SifR is an Rrf2-family quinone sensor associated with catechol iron uptake in Streptococcus pneumoniae D39.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive commensal and human respiratory pathogen. How this bacterium satisfies its nutritional iron (Fe) requirement in the context of endogenously produced hydrogen peroxide is not well understood. Here, we characterize a novel virulence-associated Rrf2-family transcriptional repressor that we term SifR (streptococcal IscR-like family transcriptional repressor) encoded by spd_1448 and conserved in Streptococci. Global transcriptomic analysis of a ΔsifR strain defines the SifR regulon as genes encoding a candidate catechol dioxygenase CatE, an uncharacterized oxidoreductase YwnB, a candidate flavin-dependent ferric reductase YhdA, a candidate heme-based ferric reductase domain-containing protein and the Piu (pneumococcus iron uptake) Fe transporter (piuBCDA). Previous work established that membrane-anchored PiuA binds FeIII-bis-catechol or monocatechol complexes with high affinity, including the human catecholamine stress hormone, norepinephrine. We demonstrate that SifR senses quinone via a single conserved cysteine that represses its regulon when in the reduced form. Upon reaction with catechol-derived quinones, we show that SifR dissociates from the DNA leading to regulon derepression, allowing the pneumococcus to access a catechol-derived source of Fe while minimizing reactive electrophile stress induced by quinones. Consistent with this model, we show that CatE is an FeII-dependent 2,3-catechol dioxygenase with broad substrate specificity, YwnB is an NAD(P)H-dependent quinone reductase capable of reducing the oxidized and cyclized norepinephrine, adrenochrome, and YhdA is capable of reducing a number of FeIII complexes, including PiuA-binding transport substrates. These findings are consistent with a model where FeIII-catechol complexes serve as significant nutritional Fe sources in the host.

Cellular Mn/Zn Ratio Influences Phosphoglucomutase Activity and Capsule Production in Streptococcus pneumoniae D39.

Capsular polysaccharide (CPS) is a major virulence determinant for many human-pathogenic bacteria. Although the essential functional roles for CPS in bacterial virulence have been established, knowledge of how CPS production is regulated remains limited. Streptococcus pneumoniae (pneumococcus) CPS expression levels and overall thickness change in response to available oxygen and carbohydrate. These nutrients in addition to transition metal ions can vary significantly between host environmental niches and infection stage. Since the pneumococcus must modulate CPS expression among various host niches during disease progression, we examined the impact of the nutritional transition metal availability of manganese (Mn) and zinc (Zn) on CPS production. We demonstrate that increased Mn/Zn ratios increase CPS production via Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway in a transcription-independent manner. Furthermore, we find that the downstream CPS protein CpsB, an Mn-dependent phosphatase, does not promote aberrant dephosphorylation of its target capsule-tyrosine kinase CpsD during Mn stress. Together, these data reveal a direct role for cellular Mn/Zn ratios in the regulation of CPS biosynthesis via the direct activation of Pgm. We propose a multilayer mechanism used by the pneumococcus in regulating CPS levels across various host niches. IMPORTANCE Evolving evidence strongly indicates that maintenance of metal homeostasis is essential for establishing colonization and continued growth of bacterial pathogens in the vertebrate host. In this study, we demonstrate the impact of cellular manganese/zinc (Mn/Zn) ratios on bacterial capsular polysaccharide (CPS) production, an important virulence determinant of many human-pathogenic bacteria, including Streptococcus pneumoniae. We show that higher Mn/Zn ratios increase CPS production via the Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway. The findings provide a direct role for Mn/Zn homeostasis in the regulation of CPS expression levels and further support the ability of metal cations to act as important cellular signaling mediators in bacteria.

A Mn-sensing riboswitch activates expression of a Mn2+/Ca2+ ATPase transporter in Streptococcus.

Maintaining manganese (Mn) homeostasis is important for the virulence of numerous bacteria. In the human respiratory pathogen Streptococcus pneumoniae, the Mn-specific importer PsaBCA, exporter MntE, and transcriptional regulator PsaR establish Mn homeostasis. In other bacteria, Mn homeostasis is controlled by yybP-ykoY family riboswitches. Here, we characterize a yybP-ykoY family riboswitch upstream of the mgtA gene encoding a PII-type ATPase in S. pneumoniae, suggested previously to function in Ca2+ efflux. We show that the mgtA riboswitch aptamer domain adopts a canonical yybP-ykoY structure containing a three-way junction that is compacted in the presence of Ca2+ or Mn2+ at a physiological Mg2+ concentration. Although Ca2+ binds to the RNA aptamer with higher affinity than Mn2+, in vitro activation of transcription read-through of mgtA by Mn2+ is much greater than by Ca2+. Consistent with this result, mgtA mRNA and protein levels increase ≈5-fold during cellular Mn stress, but only in genetic backgrounds of S. pneumoniae and Bacillus subtilis that exhibit Mn2+ sensitivity, revealing that this riboswitch functions as a failsafe 'on' signal to prevent Mn2+ toxicity in the presence of high cellular Mn2+. In addition, our results suggest that the S. pneumoniae yybP-ykoY riboswitch functions to regulate Ca2+ efflux under these conditions.

Perturbation of manganese metabolism disrupts cell division in Streptococcus pneumoniae.

Manganese (Mn) is an essential micronutrient and required cofactor in bacteria. Despite its importance, excess Mn can impair bacterial growth, the mechanism of which remains largely unexplored. Here, we show that proper Mn homeostasis is critical for cellular growth of the major human respiratory pathogen Streptococcus pneumoniae. Perturbations in Mn homeostasis genes, psaBCA, encoding the Mn importer, and mntE, encoding the Mn exporter, lead to Mn sensitivity during aerobiosis. Mn-stressed cells accumulate iron and copper, in addition to Mn. Impaired growth is a direct result of Mn toxicity and does not result from iron-mediated Fenton chemistry, since cells remain sensitive to Mn during anaerobiosis or when hydrogen peroxide biogenesis is significantly reduced. Mn-stressed cells are significantly elongated, whereas Mn-limitation imposed by zinc addition leads to cell shortening. We show that Mn accumulation promotes aberrant dephosphorylation of cell division proteins via hyperactivation of the Mn-dependent protein phosphatase PhpP, a key enzyme involved in the regulation of cell division. We discuss a mechanism by which cellular Mn:Zn ratios dictate PhpP specific activity thereby regulating pneumococcal cell division. We propose that Mn-metalloenzymes are particularly susceptible to hyperactivation or mismetallation, suggesting the need for exquisite cellular control of Mn-dependent metabolic processes.

The zinc efflux activator SczA protects Streptococcus pneumoniae serotype 2 D39 from intracellular zinc toxicity.

Zinc is an essential trace element that serves as a catalytic cofactor in metalloenzymes and a structural element in proteins involved in general metabolism and cellular defenses of pathogenic bacteria. Despite its importance, high zinc levels can impair cellular processes, inhibiting growth of many pathogenic bacteria, including the major respiratory pathogen Streptococcus pneumoniae. Zinc intoxication is prevented in S. pneumoniae by expression of the zinc exporter CzcD, whose expression is activated by the novel TetR-family transcriptional zinc-sensing regulator SczA. How zinc bioavailability triggers activation of SczA is unknown. It is shown here through functional studies in S. pneumoniae that an unannotated homodimeric TetR from S. agalactiae (PDB 3KKC) is the bona fide zinc efflux regulator SczA, and binds two zinc ions per protomer. Mutagenesis analysis reveals two metal binding sites, termed A and B, located on opposite sides of the SczA C-terminal regulatory domain. In vivo, the A- and B-site SczA mutant variants impact S. pneumoniae resistance to zinc toxicity and survival in infected macrophages. A model is proposed for S. pneumoniae SczA function in which both A- and B-sites were required for transcriptional activation of czcD expression, with the A-site serving as the evolutionarily conserved intracellular sensing site in SczAs.

The Escherichia coli small protein MntS and exporter MntP optimize the intracellular concentration of manganese.

Escherichia coli does not routinely import manganese, but it will do so when iron is unavailable, so that manganese can substitute for iron as an enzyme cofactor. When intracellular manganese levels are low, the cell induces the MntH manganese importer plus MntS, a small protein of unknown function; when manganese levels are high, the cell induces the MntP manganese exporter and reduces expression of MntH and MntS. The role of MntS has not been clear. Previous work showed that forced MntS synthesis under manganese-rich conditions caused bacteriostasis. Here we find that when manganese is scarce, MntS helps manganese to activate a variety of enzymes. Its overproduction under manganese-rich conditions caused manganese to accumulate to very high levels inside the cell; simultaneously, iron levels dropped precipitously, apparently because manganese-bound Fur blocked the production of iron importers. Under these conditions, heme synthesis stopped, ultimately depleting cytochrome oxidase activity and causing the failure of aerobic metabolism. Protoporphyrin IX accumulated, indicating that the combination of excess manganese and iron deficiency had stalled ferrochelatase. The same chain of events occurred when mutants lacking MntP, the manganese exporter, were exposed to manganese. Genetic analysis suggested the possibility that MntS exerts this effect by inhibiting MntP. We discuss a model wherein during transitions between low- and high-manganese environments E. coli uses MntP to compensate for MntH overactivity, and MntS to compensate for MntP overactivity.

Functional Determinants of Metal Ion Transport and Selectivity in Paralogous Cation Diffusion Facilitator Transporters CzcD and MntE in Streptococcus pneumoniae.

UNLABELLED: Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens. Streptococcus pneumoniae expresses two homologous CDF efflux transporters, MntE and CzcD. Cells lacking mntE or czcD are sensitive to manganese (Mn) or zinc (Zn) toxicity, respectively, and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology to Escherichia coli YiiP predicts that both MntE and CzcD are dimeric and each protomer harbors four pairs of conserved metal-binding sites, termed the A site, the B site, and the C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A site and the B site in both CDFs result in a cellular metal sensitivity similar to that of the corresponding null mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD, which do have on impact on cellular Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in the ND-DD A-site motif of MntE and the most N-terminal His in the HD-HD site A of CzcD (the specified amino acids are underlined) play key roles in transporter metal selectivity. IMPORTANCE: Cation diffusion facilitator (CDF) proteins are divalent metal ion transporters that are conserved in organisms ranging from bacteria to humans and that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates. The respiratory pathogen Streptococcus pneumoniae expresses two metal CDF transporters, CzcD and MntE. How CDFs achieve metal selectivity is unclear. We show here that CzcD and MntE are true paralogs, as CzcD transports zinc, while MntE selectively transports manganese. Through the use of an extensive collection of pneumococcal variants, we show that a primary determinant for metal selectivity is the A site within the transmembrane domain. This extends our understanding of how CDFs discriminate among transition metals.

Electrostatic occlusion and quaternary structural ion pairing are key determinants of Cu(I)-mediated allostery in the copper-sensing operon repressor (CsoR).

The copper-sensing operon repressor (CsoR) is an all-α-helical disc-shaped D2-symmetric homotetramer that forms a 2:1 tetramer/DNA operator complex and represses the expression of copper-resistance genes in a number of bacteria. A previous bioinformatics analysis of CsoR-family repressors distributes Cu(I)-sensing CsoRs in four of seven distinct clades on the basis of global sequence similarity. In this work, we define energetically important determinants of DNA binding in the apo-state (ΔΔGbind), and for allosteric negative coupling of Cu(I) binding to DNA binding (ΔΔGc) in a model clade IV CsoR from Geobacillus thermodenitrificans (Gt) of known structure, by selectively targeting for mutagenesis those charged residues uniquely conserved in clade IV CsoRs. These include a folded N-terminal "tail" and a number of Cu(I)-sensor and clade-specific residues that when mapped onto a model of Cu(I)-bound Gt CsoR define a path across one face of the tetramer. We find that Cu(I)-binding prevents formation of the 2:1 "sandwich" complex rather than DNA binding altogether. Folding of the N-terminal tail (residues R18, E22, R74) upon Cu-binding to the periphery of the tetramer inhibits assembly of the 2:1 apoprotein-DNA complex. In contrast, Ala substitution of residues that surround the central "hole" (R65, K101) in the tetramer, as well R48, impact DNA binding. We also identify a quaternary structural ion-pair, E73-K101″, that crosses the tetramer interface, charge-reversal of which restores DNA binding activity, allosteric regulation by Cu(I), and transcriptional derepression by Cu(I) in cells. These findings suggest an "electrostatic occlusion" model, in which basic residues important for DNA binding and/or allostery become sequestered via ion-pairing specifically in the Cu(I)-bound state, and this aids in copper-dependent disassembly of a repression complex.

Calcium Rescues Streptococcus pneumoniae D39 ΔmntE Manganese-Sensitive Growth Phenotype.

Calcium (Ca2+) functions as a universal signal messenger in eukaryotes but in bacteria, the physiological roles for Ca2+ are limited. Here, we examine the role of Ca2+ in Streptococcus pneumoniae during manganese (Mn2+) intoxication. S. pneumoniae mntE mutants, lacking the Mn2+ efflux transporter, exhibit impaired growth due to accumulation of Mn2+ when exposed to elevated exogenous Mn2+. This Mn2+-sensitive growth defect is restored to wild-type growth level by exogenous Ca2+, in a Ca2+-dependent manner. Despite growth restoration of the mntE mutant to wild-type levels, cellular Mn2+ remains elevated in this strain. Bacterial capsule production is also increased for the mntE mutant, resulting in reduced adherence capacity to surfaces and poor biofilm formation, which is consistent with it experiencing Mn2+ intoxication. Ca2+ presence did not significantly impact bacterial capsule production or biofilm formation. Further analysis of the cell morphology demonstrates that Ca2+ contributes to cell division and reduces cell chain lengths. Together, these data describe the first role of Ca in S. pneumoniae that has potential implications in bacterial virulence since Ca affects cell division and likely Mn2+-associated cellular processes.

The alternative aerobic ribonucleotide reductase of Escherichia coli, NrdEF, is a manganese-dependent enzyme that enables cell replication during periods of iron starvation.

The genome of Escherichia coli encodes two class I ribonucleotide reductases. The first, NrdAB, is a well-studied iron-dependent enzyme that is essential for aerobic growth. The second, NrdEF, is not functional under routine conditions, and its role is obscure. Recent studies demonstrated that NrdEF can be activated in vitro by manganese as well as iron. Since iron enzymes are potential targets for hydrogen peroxide, and since the nrdHIEF operon is induced during H(2) O(2) stress, we hypothesized that H(2) O(2) might inactivate NrdAB and that NrdEF might be induced to compensate. This idea was tested using E. coli mutants that are chronically stressed by H(2) O(2) . Contrary to expectation, NrdAB remained active. Its resistance to H(2) O(2) depended upon YfaE, which helps to activate NrdB. The induction of NrdEF during H(2) O(2) stress was mediated by the inactivation of Fur, an iron-dependent repressor. This regulatory arrangement implied that NrdEF has a physiological role during periods of iron starvation. Indeed, NrdEF supported cell replication in iron-depleted cells. Iron bound to NrdF when it was expressed in iron-rich cells, but NrdEF was functional only in cells that were both iron-depleted and manganese-rich. Thus NrdEF supports DNA replication when iron is unavailable to activate the housekeeping NrdAB enzyme.

Regulation of Bacterial Manganese Homeostasis and Usage During Stress Responses and Pathogenesis.

Manganese (Mn) plays a multifaceted role in the survival of pathogenic and symbiotic bacteria in eukaryotic hosts, and it is also important for free-living bacteria to grow in stressful environments. Previous research has uncovered components of the bacterial Mn homeostasis systems that control intracellular Mn levels, many of which are important for virulence. Multiple studies have also identified proteins that use Mn once it is inside the cell, including Mn-specific enzymes and enzymes transiently loaded with Mn for protection during oxidative stress. Emerging evidence continues to reveal proteins involved in maintaining Mn homeostasis, as well as enzymes that can bind Mn. For some of these enzymes, Mn serves as an essential cofactor. For other enzymes, mismetallation with Mn can lead to inactivation or poor activity. Some enzymes may even potentially be regulated by differential metallation with Mn or zinc (Zn). This review focuses on new developments in regulatory mechanisms that affect Mn homeostasis and usage, additional players in Mn import that increase bacterial survival during pathogenesis, and the interplay between Mn and other metals during Mn-responsive physiological processes. Lastly, we highlight lessons learned from fundamental research that are now being applied to bacterial interactions within larger microbial communities or eukaryotic hosts.

mSphere of Influence: Intermetal Competition during Manganese Toxicity.

Julia E. Martin works in the field of metals in biology, with a focus on manganese (Mn) homeostasis in Streptococcus pneumoniae In this mSphere of Influence article, she reflects on how the paper entitled "Role of the manganese efflux system mntE for signalling and pathogenesis in Streptococcus pneumoniae" (J. W. Rosch, G. Gao, G. Ridout, Y.-D. Wang, and E. I. Tuomanen, Mol Microbiol 72:12-25, 2009, https://doi.org/10.1111/j.1365-2958.2009.06638.x) has impacted her thinking and research direction toward investigating the molecular underpinnings of why and how bacteria maintain optimal intracellular Mn levels.

Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae.

Resistance to copper (Cu) toxicity in the respiratory pathogen Streptococcus pneumoniae is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from Staphylococcus aureus, but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the Spn CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of cop promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H2O complex formed by the same residues that coordinate Cu(i), as determined by X-ray absorption spectroscopy and ratiometric pulsed alkylation-mass spectrometry (rPA-MS). Apo- and Zn-bound CopY are homodimers by small angle X-ray scattering (SAXS); however, Zn stabilizes the dimer, narrows the conformational ensemble of the apo-state as revealed by ion mobility-mass spectroscopy (IM-MS), and activates DNA binding in vitro and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster (KCu ≈ 1016 M-1) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. These studies suggest that CopY employs conformational selection to drive Zn-activation of DNA binding, and a novel Cu(i)-mediated assembly mechanism that dissociates CopY from the DNA via ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed.

Characterization and Management of Patients with Heroin versus Nonheroin Opioid Overdoses: Experience at an Academic Medical Center.

STUDY OBJECTIVES: To characterize the differences between patients who had heroin and nonheroin opioid overdoses and to determine whether there were any significant differences in their management with regard to the naloxone use. DESIGN: Retrospective cohort study. SETTING: Large academic medical center. PATIENTS: A total of 923 patients admitted to the medical center who were identified for overdose by heroin or other opiate-related narcotics between January 2010 and September 2015; 480 patients experienced a nonheroin opioid overdose event, and 443 patients experienced a heroin overdose event. MEASUREMENTS AND MAIN RESULTS: Patients presenting with heroin overdose tended to be younger and male, with higher rates of hepatitis C virus (HCV) infection compared with those presenting with nonheroin opioid overdose (p<0.05). Patients in the heroin group were also more likely to have a previous overdose event, history of injection drug use, and history of prescription opioid abuse compared with the nonheroin group (p<0.05). Those presenting with heroin overdose were more likely to receive naloxone in the prehospital setting (p<0.05) but were less likely to receive naloxone once admitted (p<0.05). Patients with nonheroin opioid overdoses required more continuous infusions of naloxone (p<0.05) and admission to the intensive care unit (p<0.05). Of all 923 patients, 178 (19.3%) had a repeat admission for any reason, and 70 (7.6%) were readmitted over the course of the study period for another overdose event with the same drug. The proportion of patients presenting with a heroin overdose steadily increased from 2010-2015; the number of patients presenting to the emergency department with nonheroin opioid overdoses steadily decreased. As rates of heroin overdose increased each year, the incidence of HCV infection increased dramatically. CONCLUSION: This study indicates that the incidence of heroin overdoses has significantly increased over the last several years, and the rates of HCV infection 4-fold since the start of the study period. Patients admitted for nonheroin opioid overdose were more likely to be admitted to the hospital and intensive care unit compared with those admitted for heroin overdose. The rise in overdose events only further illustrates a gap in our understanding of the cycle of addiction, drug abuse, and overdose events.

Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production in Streptococcus pneumoniae D39.

The catalase-negative, facultative anaerobe Streptococcus pneumoniae D39 is naturally resistant to hydrogen peroxide (H2O2) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced H2O2. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that ΔlctO mutants produce significantly lower H2O2. In addition, both the SpxB pathway and a candidate pyruvate dehydrogenase complex (PDHC) pathway contribute to acetyl coenzyme A (acetyl-CoA) production during aerobic growth, and the pyruvate format lyase (PFL) pathway is the major acetyl-CoA pathway during anaerobic growth. Microarray analysis of the D39 strain cultured under aerobic versus strict anaerobic conditions shows upregulation of spxB, a gene encoding a rhodanese-like protein (locus tag spd0091), tpxD, sodA, piuB, piuD, and an Fe-S protein biogenesis operon under H2O2-producing conditions. Proteome profiling of H2O2-induced sulfenylation reveals that sulfenylation levels correlate with cellular H2O2 production, with endogenous sulfenylation of ≈50 proteins. Deletion of tpxD increases cellular sulfenylation 5-fold and has an inhibitory effect on ATP generation. Two major targets of protein sulfenylation are glyceraldehyde-3-phosphate dehydrogenase (GapA) and SpxB itself, but targets also include pyruvate kinase, LctO, AdhE, and acetate kinase (AckA). Sulfenylation of GapA is inhibitory, while the effect on SpxB activity is negligible. Strikingly, four enzymes of capsular polysaccharide biosynthesis are sulfenylated, as are enzymes associated with nucleotide biosynthesis via ribulose-5-phosphate. We propose that LctO/SpxB-generated H2O2 functions as a signaling molecule to downregulate capsule production and drive altered flux through sugar utilization pathways. IMPORTANCE Adaptation to endogenous oxidative stress is an integral aspect of Streptococcus pneumoniae colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H2O2 and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in S. pneumoniae.

Pharmacologic options for reducing the shivering response to therapeutic hypothermia.

Recent literature has demonstrated significant improvements in neurologic outcomes in patients who have received induced hypothermia in the setting of out-of-hospital cardiac arrest. Through multiple metabolic mechanisms, the induction of hypothermia slows the progression and devastation of transient cerebral hypoxia. Despite these benefits, the desired reduction in core temperature is often a challenging venture as the body attempts to maintain homeostasis through the induction of thermoregulatory processes aimed at elevating body temperature. Shivering is an involuntary muscular activity that enhances heat production in an attempt to restore homeostasis. For successful induction and maintenance of induced hypothermia, shivering, as well as other thermoregulatory responses, must be overcome. Several pharmacologic options are available, either used alone or in combination, that safely and effectively prevent or treat shivering after the induction of hypothermia. We conducted a PubMed search (1966-March 2009) to identify all human investigations published in English that discussed pharmacologic mechanisms for the control of shivering. Among these options, clonidine, dexmedetomidine, and meperidine have demonstrated the greatest and most clinically relevant impact on depression of the shivering threshold. More research in this area is needed, however, and the role of the clinical pharmacist in the development and implementation of this therapy needs to be defined.