Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism

Abstract

RNAIII is the intracellular effector of the quorum-sensing system in Staphylococcus aureus. It is one of the largest regulatory RNAs (514 nucleotides long) that are known to control the expression of a large number of virulence genes. Here, we show that the 3' domain of RNAIII coordinately represses at the post-transcriptional level, the expression of mRNAs that encode a class of virulence factors that act early in the infection process. We demonstrate that the 3' domain acts primarily as an antisense RNA and rapidly anneals to these mRNAs, forming long RNA duplexes. The interaction between RNAIII and the mRNAs results in repression of translation initiation and triggers endoribonuclease III hydrolysis. These processes are followed by rapid depletion of the mRNA pool. In addition, we show that RNAIII and its 3' domain mediate translational repression of rot mRNA through a limited number of base pairings involving two loop-loop interactions. Since Rot is a transcriptional regulatory protein, we proposed that RNAIII indirectly acts on many downstream genes, resulting in the activation of the synthesis of several exoproteins. These data emphasize the multitude of regulatory steps affected by RNAIII and its 3' domain in establishing a network of S. aureus virulence factors.

Figure 1.

Secondary structure of RNAIII and potential base pairings with target mRNAs. (A) The secondary structure of RNAIII is from Benito et al. (2000). Deletion of the central domain (RNAIII-Δ7–9), of hairpin 13 (RNAIII-Δ13) or of hairpin 14 (RNAIII-Δ14) is delimitated by arrows. The isolated hairpins H7, H13, and H14 used in this work are shown. The first 19 bp of H13 are also present in H14 and H7. (B) Potential base pairings between the hairpin 13 and mRNAs encoding for virulence factors and for the transcriptional regulatory protein Rot. The minimum free energy for each sequence is given. The levels of several mRNAs were shown to be growth-phase dependent and were strongly decreased as soon as RNAIII was synthesized: spa (Huntzinger et al. 2005), coa (Lebeau et al. 1994), lytM (Ramadurai et al. 1999), SA1000 and SA2353 (this study). The synthesis of Rot is regulated at the translational level by RNAIII (Geisinger et al. 2006; this study). The mRNAs encoding protein A and SA2093 were also not detected in a mutant strain deleted of the agr operon that does not express RNAIII (Dunman et al. 2001). The SD sequences and the AUG codons are indicated in red. The hybrids formed between RNAIII and spa mRNA or SA1000 mRNA were analyzed by chemical and enzymatic probing, Noncanonical base pairs are denoted by circles in these duplexes.

Figure 2.

RNAIII and its 3′ domain bind to the RBS of SA1000 mRNA. (A,B) Enzymatic hydrolysis (RNases T2 and V1) and lead-induced cleavages (Pb²⁺) of 5′-end-labeled SA1000 mRNA free (−) or with a twofold excess of RNAIII (+) (A) or 5′-end-labeled 3′ domain free (−) or with a twofold excess of SA1000 mRNA (+) (B). (Lane T1) RNase T1. (Lane L) Alkaline ladders. Bars denote the main reactivity changes induced by complex formation. (C) Probing data represented on the secondary structure of SA1000 mRNA—the 3′ domain of RNAIII and of the RNAIII–SA1000 mRNA duplex. Enzymatic cleavages are as follows: RNase T1 (), RNase T2 (→) lead cleavages (), and RNase V1 moderate (▹) and strong (▸) cleavages. Reactivity changes induced by complex formation are indicated as follows: Black or empty circles indicate strong and moderate protection, respectively; enhancements are shown by asterisks; and new RNase V1 cleavages are denoted by arrows followed by an asterisk. For RNAIII–SA1000 duplex, the same symbols for lead cleavage and RNase V1, and RNase III () are used. The SD sequence is squared on SA1000 mRNA.

Figure 3.

RNAIII and its 3′ domain regulate the expression of SA1000 mRNA at the post-transcriptional level. (A) β-Galactosidase activity measured from PrpoB (+1/+200)∷lacZ fusions in S. aureus RN6390 (rnaIII⁺, agr⁺), RN6390-Δrnc (deletion of rnc gene encoding RNase III), WA400 (ΔrnaIII), WA400 + RNAIII, WA400 + 3′ domain, WA400 + RNAIII-Δ13 (RNAIII deleted of hairpin 13), and WA400 + H14 (expression of the hairpin 14 of RNAIII). The β-galactosidase activity was normalized for total cell density and is represented as a percentage of the uninhibited control (WA400). The results represented a mean of three independent experiments. (B) Northern blot analysis of SA1000 mRNA levels in different strains. RNAs from post-exponential phase cultures were hybridized with probes corresponding to RNAIII, the 3′ domain, SA1000 mRNA, and 5S rRNA as internal control: RN6390 (lane 1), RN6390-Δrnc (lane 2), RN6390-Δhfq (deletion of the hfq gene) (lane 3), WA400 (lane 4), WA400 + RNAIII (lane 5), WA400 + 3′ domain (lane 6), WA400 + RNAIII-Δ13 (lane 7), WA400 + 3′ dom-ΔL14 (AACCCUCCC was changed by GAGA in the hairpin loop 14) (lane 8), and WA400 + H14 (lane 9). (C) Formation of the complex between SA1000 mRNA (15 nM), S. aureus 30S ribosomal subunits (100 nM), and initiator tRNA (1 μM) monitored in the absence (lane 4) or presence of increasing concentrations of RNAIII or RNAIII-Δ13 (15, 75, and 150 nM). The toeprint is indicated. Incubation controls of SA1000 mRNA alone (lane 1) and with RNAIII (lane 2) or RNAIII-Δ13 (lane 3). (Lanes U,G,C,A) Dideoxy-sequencing reactions. (D) RNase III hydrolysis of 5′-end-labeled SA1000 mRNA, free (−) or with RNAIII (+). (Lane T) RNase T1. (Lane L) Alkaline ladders. Arrows indicate the RNase III cleavages.

Figure 4.

RNAIII-dependant regulation of SA2353 mRNA. (A) Northern blot analysis of SA2353 mRNA levels in different strains. RNAs were prepared from early exponential phase (OD 0.5 at 600 nm) (lanes 1–3) and from stationary phase (OD 5.0 at 600 nm) (lanes 4–11) cultures hybridized with probes corresponding to SA2353 mRNA, RNAIII, and 5S rRNA: RN6390 (lanes 1,4), RN6390-Δrnc (lanes 2,5), RN6390-Δhfq (lanes 3,6), WA400 + H14 (lane 7), WA400 + RNAIII (lane 8), WA400 + 3′ domain (lane 9), WA400 + 3′ dom-ΔL14 (lane 10) (see Fig. 2B), and WA400 transformed with the empty plasmid pE194 (lane 11). (B) Binding of RNAIII to SA2353 mRNA prevents ribosome binding. Ternary (mRNA–tRNA_f^Met–ribosome) complex incubated in the absence (lane 3) or presence of increasing concentrations of RNAIII (100 nM, lane 4; 500 nM, lane 5), the 3′ domain (100 nM, lane 6; 400 nM, lane 7), or RNAIII-Δ13 (100 nM, lane 8; 600 nM, lane 9). (Lanes U,G) Dideoxy-sequencing reactions. (C) Potential interactions between SA2353 mRNA and the hairpin 13 of RNAIII.

Figure 5.

RNAIII binds to rot mRNA. (A) RNase T1 hydrolysis of 5′-end-labeled rot mRNA either free (lane 3) or in the presence of increasing concentrations of RNAIII and of the isolated hairpins H7, H13, or H14. (Lanes 4–8) Shown are 1, 10, 50, 100, and 250 nM, respectively. (Lanes 1,2) Incubation controls in the absence (lane 1) or presence (lane 2) of RNAIII. (Lane T) RNase T1. (Lane L) Alkaline ladders. (B) Probing data shown on the secondary structure of rot mRNA. Enzymatic cleavages are given as follows: RNase T1 (), RNase T2 (), and RNase V1 moderate (▹) and strong (▸) cleavage. Reactivity changes induced by complex formation are indicated as follows: Black or empty circles denote strong and moderate protection, respectively. (C) Models of the RNAIII–rot mRNA complex with the RNase III cleavages denoted by arrows.

Figure 6.

RNAIII regulates the expression of rot mRNA at the post-transcriptional level. (A) β-Galactosidase activity measured from PrpoB (+1/+290)∷rot fusions in S. aureus RN6390 (rnaIII⁺, agr⁺), RN6390-Δrnc, WA400 (ΔrnaIII), WA400 + RNAIII, and WA400 + 3′ domain, and WA400 + 3′ domain mutated in loop 14 (ΔL14). The legend is identical to Figure 3A. (B) Northern blot analysis of rot mRNA levels in different strains. RNAs from late-exponential phase cultures were hybridized with probes corresponding to rot mRNA and 5S rRNA: RN6390, WA400, and RN6390-Δrnc. Full and empty arrows denote full-length mRNA and a fragment, respectively. (C) Formation of the complex between rot mRNA, S. aureus 30S ribosomal subunits, and tRNA_f^Met monitored in the absence (lane 3) or presence of increasing concentrations of RNAIII, the 3′ domain (0.1 μM, lane 4; 0.25 μM, lane 5), and the hairpin 13 or 14 (0.5 μM, lane 4; 1 μM, lane 5). (Lanes U,G) Dideoxy-sequencing reactions. An arrow denotes the toeprint. (D) RNase III hydrolysis of 5′-end-labeled rot mRNA in the absence (lane 3) or presence of an excess of RNAIII variants. (Lanes 4–8) RNAIII, RNAIII-Δ13 (ΔH13), RNAIII-Δ14 (ΔH14), 3′ domain (3′ dom), and RNAIII-Δ7–9 (ΔH7–9) at 100 nM, respectively. (Lanes 9–11) Isolated hairpins 7 (H7), 13 (H13), and 14 (H14) at 200 nM, respectively. (Lanes 1,2) Incubation controls in the absence (lane 1) or presence (lane 2) of RNAIII. (Lane T) RNase T1. (Lane L) Alkaline ladders. Arrows show cleavages, which occur in rot–RNAIII complex.

Figure 7.

Schematic view of the antisense regulatory mechanisms. (A) RNAIII binds to its target mRNAs via one (top panel) or two (bottom panel) loop–loop interactions. In the first case, the initial base pairings are propagated, leading to the formation of an extended irregular duplex. In both cases, binding of RNAIII hinders ribosome binding and promotes the access to RNase III. For rot mRNA, total depletion of the mRNA pool is not observed. SD and AUG are in green. RNAIII is in red, and the mRNA target in black. (B) RNAIII represses directly the synthesis of adhesins and surface proteins and of Rot, which in turn represses the synthesis of exotoxins and activates the synthesis of adhesin proteins.