Functional asymmetry and chemical reactivity of CsoR family persulfide sensors.

CstR is a persulfide-sensing member of the functionally diverse copper-sensitive operon repressor (CsoR) superfamily. While CstR regulates the bacterial response to hydrogen sulfide (H2S) and more oxidized reactive sulfur species (RSS) in Gram-positive pathogens, other dithiol-containing CsoR proteins respond to host derived Cu(I) toxicity, sometimes in the same bacterial cytoplasm, but without regulatory crosstalk in cells. It is not clear what prevents this crosstalk, nor the extent to which RSS sensors exhibit specificity over other oxidants. Here, we report a sequence similarity network (SSN) analysis of the entire CsoR superfamily, which together with the first crystallographic structure of a CstR and comprehensive mass spectrometry-based kinetic profiling experiments, reveal new insights into the molecular basis of RSS specificity in CstRs. We find that the more N-terminal cysteine is the attacking Cys in CstR and is far more nucleophilic than in a CsoR. Moreover, our CstR crystal structure is markedly asymmetric and chemical reactivity experiments reveal the functional impact of this asymmetry. Substitution of the Asn wedge between the resolving and the attacking thiol with Ala significantly decreases asymmetry in the crystal structure and markedly impacts the distribution of species, despite adopting the same global structure as the parent repressor. Companion NMR, SAXS and molecular dynamics simulations reveal that the structural and functional asymmetry can be traced to fast internal dynamics of the tetramer. Furthermore, this asymmetry is preserved in all CstRs and with all oxidants tested, giving rise to markedly distinct distributions of crosslinked products. Our exploration of the sequence, structural, and kinetic features that determine oxidant-specificity suggest that the product distribution upon RSS exposure is determined by internal flexibility.

Allosteric control of a bacterial stress response system by an anti-σ factor.

Bacterial signal transduction systems commonly use receiver (REC) domains, which regulate adaptive responses to the environment as a function of their phosphorylation state. REC domains control cell physiology through diverse mechanisms, many of which remain understudied. We have defined structural features that underlie activation of the multi-domain REC protein, PhyR, which functions as an anti-anti-σ factor and regulates transcription of genes required for stress adaptation and host-microbe interactions in Alphaproteobacteria. Though REC phosphorylation is necessary for PhyR function in vivo, we did not detect expected changes in inter-domain interactions upon phosphorylation by solution X-ray scattering. We sought to understand this result by defining additional molecular requirements for PhyR activation. We uncovered specific interactions between unphosphorylated PhyR and an intrinsically disordered region (IDR) of the anti-σ factor, NepR, by solution NMR spectroscopy. Our data support a model whereby nascent NepR(IDR)-PhyR interactions and REC phosphorylation coordinately impart the free energy to shift PhyR to an open, active conformation that binds and inhibits NepR. This mechanism ensures PhyR is activated only when NepR and an activating phosphoryl signal are present. Our study provides new structural understanding of the molecular regulatory logic underlying a conserved environmental response system.

Sulfide Homeostasis and Nitroxyl Intersect via Formation of Reactive Sulfur Species in Staphylococcus aureus.

Staphylococcus aureus is a commensal human pathogen and a major cause of nosocomial infections. As gaseous signaling molecules, endogenous hydrogen sulfide (H2S) and nitric oxide (NO·) protect S. aureus from antibiotic stress synergistically, which we propose involves the intermediacy of nitroxyl (HNO). Here, we examine the effect of exogenous sulfide and HNO on the transcriptome and the formation of low-molecular-weight (LMW) thiol persulfides of bacillithiol, cysteine, and coenzyme A as representative of reactive sulfur species (RSS) in wild-type and ΔcstR strains of S. aureus. CstR is a per- and polysulfide sensor that controls the expression of a sulfide oxidation and detoxification system. As anticipated, exogenous sulfide induces the cst operon but also indirectly represses much of the CymR regulon which controls cysteine metabolism. A zinc limitation response is also observed, linking sulfide homeostasis to zinc bioavailability. Cellular RSS levels impact the expression of a number of virulence factors, including the exotoxins, particularly apparent in the ΔcstR strain. HNO, like sulfide, induces the cst operon as well as other genes regulated by exogenous sulfide, a finding that is traced to a direct reaction of CstR with HNO and to an endogenous perturbation in cellular RSS, possibly originating from disassembly of Fe-S clusters. More broadly, HNO induces a transcriptomic response to Fe overload, Cu toxicity, and reactive oxygen species and reactive nitrogen species and shares similarity with the sigB regulon. This work reveals an H2S/NO· interplay in S. aureus that impacts transition metal homeostasis and virulence gene expression. IMPORTANCE Hydrogen sulfide (H2S) is a toxic molecule and a recently described gasotransmitter in vertebrates whose function in bacteria is not well understood. In this work, we describe the transcriptomic response of the major human pathogen Staphylococcus aureus to quantified changes in levels of cellular organic reactive sulfur species, which are effector molecules involved in H2S signaling. We show that nitroxyl (HNO), a recently described signaling intermediate proposed to originate from the interplay of H2S and nitric oxide, also induces changes in cellular sulfur speciation and transition metal homeostasis, thus linking sulfide homeostasis to an adaptive response to antimicrobial reactive nitrogen species.

Cysteine sulfur chemistry in transcriptional regulators at the host-bacterial pathogen interface.

Hosts employ myriad weapons to combat invading microorganisms as an integral feature of the host-bacterial pathogen interface. This interface is dominated by highly reactive small molecules that collectively induce oxidative stress. Successful pathogens employ transcriptional regulatory proteins that sense these small molecules directly or indirectly via a change in the ratio of reduced to oxidized low-molecular weight (LMW) thiols that collectively comprise the redox buffer in the cytoplasm. These transcriptional regulators employ either a prosthetic group or reactive cysteine residue(s) to effect changes in the transcription of genes that encode detoxification and repair systems that is driven by regulator conformational switching between high-affinity and low-affinity DNA-binding states. Cysteine harbors a highly polarizable sulfur atom that readily undergoes changes in oxidation state in response to oxidative stress to produce a range of regulatory post-translational modifications (PTMs), including sulfenylation (S-hydroxylation), mixed disulfide bond formation with LMW thiols (S-thiolation), di- and trisulfide bond formation, S-nitrosation, and S-alkylation. Here we discuss several examples of structurally characterized cysteine thiol-specific transcriptional regulators that sense changes in cellular redox balance, focusing on the nature of the cysteine PTM itself and the interplay of small molecule oxidative stressors in mediating a specific transcriptional response.

Conformational analysis and chemical reactivity of the multidomain sulfurtransferase, Staphylococcus aureus CstA.

The cst operon of the major human pathogen Staphylococcus aureus (S. aureus) is under the transcriptional control of CsoR-like sulfurtransferase repressor (CstR). Expression of this operon is induced by hydrogen sulfide, and two components of the cst operon, cstA and cstB, protect S. aureus from sulfide toxicity. CstA is a three-domain protein, and each domain harbors a single cysteine that is proposed to function in vectorial persulfide shuttling. We show here that single cysteine substitution mutants of CstA fail to protect S. aureus against sulfide toxicity in vivo. The N-terminal domain of CstA exhibits thiosulfate sulfurtransferase (TST; rhodanese) activity, and a Cys66 (34)S-persulfide is formed as a catalytic intermediate in both the presence and absence of the adjacent TusA-like domain using (34)S-SO3(2-) as a substrate. Cysteine persulfides can be trapped on both C66 in CstA(Rhod) and on C66 and C128 in CstA(Rhod-TusA) when incubated with thiosulfate, sodium tetrasulfide (Na2S4), and in situ persulfurated SufS. C66A substitution in CstA(Rhod-TusA) abolishes C128 S-sulfhydration, consistent with directional persulfide shuttling in CstA. Fully reduced CstA(Rhod-TusA) is predominately monomeric, and high resolution tandem mass spectrometry reveals that Cys66 and Cys128 can form a C66-C128 disulfide bond using a number of oxidants, which leads to a significant change in conformation. A competing intermolecular C128-C128' disulfide bond is also formed. Small-angle X-ray scattering measurements and gel filtration chromatography of reduced CstA(Rhod-TusA) reveal an elongated molecule (Rg ≈ 30 Å, 21.6 kDa) where the two domains pack "side-by-side" that likely places Cys66 and Cys128 far apart. These studies are consistent with the low yield of C66-C128 cross-link as a mimic of a persulfide transfer intermediate in CstA, and small, but measurable persulfide transfer from Cys66 to Cys128 within the CstA(Rhod-TusA) with inorganic sulfur donors.

The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.

How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR [Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor] represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high-resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60' interprotomer cross-links, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR.

Selenite and tellurite form mixed seleno- and tellurotrisulfides with CstR from Staphylococcus aureus.

Staphylococcus aureus CstR (CsoR-like sulfur transferase repressor) is a member of the CsoR family of transition metal sensing metalloregulatory proteins. Unlike CsoR, CstR does not form a stable complex with transition metals but instead reacts with sulfite to form a mixture of di- and trisulfide species, CstR2(RS-SR') and CstR2(RS-S-SR')n)n=1 or 2, respectively. Here, we investigate if CstR performs similar chemistry with related chalcogen oxyanions selenite and tellurite. In this work we show by high resolution tandem mass spectrometry that CstR is readily modified by selenite (SeO3(2-)) or tellurite (TeO3(2-)) to form a mixture of intersubunit disulfides and selenotrisulfides or tellurotrisulfides, respectively, between Cys31 and Cys60'. Analogous studies with S. aureus CsoR reveals no reaction with selenite and minimal reaction with tellurite. All cross-linked forms of CstR exhibit reduced DNA binding affinity. We show that Cys31 initiates the reaction with sulfite through the formation of S-sulfocysteine (RS-SO3(2-)) and Cys60 is required to fully derivatize CstR to CstR2(RS-SR') and CstR2(RS-S-SR'). The modification of Cys31 also drives an allosteric switch that negatively regulates DNA binding while derivatization of Cys60 alone has no effect on DNA binding. These results highlight the differences between CstRs and CsoRs in chemical reactivity and metal ion selectivity and establish Cys31 as the functionally important cysteine residue in CstRs.

Glycemic responses to sweetened dried and raw cranberries in humans with type 2 diabetes.

This study assessed the metabolic response to sweetened dried cranberries (SDC), raw cranberries (RC), and white bread (WB) in humans with type 2 diabetes. Development of palatable cranberry preparations associated with lower glycemic responses may be useful for improving fruit consumption and glycemic control among those with diabetes. In this trial, type 2 diabetics (n= 13) received WB (57 g, 160 cal, 1 g fiber), RC (55 g, 21 cal, 1 g fiber), SDC (40 g, 138 cal, 2.1 g fiber), and SDC containing less sugar (SDC-LS, 40 g, 113 cal, 1.8 g fiber + 10 g polydextrose). Plasma glucose (mmol/L) peaked significantly at 60 min for WB, and at 30 min for RC, SDC, and SDC-LS at 9.6 ± 0.4, 7.0 ± 0.4, 9.6 ± 0.5, and 8.7 ± 0.5, respectively, WB remained significantly elevated from the other treatments at 120 min. Plasma insulin (pmol/mL) peaked at 60 min for WB and SDC and at 30 min for RC and SDC-LS at 157 ± 15, 142 ± 27, 61 ± 8, and 97 ± 11, respectively. Plasma insulin for SDC-LS was significantly lower at 60 min than either WB or SDC. Insulin area under the curve (AUC) values for RC and SDC-LS were both significantly lower than WB or SDC. Phenolic content of SDC and SDC-LS was determined following extraction with 80% acetone prior to high-performance liquid chromatography (HPLC) and electronspray ionization-mass spectrometry (ESI-MS) and found to be rich in 5-caffeoylquinic cid, quercetin-3-galactoside, and quercetin-3-galactoside, and the proanthocyanidin dimer epicatechin. In conclusion, SDC-LS was associated with a favorable glycemic and insulinemic response in type 2 diabetics. Practical Application: This study compares phenolic content and glycemic responses among different cranberry products. The study seeks to expand the palatable and portable healthy food choices for persons with type 2 diabetes. The novel use of polydextrose as a bulking agent making possible a reduction in caloric content and potential glycemic response is also characterized in this study.