Staphylococcal sRNA IsrR downregulates methylthiotransferase MiaB under iron-deficient conditions

Abstract

Staphylococcus aureus is a major contributor to bacterial-associated mortality, owing to its exceptional adaptability across diverse environments. Iron is vital to most organisms but can be toxic in excess. To manage its intracellular iron, S. aureus, like many pathogens, employs intricate systems. We have recently identified IsrR as a key regulatory RNA induced during iron starvation. Its role is to reduce the synthesis of non-essential iron-containing proteins under iron-depleted conditions. In this study, we unveil IsrR's regulatory action on MiaB, an enzyme responsible for methylthio group addition to specific sites on transfer RNAs (tRNAs). We use predictive tools and reporter fusion assays to demonstrate IsrR's binding to the Shine-Dalgarno sequence of miaB RNA, thereby impeding its translation. The effectiveness of IsrR hinges on the integrity of a specific C-rich region. As MiaB is non-essential and has iron-sulfur clusters, IsrR induction spares iron by downregulating miaB. This may improve S. aureus fitness and aid in navigating the host's nutritional immune defenses.IMPORTANCEIn many biotopes, including those found within an infected host, bacteria confront the challenge of iron deficiency. They employ various strategies to adapt to this scarcity of nutrients, one of which involves regulating iron-containing proteins through the action of small regulatory RNAs. Our study shows how IsrR, a small RNA from S. aureus, prevents the production of MiaB, a tRNA-modifying enzyme containing iron-sulfur clusters. With this illustration, we propose a new substrate for an iron-sparing small RNA, which, when downregulated, should reduce the need for iron and save it to essential functions.

Keywords: IsrR; Staphylococcus aureus; [Fe-S] clusters; iron homeostasis; small regulatory RNA; translational regulation.

Fig 1

Computer prediction of IsrR and mutated IsrR pairing with miaB mRNA. (A) IntaRNA (17) pairing prediction of IsrR and IsrR derivatives deleted for CRR1, CRR2, or CRR3 with miaB mRNA. Blue sequences, SD; red sequences, CRRs; bold underlined characters, GUG start codon; E, energy; HE, hybridization energy. (B) IntaRNA pairing prediction of IsrR and miaB mRNA in different species from the Staphylococcus genus. IsrR sequences from the indicated species were obtained with GLASSgo (29). For legends, see Fig. 1A.

Fig 2

MiaB-Flag reporter detection. (A) Schematic representation of the MiaB-Flag reporter fusion. Blue, 3’ end of miaB ORF; orange, flag sequence; yellow, native stop codon; gray, first nts of miaB 3’ UTR. (B) Western blot experiment with anti-Flag antibodies in the presence or absence of iron chelators (N = 4). Genotypes and growth conditions are indicated. 75 kDa, non-specific signal present in all samples including Flag-less strain. 60 kDa, signals corresponding to MiaB-Flag. (C) Western blot experiment with anti-Flag antibodies with fur⁺ and fur::tet strains (N = 3) as explained in Fig. 2B. (D) Histograms. MiaB-Flag signals from Western blot (Fig. 2C) were normalized to the 75 kDa signals from their corresponding lane. Histogram y-axis indicates the values of normalized MiaB-Flag signals of indicated strains divided by the normalized MiaB-Flag signal from the wild-type background strain (fur⁺). Error bars indicate the standard deviation of three biological replicates. ***, P-value < 0,001 for student test (N = 3). (E) The stability of miaB mRNA is not significantly affected by IsrR. HG003 and its isogenic ΔisrR derivative were grown in a rich medium supplemented with DIP. At t0, rifampicin (200 µg/mL) was added to the growth medium. Cultures were sampled at t0, 2, 5, and 10 min after the addition of rifampicin, total RNA was extracted, and the amounts of miaB mRNA, rrsA rRNA (control), and IsrR were determined by RT-qPCR. Histograms Y-axis show the quantification of miaB mRNA, rrsA sRNA, and IsrR normalized to t0. Error bars indicate the standard deviation of biological triplicates (N = 3).

Fig 3

Translational repression of miaB reporter fusion by IsrR. (A) Chromosomal reporter fusion for detection of IsrR activity. 17c, 17 first codon of miaB; red nt, transcription start site; blue bold nts, RBS; black bold nts, start codon; underlined nts, nts in interaction with IsrR as predicted by IntaRNA. (B) Translational activity of the miaB-mAm reporter fusion (mAmetrine fluorescence signal) in HG003 and its ΔisrR derivative grown in BHI and BHI supplemented with DIP as shown. Results (N = 3) are normalized to OD₆₀₀ = 1. (C) Expression of isrR and its derived alleles expressed from the indicated plasmids in stationary phase normalized to wild-type isrR expression (first histogram). The amount was determined by RT-qPCR. Error bars indicate the standard deviation of four independent biological replicates, with the exception of pIsrRΔC3, which has three. (D) The ΔisrR strain harboring the miaB-mAm reporter fusion was transformed with plasmids expressing different versions of IsrR and cultured in BHI supplemented with DIP. The translational activity of the reporter fusion was quantified by measuring the fluorescence. The significance of the difference in fluorescence between IsrR and IsrRΔC3 groups is supported by ANOVA: results (N = 3) are normalized to OD₆₀₀ = 1. **, P-value < 0.01; ***, P-value < 0.001. (E) The ΔisrR strains harboring the miaB-mAm reporter fusion or its mutated derivative in its three G motif (GGG to TAT) were transformed with an empty plasmid, a plasmid expressing IsrR or a plasmid expressing isrR mutated in CRR3 (CCC change to ATA). Mutations in the three G motif and CCR3 were designed to be complementary as shown below the histograms. Strains were cultured in BHI supplemented with DIP. The translational activity of the reporter fusions, represented by histograms, was quantified by measuring the fluorescence (N = 5). The significance of the difference in fluorescence between the optimum pairing and altered pairing is supported by ANOVA and Welch tests.

Fig 4

Contribution of IsrR CRRs to downregulate different mRNA targets. Negative arrows starting with CRR1, 2, or 3 and pointing to mRNA targets indicate CRRs required for IsrR activity toward the corresponding targets. Boolean symbols indicate that regulation requires the integrity of two CRRs (AND gate, empty symbol) or that two CRRs can act independently of each other (OR gate, solid symbol). Data were derived from results obtained with fluorescent reporters in (3, 4) and this work.