sequences within the UTR affect the RNAIII fragments, we showed that deletions of the segments expression of the mgrA gene, we performed deletion analysis. As containing either one of the complementary regions in RNAIII still shown in Fig. 1, a deletion of 95 bp within the P1 UTR reduced the resulted in a shifted band, but deletions of both regions abolished reporter activity by about 33%, whereas a deletion of 138 bp within the shift (Fig. 3A and Fig. S5). These results suggest that both the the P2 UTR reduced by about 47%, suggesting that the sequences 28-nt and 14-nt regions of RNAIII contribute to the interaction of within the UTRs are important for mgrA transcription. RNAIII with mgrA P2 UTR and that the interaction is specific. One mechanism whereby the 5′ UTR could affect gene tran- To further confirm the interaction between RNAIII and mgrA scription is by influencing mRNA stability. To determine whether mRNA using EMSA, we labeled RNAIII. As shown in Fig. 3B, this mechanism is involved, we first constructed a 35-bp deletion in fragments containing both the 33-nt and the 14-nt regions of the P1 UTR (ΔP1 UTR) and a 138-bp deletion in the P2 UTR (ΔP2 mgrA UTR resulted in a shifted band. However, deletions of the UTR) in the chromosome. The mutants were tested for mgrA 14-nt region, but not the 33-nt region, abolished the shift. These

mRNA stability by real-time RT-PCR (qRT-PCR) upon rifampicin- results did not totally conform to those of Fig. 3A and Fig. S5,in MICROBIOLOGY mediated transcription arrest. We found that the ΔP2 UTR muta- which both the 5′ and the 3′ domains of RNAIII contributed to tion significantly reduced the mgrA mRNA stability, whereas the ΔP1 the binding. However, close examination of the shifted bands UTR mutation had no significant effect on mRNA stability (Fig. 2A). revealed a much-weaker band intensity when the 14-nt region was deleted compared with that of the deletion of the 28-nt re- RNAIII Stabilizes mgrA mRNA by Interacting with mgrA P2 UTR. The gion (Fig. 3A and Fig. S5). Thus, based on these results, we above results suggest that sequences in the P2 UTR are involved speculate that the perfectly complementary 14-nt domain may in stabilization of mgrA mRNA. To investigate the mechanism, play a more important role than the 28-nt/33-nt domain in the we searched for known S. aureus sRNAs using IntaRNA (26) and interaction between RNAIII and mgrA mRNA. identified RNAIII as a potential sRNA that could interact with the UTR of mgrA mRNA by base paring at two regions (Fig. S2). RNA SHAPE-Directed Chemical Probing of Interaction of mgrA 5′ UTR Both the 33-nt and 14-nt regions in the mgrA transcript are lo- with RNAIII. We then used the RNA SHAPE (selective 2′-hydroxyl cated within the P2 UTR, whereas the two corresponding com- acylation analyzed by primer extension) method (28) to analyze plementary regions in RNAIII are located at the ends of the the mgrA UTR structure, with or without the presence of RNAIII. molecule (the 5′ end 28 nt and the 3′ end 14 nt). To test whether N-methylisatoic anhydride (NMIA) was used to form a 2′-O-adduct, RNAIII is involved in stabilizing mgrA mRNA by interacting with flexible nucleotides representing unpaired sequences, which with P2 UTR, we first compared the effect of agr deletion on were then detected by primer extension. The in vitro synthesized mgrA mRNA stability in the wild-type, ΔP1 UTR, and ΔP2 UTR 303-nt mgrA UTR with adaptor sequences at both 5′ and 3′ ends strains. The results showed that the deletion of agr (CYL11346) was used as the template for the reaction and primer extension. The decreased the half-life of mgrA mRNA (Fig. 2A). Furthermore, the raw data were analyzed by QuShape software (28–30). This ex- deletion of agr in the ΔP1 UTR strain reduced stability to about the perimental design resulted in highly reproducible SHAPE reactivity samelevelasthatoftheagr deletion alone (compare 11346 to data of the mgrA UTR (Fig. 4A). The reactivity data were then used 12501) but had no effect on mRNA stability in the ΔP2 UTR strain to predict the structure of mgrA UTR (Fig. 4B) by using RNAs- (compare 1845 to 12502). These results suggest that agr has an effect tructure and XRNA software programs (31, 32). The proposed on mgrA mRNA stability by targeting the P2 UTR. To further structure indicates some of the nucleotides in the 14-nt (nucleotides confirm that RNAIII of the agr system is involved in the regulation, we expressed RNAIII under the control of an anhydrotetracycline (aTc)-inducible promoter. As shown in Fig. 2B, the induction of RNAIII increased (though not statistically significant) the mgrA A B Δ 6 6 mRNA stability in the P1 UTR mutant in which the P2 UTR is * * * 5 ** ** * 5 * intact, whereas the stability in the ΔP2 UTR strain remained the 4 4 same with or without RNAIII induction. Because the stability test 3 3 2 2 above using qRT-PCR was unable to distinguish between P1- and 1 1

Half-life (min) 0 0 P2-derived mgrA mRNA species, we further carried out Northern Half-life (min) agr agr agr Δ analyses and confirmed that agr affected only the P2-derived mRNA Δ Δ (11346) species (Fig. S3A). In addition, to test the effect in a different genetic Newman P1 UTR(1844) P2 UTR P1 UTR P1 UTR P2 UTR P2 UTR Δ (1845) Δ Δ Δ Δ +RNAIII +pML100Δ +RNAIII (A22)5-nt mutant(12916) +pML100 (12501) (12502) background, we showed that agr deletion has similar effect in strain P1 UTR P2 UTR 5-nt revertant USA300 LAC (JE2), a strain with naturally high RNAIII level (Fig. Δ Δ S3B). These results further suggest that RNAIII is involved in sta- Fig. 2. Stability of mgrA mRNA. (A) Stability in various chromosomal mutants. bilizing mgrA mRNA by targeting P2 UTR. (B) RNAIII complementation of deletion mutations in mgrA UTR. mRNA sta- As shown in Fig. S1,themgrA P2 promoter is located within the bility expressed as half-life in minutes. The numbers in parentheses represent divergent NWMN_0656 coding sequence. Overlapping transcripts strain number. *P < 0.05, **P < 0.01 (unpaired two-tailed Student t test be- have been shown to be degraded by RNase III, thereby affecting tween Newman and each mutant, n = 3).

Gupta et al. PNAS | November 10, 2015 | vol. 112 | no. 45 | 14037 A this region is consistent with the notion that these sequences are displaced upon RNAIII interaction. Similarly, we analyzed the RNAIII structure by RNA SHAPE in the presence or absence of mgrA UTR (Fig. S6A). In the presence of mgrA UTR, we found the 14-nt (nucleotides 447–460) and the 28-nt (nucleotides 8–35) domains of RNAIII, which were predicted to interact with mgrA, became less accessible to NMIA. Few or no changes were found in the rest of the RNAIII sequences. We also B modeled the RNAIII structure based on the reactivity data (Fig. S6B), which matches well with the structure of RNAIII predicted using chemical and enzymatic probing (9). The high similarity between our predicted RNAIII structure and the previously estab- lished structure indirectly validates our RNA SHAPE results. These results from SHAPE analyses are in line with our model that mgrA UTR interacts with RNAIII at the 14-nt and 33-nt/28-nt domains, which further corroborate the EMSA study described above.

RNA Walk Reveals in Vivo Interaction of RNAIII and mgrA UTR. To investigate the interaction of RNAIII and mgrA mRNA in vivo, we Fig. 3. RNA EMSA. (A)LabeledmgrA P2 UTR reacted with various deletions modified the RNA Walk method that had been used to study of 514-nt RNAIII. clpC RNA was used as a negative control. (B)LabeledRNAIII RNA–RNA interactions in trypanosomes (33). The method uses reacted with various deletions of mgrA UTR RNA fragments A through F. Red AMT (4′-aminomethyl-trioxsalen hydrochloride) to induce cross- and green boxes represent matched sequences in each RNA species. Triangles linking under UV irradiation, which links two RNA molecules in indicate transcriptional start sites. close proximity within the cell. The cross-linked molecules were then mapped by RT-PCR, in which the primers spanning the region of an interacting domain cannot be copied efficiently 96–109) and the 33-nt (nucleotides 57–89) regions are involved because of cross-linking. S. aureus Newman, CYL1844 (ΔP1 in the formation of secondary structures. However, the majority of UTR), CYL1845 (ΔP2 UTR), and CYL11346 (Δagr mutant) were the nucleotides in these regions are moderately reactive (Fig. 4A), treated with AMT and UV-irradiated. Total RNA from the strains, suggesting that the hybrids are not stable. These regions also be- with or without treatment, were isolated, converted to cDNA, and came less accessible to NMIA upon RNAIII addition (Fig. 4A, then PCR-amplified using primers spanning the P2 UTR con- Lower,andFig.4C), suggesting that RNAIII displaces the hybrids taining the 14-nt region (the 5′ primer contains a portion of the 14-nt to form more stable duplexes at these regions. Besides the changes region sequence for specific amplification of the interacting region) in reactivity at the RNAIII-interacting regions, we found little re- (Table S1). Amplification of the hu gene was included as a control. activity change in the rest of the mgrA UTR molecule except two AsshowninFig.5A, a 170-bp PCR fragment could be detected in short regions: one near the 5′-end (nucleotides 23–27) and the strainswithanintactP2UTRandanintactagr locus when the other near the 3′-end (nucleotides 263–278). The former is near the cultures were not treated. However, no PCR product was detected in regions that interact with RNAIII suggesting that the interaction treated cultures (Fig. 5A, lanes 1–4). On the other hand, the 170-bp with RNAIII induces some changes in secondary structure at PCR fragment was detected in the agr-deleted strain but not in the nearby sequences. The latter includes the region that is predicted ΔP2 UTR strain, with or without AMT and UV (Fig. 5A, lanes 5–8). to form a long-distance hybrid with the 33-nt domain within the In addition, treatment did not affect amplification of the hu gene in mgrA UTR (Fig. 4B) and, therefore, the increase of reactivity at any strain. These results demonstrated that the presence of both agr

A 3 33nt 14nt 2 UTR B 170 mgrA 180 1 0 1 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 221 231 241 251 261 271 281 291 301 GAU AUAU 11 211 150 160 140 190 CUA UAUA 3 UTR + RNAIII 2 mgrA 130 1 100 GAUAAC 0 1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 211 221 231 241 251 261 271 281 291 301

UUAUUG 260 20 14 nt GC U A C A U A U U U 210 33nt U A 90 U A A C 14nt U A 110 U A A GAAUCA U A C AA C G 220 U A U C GAAAGUUG 10 G U 30 A AUUAGU G C G C U A UUUUCAAC UG C 50 U U A 250 C G U A U 80 230 A U U 280 A 240 A UA A U A 33 nt AG AAU U U G U U G G U ACGGAU UAU A A 290 G U 40 60 GUCU G 70 A 300

Fig. 4. SHAPE analysis of mgrA UTR. (A) Average reactivity of each nucleotide of mgrA UTR in the absence (Upper) or presence (Lower) of RNAIII. Red, highly reactive (>0.80 unit); orange, moderately reactive (0.40–0.80 unit); black, unreactive (<0.40 unit). n = 2. (B) Predicted structure of mgrA UTR RNA. The sequences of the 14-nt domain are in red and the 33-nt domain in green. Solid and open arrowheads indicate increase and decrease, respectively, in nucleotide reactivity in the presence of RNAIII. (C) Comparison of reactivity of each nucleotide in the RNAIII-binding domains in mgrA P2 UTR. Red trace, with RNAIII; Blue trace, without RNAIII.

14038 | www.pnas.org/cgi/doi/10.1073/pnas.1509251112 Gupta et al. RNAIII and the P2 UTR was required to form a cross-link adduct, function, we tested a phenotype of CYL12916 containing the 5-nt suggesting their specific interaction in vivo. mutation in the mgrA UTR. We found that hla, which encodes α-toxin, was affected by the 5-nt mutation to the same degree as Mutation in the 14-nt Site Confirms Interaction of RNAIII and mgrA the agr deletion (Fig. 6A), suggesting that agr activates hla primarily UTR. To further demonstrate that the RNAIII interacts with mgrA through mgrA in Newman. We also found that abcA,whichen- mRNA, we constructed a 5-bp substitution mutation within the codes an ABC transporter involvedinantibioticresistance(34), 14-bp domain, in the Newman chromosome. These 5 bp were and capD, which is one of the capsule genes in the cap operon, chosen because of their high GC content and relative central lo- were significantly affected by the 5-nt mutation but not as drasti- cation in the 14-nt domain (Fig. S2C). The mutation did not affect cally as the agr deletion (Fig. 6A), suggesting that agr affects these bacterial growth (Fig. S7). We then assessed whether the mutation genes, in part, through interaction with the mgrA mRNA. Ex- affected mgrA mRNA stability. As shown in Fig. 2, the 5-bp mu- pression of all these genes was restored to the wild-type level in the tation (CYL12916) resulted in a significant reduction in mRNA 5-nt revertant strain. In addition, we also tested the effect of the stability, similar to that of the agr deletion. These results suggest 5-nt mutation on hemolysin activity and capsule production. As that the 14-nt domain in the P2 UTR is critical for mgrA mRNA showninFig.6A,mutationinagr reduced the hemolysin activity to stability. Because the 5-bp mutation within the 14-bp domain can- an undetectable level, whereas the 5-nt mutation in the mgrA P2 not be complemented with a plasmid, we sought to revert the UTR had a moderate reduction, which is consistent with a previous mutation to the wild-type sequence to ensure that the mutant study that agr could affect hla at the translational level (10). On the phenotype was not a result of inadvertent secondary mutations other hand, the effect of the mutation on capsule production was during the construction of the mutant. The mgrA mRNA stability comparable to the transcriptional effect measured by qRT-PCR. of the resultant revertant (CYLA22) was similar to that of the MgrA has been shown to inhibit cell lysis (24). As shown in Fig. Newman (Fig. 2A), indicating that the mutant phenotype was a 6B, we found increased Triton X-100–induced cell autolysis in result of the 5-bp mutation. CYL12916 compared with Newman. Deletion of agr (CYL11346) As the 14-nt domain affects stability of mRNA transcribed from increased the autolysis to the same extent as the 5-bp mutation, the P2 promoter, but not from the P1 promoter, we expected that suggesting that agr could affect autolysis through the 14-nt element. MgrA production would be partially affected by the mutation in The revertant of the 5-bp mutation had a rate of lysis similar to the 14-nt domain or the agr regulatory system under our ex- Newman. To test this further, we complemented an agr deletion perimental conditions. By Western analyses, we found that the mutant (CYL13056) with pLL48 expressing either intact RNAIII amounts of MgrA were similar between the 5-bp mutation (pLL48-RNAIII) or mutated RNAIII (pLL48-RNAIIIm9, which mutant and the Δagr mutant, which were both about 60% of contains a substitution of 9-nt in the 14-nt domain in RNAIII). Our MICROBIOLOGY the wild-type (Fig. 5B), suggesting that agr affects mgrA expression results (Fig. 6C) showed that pLL48-RNAIIIm9 could not com- through the 14-nt domain in the P2 UTR. As expected, the revertant plement the agr deletion, whereas pLL48-RNAIII complemented produced a similar amount of MgrA as Newman and the ΔmgrA the deletion although not to the level of Newman. These results mutant produced, essentially, an undetectable amount of protein. suggest that RNAIII affects autolysis by stabilizing mgrA mRNA To directly confirm the interaction, we constructed a 5-nt mu- through the 14-nt domain. tation in RNAIII such that the 14-ntregionoftheRNAIIIis The above results demonstrated that the stabilization of mgrA complementary to the 5-bp mutation in the 14-nt region of mgrA mRNA by RNAIII could lead to significant biological effects. mRNA in strain CYL12916 described above. The 5-bp mutant However, we found that the minimum inhibitory concentration RNAIII fragment was cloned in pML100 under an aTc-inducible (MIC) of oxacillin or ciprofloxacin was not affected by the 5-nt promoter (pRGE23). As shown in Fig. 5B, the mutation in RNAIII mutation, although others have reported that mgrA mutants have restored the MgrA protein production in CYLA15 (CYL12916 reduced MIC to ciprofloxacin (23) and that overexpression of Δagr) to a similar level produced in Newman. These results strongly mgrA increased the MIC to oxacillin in strain N315 (35). Thus, suggestthatRNAIIIdirectlyinteractswithmgrA mRNA at the not all phenotypic changes associated with MgrA regulation are 14-nt domain to stabilize the mgrA mRNA stability. affected by RNAIII. Discussion Phenotypic Effects of a 14-nt Domain Mutation in mgrA P2 UTR. MgrA has been shown to affect capsule, surface proteins, toxins, autolysis, Microarray studies showed that Agr and MgrA have similar effects and antibiotic resistance (21–24, 34). To determine whether on many virulence genes, suggesting that the two regulators may RNAIII–mgrA mRNA interaction has an effect on biological coregulateasubsetoftargetgenes(25).HerewefoundthatRNAIII directly interacts with the mgrA mRNA. Our results suggest that RNAIII stabilizes the mgrA mRNA primarily through base pairing between the two RNA molecules at two domains. We provide several lines of evidence supporting this conclusion. RNAIII, as a reg- AB ulatory effector of the agr system, has so far been shown to affect its target genes at the translational level by RNA base pairing that either masks or unmasks the translation initiation site (10, 11, 13, 14). To our knowledge, the findings presented here are the first to show that S. aureus RNAIII affects a target gene by mRNA stabilization. The agr system regulates its targets mostly through an RNAIII- dependent pathway, but direct transcriptional activation of the psm genes by AgrA, the response regulator of the agr system, has been reported (8). Among the target genes, rot is the only regulatory gene Fig. 5. Interaction of RNAIII and mgrA UTR in vivo. (A) RNA Walk. Cross-linked found to be directly targeted by RNAIII. As such, Rot serves as an RNAs were amplified by RT-PCR with primers specific for the 14-nt region in mgrA intermediary regulator of RNAIII (12, 13). In this study, our findings UTR. Cross-linked adducts prevents amplification. hu primers were used as positive controls. (B) MgrA Western blots were quantified by densitometry, normalized to strongly suggest that MgrA is another intermediary regulator of a Coomassie blue-stained protein band (arrow), and expressed relative to New- RNAIII. However, we found that RNAIII only affected the tran- man (Nm). n = 3. CYL12916 carries the 5-nt mutation in mgrA UTR and A22 carries script initiated from the P2 promoter, indicating that it has only the corresponding reversion. MgrA complementation of the 5-nt mutation in partial control on mgrA expression. Interestingly, in addition to mgrA UTR (Right) was performed in A15 (CYL12916 Δagr) using pML100 carrying the P2 UTR, expression of mgrA is also affected by the sequences either the wild-type RNAIII or RNAIII with a 5-nt mutation (RNAIIIm5) that is within the P1 UTR, suggesting a different regulatory factor (or complementary to the five mutated nucleotides in CYL12916. *P < 0.05, **P < factors) is involved (Fig. 1). A recent report has shown that RsaA, 0.01 (unpaired two-tailed Student’s t test between Newman and each mutant). a SigB-dependent sRNA, represses translation of MgrA by

Gupta et al. PNAS | November 10, 2015 | vol. 112 | no. 45 | 14039 A 1.2 hla abcA capD capsule 1.0 α-toxin .8 .6 .4

.2 )

Relative 0 expression

(11346)

Newman (11346) agr agr agr 5nt rt 5nt rt Newman 1346) 5nt rt 5nt rt(A22) Δ (A22) 1346) (A22) agr Δ 1346) (A22) Δ 5nt mt 5nt mt 5nt mt 5nt rt(A22) Δ agr (12916) (1 (1 (12916) (12916) (1 Newman Newman Δ Newman 5nt mt(12916)

5nt mt(12916) B100 C Fig. 6. Biological effect of the RNAIII-interacting 14-nt without region. (A) Effect of mgrA P2 UTR 5-nt mutation without Δ 80 Triton (CYL12916) and agr (CYL11346) on hla, abcA,orcapD X-100 Triton (gray bars) assayed by qRT-PCR and expressed relative X-100 ≥ α 60 to the wild-type (n 2). Effects on -toxin on 5% rabbit blood agar and capsule production are shown to the with right. (B) Autolysis assays (n = 3) of Newman, 5-nt 40 Newman with Triton mutant, and Δagr in the presence or absence of 5nt mt(12916) Newman Triton X-100 Δ (pLL48) Triton X-100 (0.05%). Note that some curves overlap. Δagr (11346) agr X-100

20% Initial OD 580nm Δagr(pLL48-RNA3) (C) Autolysis assays (n = 3) of Newman and the Δagr

5nt rt(A22) % Initial OD 580nm Δagr(pLL48-RNA3m9) strain CYL13056 with pLL48, pLL48-RNAIIIm9, or pLL48- 0 0' 30' 60' 90' 120' 150' 180' RNAIII in the presence or absence of Triton X-100 0' 30' 60' 120' 150' 180' 240' 300' (0.05%). *P < 0.05, **P < 0.01 (unpaired two-tailed Time (minutes) Time (minutes) Student’s t test between Newman and each mutant). blocking the SD sequence and the initiation codon of mgrA mRNA however, requires further investigations. Our studies further un- (36). This regulatory mechanism is unlikely to explain what we derscore the complexity of virulence gene regulation in S. aureus. observed in the P1 UTR deletion because the deletion is further Our SHAPE analyses demonstrate that mgrA UTR directly in- upstream of the SD sequence and its effect is opposite to that of teracts with RNAIII at the two short regions. Importantly, our RsaA. Likely, there exists another regulatory element that could in- RNAIII structure prediction is in close agreement with the pub- teract with P1 UTR. Thus, taken together, we envision that MgrA lished RNAIII structure by Benito et al. (9), with only minor dif- production could be influenced by multiple signals in which tran- ferences, which validates our SHAPE methodology. Based on the scripts initiated from the P2 promoter could be controlled by quorum predicted structure, the 14-nt sequence and the adjacent 33-nt se- sensing through RNAIII, whereas transcripts from both promoters quence of the mgrA UTR are not totally unpaired in the absence of could be controlled by different stimuli by interacting with RsaA RNAIII. However, many of the nucleotides within these regions are through SigB, as well as an unknown regulatory element. The various moderately reactive, and some are highly reactive, suggesting they modesofregulationatthemRNAlevelandthefactthattheregu- can readily form hybrids with RNAIII to form stable complexes latory function of MgrA can be modulated by oxidation or phos- when RNAIII is present. By using the RNA-Fold RNAhybrid phorylation at a conserved cysteine residue (37–39) indicate that (bibiserv.techfak.uni-bielefeld.de/rnahybrid) program, we calculated MgrA regulatory function is likely modulated by multiple signals. that the predicted hybridization minimum free energy of the com- Because MgrA has been shown to affect more than 350 genes and plex between the 14-nt domains in the mgrA P2 UTR and RNAIII plays an important role in virulence regulation (25, 37, 40, 41), it is is −18.8 kcal/mol, whereas that of the 33-nt/28-nt is −25 kcal/mol. In not surprising that the activity of MgrA is affected by multiple ele- comparison, the predicted minimum free energy of RNAIII com- ments. This would allow MgrA to achieve optimal regulation of its plexes with its known RNA targets ranges from −15 to −34.5 kcal/mol regulon in response to various environmental conditions. (13). Thus, the estimation of free energy suggests that these com- Although RNAIII regulation of MgrA had no effect on resistance plexes are quite thermodynamically stable. to oxacillin or ciprofloxacin, we found that the regulation could lead RNAIII is a large sRNA with 14 stem-loops in which the C-rich to other biological effects. The effect of the 5-nt mutation in the sequences in H7, H13, and H14 hairpin loops have been shown to mgrA 14-nt domain on capD and abcA expression and capsule be the primary regulatory sites that interact with target mRNAs of production is less than that of the agr mutation, suggesting that agr several genes (15). However, a large region at the 5′ end of RNAIII could regulate these genes through alternative pathways. On the including the H2 hairpin loop has been shown to interact with hla other hand, the effect of the 5-nt mgrA mutation on hla expression mRNA (10). More recently, it was demonstrated that three distant andTritonX-100–induced autolysis was comparable to that of the RNAIII domains—none of which involve the loop structures H7, agr deletion alone, suggesting that agr may regulate hla and autolysis H13, or H14—were involved in the regulation of sbi by agr (18). In primarily through stabilization of mgrA mRNA. However, it has this study, we showed that the sequences of the 14-nt at the 3′ end been shown that RNAIII can activate hla transcription through Rot and the 28-nt at the 5′ end of RNAIII were involved in the in- regulation (12, 13), which was not revealed in our results. This teraction with the mgrA mRNA P2 UTR. These sequences are not discrepancy could be because of differences in experimental con- within the loop domains of RNAIII and have not been previously ditions or strains used. Additionally, RNAIII could bind to hla shown to be involved in regulation, although they are directly adja- mRNA and activates hla translation (10), which agrees with our cent to two of the three domains involved in sbi regulation (18). The finding that agr deletion had more effect on α-toxin production than regions located near both ends of the RNAIII molecule have been the 5-nt mgrA mutation. Thus, it is apparent that not all genes within proposed to be involved in a long-distance interaction forming a helix the MgrA regulon are regulated by agr through mgrA.The5-nt (Helix C). However, our RNA SHAPE analysis (Fig. S6)indicated mutation resulted in about a 40% reduction in MgrA protein pro- that Helix C is unstable, in agreement with the finding of Benito et al. duction. Likely, those genes in the MgrA regulon that are responsive (9). Nonetheless, because the 14-nt and 33-nt domains are juxtaposed to RNAIII may require a greater quantity of MgrA for regulation in the mgrA UTR, it is tempting to speculate that the unstable Helix and that regulation of genes that are not responsive requires less C of RNAIII may serve to bring the two distant domains of RNAIII MgrA. These results suggest that the regulation by RNAIII through close together to promote efficient interaction with mgrA UTR. MgrA could fine-tune MgrA regulation in response to quorum- Based on the results presented herein, we propose a model sensing signals. The biological significance of this fine regulation, depicted in Fig. S8. We hypothesize that the 3′ RNAIII domain of

14040 | www.pnas.org/cgi/doi/10.1073/pnas.1509251112 Gupta et al. 14-nt first interacts with the 14-nt sequence of mgrA UTR by base RNA EMSA. RNA EMSA reactions were performed using RNAs synthesized by pairing. After the initial interaction, the 28-nt 5′ RNAIII domain in vitro transcription. For details, see SI Materials and Methods. then pairs with the mgrA UTR 33-nt sequence. In Newman, mgrA mRNA is expressed at the highest level in postexponential phase, RNA Walk. RNA Walk was performed essentially as described previously at which time RNAIII is most abundant (7, 42), allowing for (33). Cross-linking of paired RNAs in vivo was done by adding AMT to pairing to occur readily. The complex then stabilizes mgrA mRNA, cultures and UV exposure. RT-PCR was performed to detect cross-linked likely by blocking degradation by one or more RNases, leading to RNA regions. The detailed procedures are provided in the SI Materials more MgrA protein production, thereby affecting expression of a and Methods. subset of genes in the MgrA regulon. RNA SHAPE Reactions and Analysis. RNA SHAPE experiments were performed Materials and Methods according to the previously described method using NMIA (Sigma) (43, 44). Strain and Plasmid Construction. Strains and plasmids used in this study are The detailed procedures are provided in the SI Materials and Methods. listed in Table S2. Plasmid and strain construction were carried out using standard molecular methods. For details, see SI Materials and Methods. ACKNOWLEDGMENTS. We thank David Cue and Mei Lei for critical review Primers used in this study are listed in Table S1. and helpful discussions; Mei Lei for construction of pML12570 and tech- nical help; and Wes Sanders and Allain Laederach (University of North mRNA Stability Assay. Carolina) for help with SHAPE analyses. This work was supported by RNAs from transcription-arrested cultures were isolated National Institute of Allergy and Infectious Diseases Grants AI37027 and at different time points. mRNA stability was assayed by qRT-PCR or Northern AI113766. The University of Arkansas for Medical Sciences core facilities are analysis. Half-lives were calculated by using linear regression analysis. For supported in part by National Institutes of Health Grants P20GM103450 and detailed procedures, see SI Materials and Methods. P30GM103429.

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Gupta et al. PNAS | November 10, 2015 | vol. 112 | no. 45 | 14041