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 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.

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

2019 ◽  
Author(s):  
Arjun Sukumaran ◽  
Jennifer Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Environmental Conditions ◽  
Metal Ion ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Growth And Survival ◽  
Expression Of Genes ◽  
Capsule Production ◽  
The Impact

Abstract Background: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metals ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein regulation and secretion patterns in adaptation to changing environmental conditions. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections and by using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc) modulates the cellular proteome and secretome of the bacteria. This information will inform on protein-level regulation of bacterial responses to nutritional immunity within the host and improve our understanding of the dynamic and complex relationship between host and pathogen during infection. 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 significantl increases abundance under replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validation qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, chaB that was significantly abundant under zinc-replete conditions. Phenotypic analysis of a chaBdeletion strain observe a reduction in capsule production, greater tolerance to high extracellular zinc concentrations, and unimpaired virulence when compared to the WT 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 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 Targeted Mutagenesis of the Rice FW 2.2-Like Gene Family Using the CRISPR/Cas9 System Reveals OsFWL4 as a Regulator of Tiller Number and Plant Yield in Rice

2020 ◽  
Vol 21 (3) ◽  
pp. 809
Author(s):  
Qingsong Gao ◽  
Gang Li ◽  
Hui Sun ◽  
Ming Xu ◽  
Huanhuan Wang ◽  
...  
Keyword(s):  
Metal Ion ◽  
Ion Homeostasis ◽  
Cell Number ◽  
Tiller Number ◽  
Targeted Mutagenesis ◽  
Negative Regulator ◽  
Wild Type ◽  
Phenotypic Analysis ◽  
Plant Yield ◽  
Metal Ion Homeostasis

The FW2.2-like (FWL) genes encode cysteine-rich proteins with a placenta-specific 8 domain. They play roles in cell division and organ size control, response to rhizobium infection, and metal ion homeostasis in plants. Here, we target eight rice FWL genes using the CRISPR/Cas9 system delivered by Agrobacterium-mediated transformation. We successfully generate transgenic T0 lines for 15 of the 16 targets. The targeted mutations are detected in the T0 lines of all 15 targets and the average mutation rate is found to be 81.6%. Transfer DNA (T-DNA) truncation is a major reason for the failure of mutagenesis in T0 plants. T-DNA segregation analysis reveals that the T-DNA inserts in transgenic plants can be easily eliminated in the T1 generation. Of the 30 putative off-target sites examined, unintended mutations are detected in 13 sites. Phenotypic analysis reveals that tiller number and plant yield of OsFWL4 gene mutants are significantly greater than those of the wild type. Flag leaves of OsFWL4 gene mutants are wider than those of the wild type. The increase in leaf width of the mutants is caused by an increase in cell number. Additionally, grain length of OsFWL1 gene mutants is higher than that of the wild type. Our results suggest that transgene-free rice plants with targeted mutations can be produced in the T1 generation using the Agrobacterium-mediated CRISPR/Cas9 system and that the OsFWL4 gene is a negative regulator of tiller number and plant yield.

scholarly journals The molecular basis of Acinetobacter baumannii cadmium toxicity and resistance

2020 ◽  
Author(s):  
Saleh F. Alquethamy ◽  
Felise G. Adams ◽  
Ram Maharjan ◽  
Natasha N. Delgado ◽  
Maoge Zang ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Acinetobacter Baumannii ◽  
Metal Ion ◽  
Cadmium Toxicity ◽  
Ion Homeostasis ◽  
Cadmium Stress ◽  
Primary Role ◽  
Metal Ion Homeostasis ◽  
Primary Means ◽  
The Impact

ABSTRACTAcinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, soil and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Although metal ion homeostasis in the hospital pathogen Acinetobacter baumannii is known to contribute to pathogenesis, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. Further, we show that the CzcCBA heavy metal efflux system also contributes to cadmium efflux. Analysis of the A. baumannii metallome under cadmium stress showed zinc depletion and copper enrichment, which are likely to influence cellular fitness. Overall, this work expands our understanding of the role of membrane transporters in A. baumannii metal ion homeostasis.IMPORTANCECadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, which includes Acinetobacter baumannii. Here we show that A. baumannii utilises a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux, to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be the primary means by which cadmium exerts toxicity upon the bacterium.

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 molecular basis of Acinetobacter baumannii cadmium toxicity and resistance

2021 ◽  
Author(s):  
Saleh F. Alquethamy ◽  
Felise G. Adams ◽  
Ram Maharjan ◽  
Natasha N. Delgado ◽  
Maoge Zang ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Acinetobacter Baumannii ◽  
Metal Ion ◽  
Cadmium Toxicity ◽  
Ion Homeostasis ◽  
Cadmium Stress ◽  
Primary Role ◽  
Metal Ion Homeostasis ◽  
Primary Means ◽  
The Impact

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis, however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii . Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCE Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii , which is notorious for persisting in harsh environments. Here we show that A. baumannii utilises a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be a primary means by which cadmium exerts toxicity upon the bacterium.

scholarly journals SP1433-1438 operon of Streptococcus pneumoniae regulates metal homeostasis and cellular metabolism during zinc-stress

2018 ◽  
Author(s):  
Lindsey R. Burcham ◽  
Rebecca A. Hill ◽  
Rachel C. Caulkins ◽  
Joseph P. Emerson ◽  
Bindu Nanduri ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Proteomic Analysis ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Acute Otitis Media ◽  
Cellular Metabolism ◽  
Energy Coupling ◽  
Transport Systems ◽  
Pcr Analysis ◽  
Metal Ion Homeostasis

AbstractStreptococcus pneumoniae colonizes the mucosa of the human nasopharynx and is a leading cause of community-acquired pneumonia, acute otitis media, and bacterial meningitis. Metal ion homeostasis is vital to the survival of this pathogen and contributes significantly to both colonization and invasive disease. Microarray and qRT-PCR analysis revealed an upregulation of an uncharacterized operon (SP1433-1438) in pneumococci subjected to metal-chelation by N,N,N’,N’-tetrakis-(2-Pyridylmethyl)ethylenediamine (TPEN). Supplementation of either zinc or cobalt following TPEN treatment drastically abrogated induction. BLAST analysis predicted this operon to encode two ABC-transporters, sharing homology to a multidrug resistance system (SP1434-1435) and an energy-coupling factor (ECF) transport system (SP1436-1438). Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated changes in intracellular concentrations of iron, zinc, and manganese ions in a Δ1434-8 strain compared to parental T4R. Analysis of the secreted metabolomic profile of the T4R and Δ1434-8 strains identified significant changes in pneumococcal glycolytic pathways, indicating a shift towards increased production of acetate. Additionally, proteomic analysis revealed 41 differentially expressed proteins in the Δ1434-8 strain, with roughly 20% of them regulated by the global catabolite repressor, CcpA. Based on these findings, we propose that the SP1433-1438 operon is largely involved in the central metabolism of S. pneumoniae during zinc-limitation.ImportanceMetal sequestration is a common strategy utilized by the host immune response as well as antibiotics such as vancomycin to kill invading bacterial pathogens (1). However, pneumococcus is still able to thrive under zinc-limiting conditions. This study describes a previously uncharacterized operon encoding two ABC transport systems that are strongly induced during zinc-limiting conditions. This operon was found to be regulated by a zinc-dependent regulator (SP1433) that functions independently of the overarching AdcR regulon. We have additionally utilized a 2D-NMR approach to analyze the secreted metabolome and have employed proteomic analysis to identify a role for these systems in the maintenance of cellular metabolism. This study provides new information on how Streptococcus pneumoniae responds and adapts to zinc-limiting conditions.

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 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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