Adhesin competence repressor (AdcR) from Streptococcus pyogenes controls adaptive responses to zinc limitation and contributes to virulence

Abstract

Altering zinc bioavailability to bacterial pathogens is a key component of host innate immunity. Thus, the ability to sense and adapt to the alterations in zinc concentrations is critical for bacterial survival and pathogenesis. To understand the adaptive responses of group A Streptococcus (GAS) to zinc limitation and its regulation by AdcR, we characterized gene regulation by AdcR. AdcR regulates the expression of 70 genes involved in zinc acquisition and virulence. Zinc-bound AdcR interacts with operator sequences in the negatively regulated promoters and mediates differential regulation of target genes in response to zinc deficiency. Genes involved in zinc mobilization and conservation are derepressed during mild zinc deficiency, whereas the energy-dependent zinc importers are upregulated during severe zinc deficiency. Further, we demonstrated that transcription activation by AdcR occurs by direct binding to the promoter. However, the repression and activation by AdcR is mediated by its interactions with two distinct operator sequences. Finally, mutational analysis of the metal ligands of AdcR caused impaired DNA binding and attenuated virulence, indicating that zinc sensing by AdcR is critical for GAS pathogenesis. Together, we demonstrate that AdcR regulates GAS adaptive responses to zinc limitation and identify molecular components required for GAS survival during zinc deficiency.

Figure 1.

Zinc-dependent DNA binding by AdcR. Analysis of AdcR-adc motif interactions in the absence (A) and presence (B) of 100 μM zinc by gel mobility shift assay. Oligoduplexes containing the sequences corresponding to either the putative adc motif from the adcRCB promoter were incubated with increasing concentrations (250, 500 and 750 ng) of either purified apo-AdcR (A) or zinc-bound AdcR (B). (C) Assessment of sequence-specificity of AdcR-DNA interactions. Zinc-bound AdcR was incubated with non-cognate DNA containing the per box sequence. The reaction mixtures were resolved on a 10% native PAGE and visualized by ethidium bromide staining. The positions of free probe (F) and protein-bound probe (B) are labeled and indicated by arrows.

Figure 2.

Organization of regulatory elements in target promoters negatively regulated by AdcR. The positions of predicted -10 and -35 hexamers, and the TSS (bent arrow) in the AdcR-regulated promoters containing one (A) and two (B) AdcR-binding sites are shown. The locations of adc motifs are indicated in red. Interactions between the metallated form of AdcR and operator sequences containing one (C) and two (D) adc motifs as assessed by gel mobility shift assay. Increasing concentrations of AdcR (250, 500 and 750 ng) were incubated with the respective oligoduplexes and reactions mixtures were resolved on a 10% native PAGE. The positions of free (F) and AdcR-bound (B) forms of probe are indicated.

Figure 3.

Differential gene regulation by AdcR. Strain MGAS10870 was grown to late exponential phase (A₆₀₀ ∼ 1.0) in zinc-rich growth medium and treated with 50 μM TPEN. Samples were collected at one-minute intervals for 5 min. Transcript levels of phtD (A) and adcC (B) were measured by qRT-PCR. Three biological replicates were used. Data graphed are mean ± standard deviation. Average values for untreated samples were used as reference and the fold changes in the transcript levels in the treated samples relative to untreated sample were shown above. The sensitivity of AdcR bound to two- (C) and one-site (D) operator sequences to zinc chelation was assessed by gel mobility shift assay. The metallated form of AdcR bound to DNA was challenged by increasing concentrations of TPEN (45, 55, 60, 65, 70, 75, 80 and 100 μM) and the reaction mixtures were resolved on a 10% native PAGE. Representative image from three independent experiments is shown. The positions of free (F) and AdcR-bound (B) forms of probe are indicated.

Figure 4.

AdcR directly upregulates hasABC and prtS expression. Taqman qRT-PCR analysis of hasA (A) and prtS (B) transcript levels in indicated strains relative to ΔadcR trans-complemented with empty vector, pDC123 (ΔadcR:pDC). Data graphed are mean ± standard deviation for three biological replicates. (C) AdcR-DNA interactions as assessed by gel mobility shift assay. DNA fragments of similar size were amplified from the promoters of adcRCB (padcR), non-specific control proS (pproS), hasABC (phasABC) and prtS (pprtS) genes. Increasing concentrations of apo- (left panels) and zinc-bound AdcR (right panels) (50, 100 and 200 ng) were incubated with the indicated DNA fragments and reaction mixtures were analyzed on a 10% native PAGE. The positions of free (F) and AdcR-bound (B) probe are indicated. (D) Transcript level analysis of hasA gene measured by qRT PCR. Fold changes in GAS grown in zinc-rich media relative to cells grown under zinc-depleted conditions were shown.

Figure 5.

Analysis of the hasABC promoter region. The significance of hasABC promoter sequences for AdcR binding was assessed by gel mobility shift assay. (A) Genetic organization of hasABC operon on the chromosome of MGAS10870. The −10 and −35 promoter elements are indicated with black boxes, whereas the AdcR binding region, as identified in this study, is indicated in gray. Truncated promoter fragments (probes 1–4) tested for AdcR binding are shown and the numbers to the left denote the 5′ end of the deletions relative to the first nucleotide of the hasA start codon. (B) Increasing concentrations (50 and 100 ng) of purified apo- (left) or zinc-bound AdcR (right) were incubated with the indicated DNA fragments and reaction mixtures were resolved on a 10% native PAGE. The positions of free (F) and AdcR-bound (B) probe are indicated. (C) Architecture of the hasABC promoter. The locations of −10 hexamer, −35 hexamer and TSS, as identified by Ashbaugh et al. (56) are marked and labeled. The positions of the short and long inverted repeats within the hasABC promoter are indicated by arrows above and below the sequence, respectively. The putative CovR-binding site with the characteristic 5′ATTARA 3′ sequence located within the inverted repeat is boxed and shaded in gray.

Figure 6.

Identification of AdcR binding site in the hasABC promoter. (A) The nucleotide sequence of the two inverted repeats, IR1 (thin lined box) and IR2 (bold box), that has the putative AdcR binding site within the hasABC promoter is shown and labeled. Oligoduplexes containing either IR1 or IR2 were tested for AdcR binding by gel mobility shift assay. Increasing concentrations (50, 75 and 100 ng) of purified zinc-bound AdcR (B and D), and apo AdcR (C) were incubated with the indicated oligoduplexes and reaction mixtures were resolved on a 10% native PAGE. (E) The ability of zinc-bound AdcR (50 and 100 ng) to bind WT IR2 (left) and mutant IR2 (right) was assessed by gel mobility shift assay. The nucleotide sequences of the WT (PhasA-WT) and mutant (PhasA-mutant) IR2 are shown and the mutagenized bases are underlined. The positions of free (F) and AdcR-bound (B) probe are indicated.

Figure 7.

Metal binding ligands of AdcR are critical for AdcR-DNA interactions. (A) DNA binding activites of WT and mutant derivatives of AdcR, as assessed by gel mobility shift assay. Increasing concentrations of purified WT or AdcR mutant proteins were incubated with oligoduplexes containing the adc motif and resolved on native PAGE. The positions of unbound (F) and AdcR-bound (B) probes are indicated. Coomassie-stained bands corresponding to purified WT and mutant derivatives of AdcR are shown. Taqman qRT-PCR analysis of adcC (B), phtD (C) and hasA (D) transcript levels in indicated strains relative to constitutively expressed endogenous control, tufA. Data graphed are mean ± standard deviation for three biological replicates.

Figure 8.

Zinc sensing and gene regulation by AdcR is critical for GAS virulence. Fifteen CD-1 mice were infected with indicated strains intraperitoneally and near-mortality was recorded. The Kaplan–Meier survival curve with P-values derived by log rank test is shown.