scholarly journals Mechanism of metal ion-induced activation of a two-component sensor kinase

2019 ◽  
Vol 476 (1) ◽  
pp. 115-135 ◽  
Author(s):  
Trisiani Affandi ◽  
Megan M. McEvoy
Keyword(s):  
Metal Ion ◽  
Ion Homeostasis ◽  
Cytoplasmic Domain ◽  
Metal Resistance ◽  
Phosphoryl Group ◽  
Metal Ion Binding ◽  
Metal Ion Homeostasis ◽  
Two Component

Abstract Two-component systems (TCSs) are essential for bacteria to sense, respond, and adapt to changing environments, such as elevation of Cu(I)/Ag(I) ions in the periplasm. In Escherichia coli, the CusS–CusR TCS up-regulates the cusCFBA genes under increased periplasmic Cu(I)/Ag(I) concentrations to help maintain metal ion homeostasis. The CusS histidine kinase is a homodimeric integral membrane protein that binds to periplasmic Cu(I)/Ag(I) and transduces a signal to its cytoplasmic kinase domain. However, the mechanism of how metal binding in the periplasm activates autophosphorylation in the cytoplasm is unknown. Here, we report that only one of the two metal ion-binding sites in CusS enhances dimerization of the sensor domain. Utilizing nanodisc technology to study full-length CusS, we show that metal-induced dimerization in the sensor domain triggers kinase activity in the cytoplasmic domain. We also investigated autophosphorylation in the cytoplasmic domain of CusS and phosphotransfer between CusS and CusR. In vitro analyses show that CusS autophosphorylates its conserved H271 residue at the N1 position of the histidine imidazole. The phosphoryl group is removed by the response regulator CusR in a reaction that requires a conserved aspartate at position 51. Functional analyses in vivo of CusS and CusR variants with mutations in the autophosphorylation or phosphoacceptor residues suggest that the phosphotransfer event is essential for metal resistance in E. coli. Biochemical analysis shows that the CusS dimer autophosphorylates using a cis mechanism. Our results support a signal transduction model in which rotation and bending movements in the cytoplasmic domain maintain the mode of autophosphorylation.

scholarly journals The First Histidine Triad Motif of PhtD Is Critical for Zinc Homeostasis inStreptococcus pneumoniae

2015 ◽  
Vol 84 (2) ◽  
pp. 407-415 ◽  
Author(s):  
Bart A. Eijkelkamp ◽  
Victoria G. Pederick ◽  
Charles D. Plumptre ◽  
Richard M. Harvey ◽  
Catherine E. Hughes ◽  
...  
Keyword(s):  
Metal Ion ◽  
Ion Homeostasis ◽  
Zinc Homeostasis ◽  
Zinc Binding ◽  
Content Type ◽  
Metal Ion Homeostasis ◽  
Expression Studies ◽  
The Individual

Streptococcus pneumoniaeis the world's foremost human pathogen. Acquisition of the first row transition metal ion zinc is essential for pneumococcal colonization and disease. Zinc is acquired via the ATP-binding cassette transporter AdcCB and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that AdcAII is reliant upon the polyhistidine triad (Pht) proteins to aid in zinc recruitment. Pht proteins generally contain five histidine (His) triad motifs that are believed to facilitate zinc binding and therefore play a significant role in pneumococcal metal ion homeostasis. However, the importance and potential redundancy of these motifs have not been addressed. We examined the effects of mutating each of the five His triad motifs of PhtD. The combination ofin vitrogrowth assays, active zinc uptake, and PhtD expression studies show that the His triad closest to the protein's amino terminus is the most important for zinc acquisition. Intriguingly,in vivocompetitive infection studies investigating the amino- and carboxyl-terminal His triad mutants indicate that the motifs have similar importance in colonization. Collectively, our new insights into the contributions of the individual His triad motifs of PhtD, and by extension the other Pht proteins, highlight the crucial role of the first His triad site in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence.

scholarly journals Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
A. Sukumaran ◽  
S. Pladwig ◽  
J. Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Metal Ion Homeostasis ◽  
Capsule Production ◽  
The Impact

Abstract Background Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. Results Analysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. Conclusions This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

scholarly journals Functional reconstitution of the Salmonella typhimurium PhoQ histidine kinase sensor in proteoliposomes

2005 ◽  
Vol 390 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Sarah Sanowar ◽  
Hervé Le Moual
Keyword(s):  
Salmonella Typhimurium ◽  
Histidine Kinase ◽  
Catalytic Domain ◽  
Response Regulator ◽  
Phosphoryl Group ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
High Concentrations

Two-component signal-transduction systems are widespread in bacteria. They are usually composed of a transmembrane histidine kinase sensor and a cytoplasmic response regulator. The PhoP/PhoQ two-component system of Salmonella typhimurium contributes to virulence by co-ordinating the adaptation to low concentrations of environmental Mg2+. Limiting concentrations of extracellular Mg2+ activate the PhoP/PhoQ phosphorylation cascade modulating the transcription of PhoP-regulated genes. In contrast, high concentrations of extracellular Mg2+ stimulate the dephosphorylation of the response regulator PhoP by the PhoQ kinase sensor. In the present study, we report the purification and functional reconstitution of PhoQHis, a PhoQ variant with a C-terminal His tag, into Escherichia coli liposomes. The functionality of PhoQHis was essentially similar to that of PhoQ as shown in vivo and in vitro. Purified PhoQHis was inserted into liposomes in a unidirectional orientation, with the sensory domain facing the lumen and the catalytic domain facing the extraluminal environment. Reconstituted PhoQHis exhibited all the catalytic activities that have been described for histidine kinase sensors. Reconstituted PhoQHis was capable of autokinase activity when incubated in the presence of Mg2+-ATP. The phosphoryl group could be transferred from reconstituted PhoQHis to PhoP. Reconstituted PhoQHis catalysed the dephosphorylation of phospho-PhoP and this activity was stimulated by the addition of extraluminal ADP.

scholarly journals Autoregulation of theS. mutansSloR metalloregulator is constitutive and driven by an independent promoter

2018 ◽  
Author(s):  
Patrick Monette ◽  
Richard Brach ◽  
Annie Cowan ◽  
Roger Winters ◽  
Jazz Weisman ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Metal Ion ◽  
Bacterial Species ◽  
Ion Homeostasis ◽  
Tooth Decay ◽  
Mobility Shift ◽  
Metal Ion Homeostasis ◽  
Metalloregulatory Protein ◽  
Dental Cavities

AbstractStreptococcus mutans, one of ∼600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries,S. mutansharbors a 25kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives, in part, from transcriptional readthrough of thesloABCoperon which encodes a Mn2+/Fe2+ABC transport system. Herein, we describe the details of thesloABCpromoter that drives this transcription, as well as a novel independent promoter in an intergenic region (IGR) that contributes to downstreamsloRexpression. RT-PCR studies supportsloRtranscription that is independent ofsloABCexpression, and the results of 5′ RACE revealed asloRtranscription start site in the IGR from which the −10 and −35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with lower affinity than SloR binding to thesloABCRpromoter. Function of thesloRpromoter was validated in qRT-PCR experiments. Interestingly,sloRexpression was not significantly impacted when grown in the presence of high manganese, whereas expression of thesloABCoperon was repressed under these conditions. The results ofin vitrotranscription studies support SloR-mediated transcriptional-activation ofsloRand -repression ofsloABC.Taken together, these findings implicate SloR as a bifunctional regulator that repressessloABCpromoter activity and encouragessloRtranscription from an independent promoter.ImportanceTooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide.S. mutans-induced carious lesions cause functional, physical, and/or aesthetic impairment in the vast majority of adults, and in 60-90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviatingS. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health which is inextricably linked to oral health. Research focused on theS. mutansSloR metalloregulatory protein can guide the development of novel therapeutics and so alleviate the burden of dental cavities.

scholarly journals Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

2021 ◽  
Author(s):  
Arjun Sukumaran ◽  
Samanta Pladwig ◽  
Jennifer Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Metal Ion Homeostasis ◽  
Capsule Production ◽  
The Impact

Abstract Background: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. Results: Analysis of intra- and extracellular changes identified 2,380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. Conclusions: This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

Exploiting Cancer Metal Metabolism using Anti-Cancer Metal- Binding Agents

2019 ◽  
Vol 26 (2) ◽  
pp. 302-322 ◽  
Author(s):  
Angelica M. Merlot ◽  
Danuta S. Kalinowski ◽  
Zaklina Kovacevic ◽  
Patric J. Jansson ◽  
Sumit Sahni ◽  
...  
Keyword(s):  
Metal Ion ◽  
Antitumor Agents ◽  
Metal Ion Binding ◽  
Toxicological Assessment ◽  
Metal Metabolism ◽  
Metal Levels ◽  
Anti Cancer ◽  
Binding Agents

Metals are vital cellular elements necessary for multiple indispensable biological processes of living organisms, including energy transduction and cell proliferation. Interestingly, alterations in metal levels and also changes in the expression of proteins involved in metal metabolism have been demonstrated in a variety of cancers. Considering this and the important role of metals for cell growth, the development of drugs that sequester metals has become an attractive target for the development of novel anti-cancer agents. Interest in this field has surged with the design and development of new generations of chelators of the thiosemicarbazone class. These ligands have shown potent anticancer and anti-metastatic activity in vitro and in vivo. Due to their efficacy and safe toxicological assessment, some of these agents have recently entered multi-center clinical trials as therapeutics for advanced and resistant tumors. This review highlights the role and changes in homeostasis of metals in cancer and emphasizes the pre-clinical development and clinical assessment of metal ion-binding agents, namely, thiosemicarbazones, as antitumor agents.

Structural analysis and insight into metal-ion activation of the iron-dependent regulator fromThermoplasma acidophilum

2014 ◽  
Vol 70 (5) ◽  
pp. 1281-1288 ◽  
Author(s):  
Hyun Ku Yeo ◽  
Young Woo Park ◽  
Jae Young Lee
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Binding Property ◽  
Metal Ion Binding ◽  
Mobility Shift ◽  
Iron Cluster ◽  
Metal Binding Site ◽  
Metal Ion Homeostasis

The iron-dependent regulator (IdeR) is a metal ion-activated transcriptional repressor that regulates the expression of genes encoding proteins involved in iron uptake to maintain metal-ion homeostasis. IdeR is a functional homologue of the diphtheria toxin repressor (DtxR), and both belong to the DtxR/MntR family of metalloregulators. The structure of Fe2+-bound IdeR (TA0872) fromThemoplasma acidophilumwas determined at 2.1 Å resolution by X-ray crystallography using single-wavelength anomalous diffraction. The presence of Fe2+, which is the true biological activator of IdeR, in the metal-binding site was ascertained by the use of anomalous difference electron-density maps using diffraction data collected at the Fe absorption edge. Each DtxR/IdeR subunit contains two metal ion-binding sites separated by 9 Å, labelled the primary and ancillary sites, whereas the crystal structures of IdeR fromT. acidophilumshow a binuclear iron cluster separated by 3.2 Å, which is novel toT. acidophilumIdeR. The metal-binding site analogous to the primary site in DtxR was unoccupied, and the ancillary site was occupied by binuclear clustered ions. This difference suggests thatT. acidophilumIdeR and its closely related homologues are regulated by a mechanism distinct from that of either DtxR or MntR.T. acidophilumIdeR was also shown to have a metal-dependent DNA-binding property by electrophoretic mobility shift assay.

scholarly journals An ArsR Transcriptional Regulator Facilitates Brucella sp. Survival via Regulating Self and Outer Membrane Protein

2021 ◽  
Vol 22 (19) ◽  
pp. 10860
Author(s):  
Feijie Zhi ◽  
Dong Zhou ◽  
Jialu Chen ◽  
Jiaoyang Fang ◽  
Weifang Zheng ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Metal Ion ◽  
Target Genes ◽  
Ion Homeostasis ◽  
Infection Model ◽  
Bacterial Survival ◽  
Metal Ion Homeostasis

The arsenic acid-resistant (ArsR) family transcriptional regulators are widely distributed in microorganisms, including in the facultative intracellular pathogen Brucella spp. ArsR proteins are implicated in numerous biological processes. However, the specific roles of ArsR family members in Brucella remain obscure. Here, we show that ArsR6 (BSS2_RS07325) is required for Brucella survival both under heat, oxidative, and osmotic stress and in a murine infection model in vivo. RNA-seq and ChIP-seq reveal that 34 potential target genes for ArsR6 protein were identified, among which eight genes were up-regulated and 26 genes were down-regulated, including outer membrane protein 25D (Omp25D). ArsR6 autoregulates its own expression to maintain bacterial intracellular Cu/Ni homeostasis to benefit bacterial survival in hostile environments. Moreover, ArsR6 also regulates the production of virulence factor Omp25D, which is important for the survival of Brucella under stress conditions. Significantly, Omp25D deletion strain attenuated in a murine infection model in vivo. Altogether, our findings reveal a unique mechanism in which the ArsR family member ArsR6 autoregulates its expression and also modulates Omp25D expression to maintain metal ion homeostasis and virulence in Brucella.

scholarly journals 1455. Potential Mechanisms of Cefiderocol MIC Increase in Enterobacterales in In Vitro Resistance Acquisition Studies

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S730-S730
Author(s):  
Yoshinori Yamano ◽  
Rio Nakamura ◽  
Miki Takemura ◽  
Roger Echols
Keyword(s):  
Iron Transport ◽  
Resistance Mechanisms ◽  
Altered Expression ◽  
Carbapenem Resistant ◽  
Mueller Hinton ◽  
Two Component ◽  
Mueller Hinton Agar ◽  
Related Proteins

Abstract Background Cefiderocol (CFDC) is a novel siderophore, iron-chelating cephalosporin, which is transported into bacteria via iron transporters. CFDC has potent in vitro and in vivo activity against all aerobic Gram-negative bacteria, including carbapenem-resistant strains. To date, clinical isolates with cefiderocol MIC >4 µg/mL have been found infrequently, in which the presence of a few β-lactamases or altered iron transport was found. We investigated potential new mechanisms causing CFDC MIC increases in non-clinical studies. Methods The mutation positions were determined by whole genome sequencing using four K. pneumoniae mutants including two KPC producers and one NDM producer that had shown CFDC MIC increases in previous in vitro resistance-acquisition studies. The mutant strains were obtained at the frequency of 10-7 to < 10-8 by spreading bacteria on standard Mueller‒Hinton agar medium containing CFDC at concentrations of 10× MIC, with or without apo-transferrin (20 μg/mL). CFDC MIC was determined by broth microdilution using iron-depleted cation-adjusted Mueller-Hinton broth based on Clinical and Laboratory Standards Institute guidelines. The emergence of MIC increase mutants was also assessed by in vitro chemostat models under humanized plasma pharmacokinetic exposures of CFDC. Results The possible resistance mechanisms were investigated. Mutation of baeS or envZ, sensors of two-component regulation systems, were found in three or two mutants among the tested four isolates, respectively, and caused the MIC to increase by 4–32-fold. The altered expression level of specific genes by the baeS or envZ mutation could affect CFDC susceptibility, but the specific genes have not been identified. In addition, the mutation of exbD, an accessory protein related to iron transport, was identified in one case and caused the MIC to increase by >8-fold. In vitro chemostat studies using two isolates (one NDM producer and one KPC producer) showed no resistance acquisition during 24-hour exposure. Table. Overview of mutation emergence in five isolates of K. pneumoniae Conclusion The mutation of two-component regulation systems (BaeSR and OmpR/EnvZ) and iron transport-related proteins were shown to be possible mechanisms causing CFDC MIC increases, but these mutants did not appear under human exposures. Disclosures Yoshinori Yamano, PhD, Shionogi & Co., Ltd. (Employee) Rio Nakamura, BSc, Shionogi & Co., Ltd. (Employee) Miki Takemura, MSc, Shionogi & Co., Ltd. (Employee) Roger Echols, MD, Shionogi Inc. (Consultant)

scholarly journals Metallothionein: A Multifunctional Protein from Toxicity to Cancer

2003 ◽  
Vol 18 (3) ◽  
pp. 162-169 ◽  
Author(s):  
S.E. Theocharis ◽  
A.P. Margeli ◽  
A. Koutselinis
Keyword(s):  
Cell Proliferation ◽  
Metal Ion ◽  
Defense Mechanisms ◽  
Ion Homeostasis ◽  
Low Molecular Weight ◽  
Multifunctional Protein ◽  
Metal Ion Homeostasis ◽  
Potential Marker ◽  
Proliferation And Apoptosis ◽  
Carcinogenic Process

The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed.

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