RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus

Cys128 is required for growth and regulation of the Piu operon and colonization.

<p>(<b>A</b>) Western blot analysis representative of three independent experiments of PiuA iron transporter lipoprotein levels in the R800 <i>ritR</i> genetic background variants grown in 5% CO₂. RitR and NanA are used as controls. Ukn, an unknown cross-reacting protein. (<b>B</b>) Piu promoter activity in the genetic background of R800 <i>ritR</i> variants in 5% CO₂ as measured by beta-galactosidase activity and expressed in Miller units. (<b>C</b>) Growth comparison of wild-type D39 cells plus <i>ritR</i> variants under static 5% CO₂ and aeration (O₂) conditions. (<b>D</b>) Streptonigrin killing assay representative of three biological repeats. (<b>E</b>) Bar graph of three independent experiments as shown in <i>D</i>. (<b>F</b>) Ability of D39 variants to colonize the murine nasopharynx. Mice were inoculated and the colonization let run for 7 days before cells were collected and plated to determine CFUs. <i>Statistics</i>. Colonization data were analyzed by analysis of variance followed by Tukey’s multiple comparisons test. Statistical significance was considered to be a p-value of < 0.05. Each point indicates the CFUs retained in a single animal. Graphs in <i>B</i> and <i>E</i> represent the mean of three independent experiments. Error bars represent +/- standard deviation. One asterisk indicates a p-value of ≤0.05, two of ≤0.01, three of ≤0.001, and four of ≤ 0.0001 as determined by one-way ANOVA followed by Tukey’s multiple comparison test in <i>B</i>, and a two-tailed students t-test in <i>E</i>. ns, not significant.</p

RitR oxidized structure.

<p>(<b>A</b>) Cartoon representation of the domain-swapped RitR_OX structure. One protomer is in color and the other protomer in grey. REC, receiver domain; DBD, DNA-binding domain. (<b>B</b>) 2|F_o|-|F_c| composite omit electron density for the inter-protomer Cys128:Cys128’ disulfide bond and surrounding residues that pins the C-terminal ends of each α5 helix together. As a consueqence, both DBDs are in close proximity. One protomer is shown in color with a pink density map, and the other protomer is shown in grey with a matching grey density map. (<b>C</b>) Image of the interface between the DBD of one protomer of the RitR_OX homodimer (bright colors) and the REC domain of the other protomer (muted colors). The interactions are almost identical to those observed for the C128S structure in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.g003" target="_blank">Fig 3C</a>.</p

Conservation of RitR in the streptococci.

<p>(<b>A</b>) Alignment of linker regions of RitR homologs (in red) and CovR homologs (in black) from the streptococci. Notice the degeneracy in the “HCS” motif in the swine zoonotic pathogen <i>S</i>. <i>suis</i>. Identical residues are colored black and similar residues are colored grey. The conserved cysteine is shaded in yellow. (<b>B</b>) Phylogenetic tree of RitR (in red) and CovR (in black) streptococcal homologs. Evolutionary distance is depicted by the length of the horizontal lines. Posterior probabilities are displayed at the branch points.</p

Structure of the ‘reduced’ RitR C128S.

<p>(<b>A</b>) RitR Clustal Omega annotated alignment of RitR and two other full-length response regulators with available structures (MtrA from <i>Mycobacterium tuberculosis</i>, PDB ID 2GWR, and Rra from <i>Deinococcus radiodurans</i>, PDB ID 3Q9S). Identity is denoted by an asterisk and similarity by dots/colons. Secondary structure depicted above the sequences is color coordinated with the 3D models presented in <i>B-D</i>. The reactive Cys128 position is shaded in yellow, and the position which normally contains the phosphorylated Asp residue shaded in blue (note in RitR it is an Asn instead). (<b>B</b>) Ribbon diagram of the full-length, monomeric ('reduced' / inactive) RitR_C128S. Ser128 (Cys128 coordinate) is labeled, colored yellow and appears in ball-and-stick format. The α4-β5-α5 face of the REC domain used by canonical response regulators for dimerization is shown in green. The remainder of the REC domain is blue. The DNA-binding domain is gold, save for the recognition helix (red) and the trans-activation loop (magenta) that interacts with RNA polymerase to direct transcription [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref048" target="_blank">48</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref049" target="_blank">49</a>]. ALR, Aspartate-less receiver domain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref030" target="_blank">30</a>]; DBD, DNA-binding domain. (<b>C</b>) Close-up of the RitR DBD-REC interface shown with the same coloring. The residues comprising the interface are shown as ball-and-stick. (<b>D</b>) Close-up of the Ser128 (Cys128 coordinate) interactions with neighboring residues and water molecules. Dotted lines denote predicted electrostatic interactions. Oxygen atoms are shown in red, nitrogens in blue and water molecules as light blue circles. Images were created using MOLSCRIPT and POVRay [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref095" target="_blank">95</a>].</p

Schematic representation of RitR regulation in <i>S</i>. <i>pneumoniae</i>.

<p>Hydrogen peroxide (H₂O₂) is primarily produced from <i>S</i>. <i>pneumoniae</i> metabolism via pyruvate oxidase (SpxB). When present, iron must be kept out of the cell, or alternatively, stored such that it cannot react to yield Fenton chemistry, thereby causing cellular damage. RitR regulates this process by oxidation though Cys128 in high H₂O₂ produced in aerobic environments such as the nasopharynx, which allows its open conformation and release of the DNA-binding domain (DBD) for the Regulatory Domain (RD) to interact with the Piu promoter, repressing iron uptake. Simultaneously, RitR is postulated to remediate iron toxicity through activation of DNA repair and iron sequestration [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref025" target="_blank">25</a>]. Conversely, when H₂O₂ concentrations are low, RitR stays in its inactive form, where the interaction of the RD with the DBD prevents its binding to the Piu regulatory region, ultimately allowing for more iron to enter the cell. The potential oxidation states of RitR (SO^-, SO^2-, SO^3-) and their regulatory consequences remain enigmatic.</p

Crystallographic data collection and refinement statistics.

<p>Crystallographic data collection and refinement statistics.</p

RitR has evolved to dimerize through Cys128-mediated oxidation by H₂O₂.

<p>(<b>A</b>) Non-denaturing SDS-PAGE gels of RitR WT, C>S and C>D mutants plus various oxidants in the presence of 3 mM DTT. D, dimeric RitR; M, monomeric RitR. MW, Molecular Weight ladder; H₂O₂, hydrogen peroxide; t-butyl, tert-butyl hydroperoxide; CHP, cumene hydroperoxide; NO, nitrous oxide; Ox-Glu, oxidized glutathione; NaOCl, sodium hypochlorite. (<b>B</b>) Alignment of REC/ALR α4-β5-α5 dimerization domains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref030" target="_blank">30</a>] from RitR homologs (red) and canonical REC domain sequences (black). The black boxes are identical residues and the grey boxes are similar residues. Residues colored purple and green represent key charged and hydrophobic residues, respectively, involved in typical REC dimerization. Note that several of these key residues are changed in RitR homologs. (<b>C</b>) SEC of wild-type RitR with (+) or without (-) addition of DTT or H₂O₂ (top graph), or RitR changed back to the canonical GADDY sequence (RitR_GADDY; bottom graph). For comparison, the RitR_L86A/V96A mutant is shown, which naturally dimerizes without addition of oxidant [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref030" target="_blank">30</a>]. mAU; milli Absorbance Units. (<b>D</b>) EMSAs of RitR wild-type (WT) and the C128S mutant in the presence (+) and absence (-) of H₂O₂. RitR proteins were added at 0, 0.22, 0.66, 2.2 and 6.6 μM concentrations (left to right) in the presence of hexofluorescein (HEX)-labeled BS1-3 double-stranded DNA oligomers. A HEX labeled control oligo was also used. P, Hex DNA probe; C, RitR-DNA shifted complex. Below is a schematic diagram of the Piu promoter and regulatory region showing the location of RitR binding sites 1–3 (BS1-3) as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref025" target="_blank">25</a>]. (<b>E</b>) Mass spectrometry (MS) analysis of the Cys128 disulfide bridge formation <i>in vivo</i>. Upper (D1), middle (D2) and lower bands (M) of RitR as identified from the anti-FLAG western blot and accompanying Coomassie stain were excised from the gel and determined to contain RitR using MS. (+) oxygen = cells were aerated, (+/-) oxygen = cells were grown statically in 5% CO₂, and (-) oxygen = cells were grown anaerobically before addition of IAA and RitR immunoprecipitation. The MS identified Cys128 linked peptide is shown with Cys128 colored in orange. Data shown in A-E are representative of at least two independent experiments.</p