Sensing and regulation of reactive sulfur species (RSS) in bacteria.

The infected host deploys generalized oxidative stress caused by small inorganic reactive molecules as antibacterial weapons. An emerging consensus is that hydrogen sulfide (H2S) and forms of sulfur with sulfur-sulfur bonds termed reactive sulfur species (RSS) provide protection against oxidative stressors and antibiotics, as antioxidants. Here, we review our current understanding of RSS chemistry and its impact on bacterial physiology. We start by describing the basic chemistry of these reactive species and the experimental approaches developed to detect them in cells. We highlight the role of thiol persulfides in H2S-signaling and discuss three structural classes of ubiquitous RSS sensors that tightly regulate cellular H2S/RSS levels in bacteria, with a specific focus on the chemical specificity of these sensors.

Polysulfide metabolizing enzymes influence SqrR-mediated sulfide-induced transcription by impacting intracellular polysulfide dynamics.

Sulfide plays essential roles in controlling various physiological activities in almost all organisms. Although recent evidence has demonstrated that sulfide is endogenously generated and metabolized into polysulfides inside the cells, the relationship between polysulfide metabolism and polysulfide-sensing mechanisms is not well understood. To better define this interplay between polysulfide metabolism and sensing in cells, we investigated the role of polysulfide-metabolizing enzymes such as sulfide:quinone oxidoreductase (SQR) on the temporal dynamics of cellular polysulfide speciation and on the transcriptional regulation by the persulfide-responsive transcription factor SqrR in Rhodobacter capsulatus. We show that disruption of the sqr gene resulted in the loss of SqrR repression by exogenous sulfide at longer culture times, which impacts the speciation of intracellular polysulfides of Δsqr vs. wild-type strains. Both the attenuated response of SqrR and the change in polysulfide dynamics of the Δsqr strain is fully reversed by the addition to cells of cystine-derived polysulfides, but not by glutathione disulfide (GSSG)-derived polysulfides. Furthermore, cysteine persulfide (CysSSH) yields a higher rate of oxidation of SqrR relative to glutathione persulfide (GSSH), which leads to DNA dissociation in vitro. The oxidation of SqrR was confirmed by a mass spectrometry-based kinetic profiling strategy that showed distinct polysulfide-crosslinked products obtained with CysSSH vs. GSSH. Taken together, these results establish a novel association between the metabolism of polysulfides and the mechanisms for polysulfide sensing inside the cells.

Protocol for using organic persulfides to measure the chemical reactivity of persulfide sensors.

Hydrogen sulfide (H2S) and downstream reactive sulfur species (RSS), including organic persulfides, protect bacterial cells against diverse oxidative stressors. Specialized dithiol-based transcriptional repressors sense persulfides directly to control cellular H2S/RSS and avoid toxicity. Here, we present a protocol to quantify the kinetics of chemical reactivity of cysteines in two bacterial persulfide sensors toward cysteine persulfide and glutathione persulfide, with a LC-ESI-MS analysis that results in a kinetic model. This protocol has potential applications to other cysteine-containing proteins and oxidants. For complete details on the use and execution of this protocol, please refer to Fakhoury et al. (2021) and Capdevila et al. (2021).

Bacterial hydrophilins promote pathogen desiccation tolerance.

Acinetobacter baumannii is a leading cause of hospital-acquired infections, where outbreaks are driven by its ability to persist on surfaces in a desiccated state. Here, we show that A. baumannii causes more virulent pneumonia following desiccation and profile the genetic requirements for desiccation. We find that desiccation tolerance is enhanced upon the disruption of Lon protease, which targets unfolded and aggregated proteins for degradation. Notably, two bacterial hydrophilins, DtpA and DtpB, are transcriptionally upregulated in Δlon via the two-component regulator, BfmR. These proteins, both hydrophilic and intrinsically disordered, promote desiccation tolerance in A. baumannii. Additionally, recombinant DtpA protects purified enzymes from inactivation and improves the desiccation tolerance of a probiotic bacterium when heterologously expressed. These results demonstrate a connection between environmental persistence and pathogenicity in A. baumannii, provide insight into the mechanisms of extreme desiccation tolerance, and reveal potential applications for bacterial hydrophilins in the preservation of protein- and live bacteria-based pharmaceuticals.

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.

Human αB-crystallin discriminates between aggregation-prone and function-preserving variants of a client protein.

BACKGROUND: The eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin (γS-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant. The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity. METHODS: Molecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A. RESULTS: γS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature. CONCLUSIONS: αB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant. GENERAL SIGNIFICANCE: Human αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature.