Zinc Transporter Research Articles

Identification and analysis of bZIP family genes in Citrus sinensis and the role of CsbZIP24 in response to Huanglongbing

Citrus Huanglongbing (HLB) is one of the most destructive citrus diseases worldwide, mainly caused by ‘Candidatus Liberibacter asiaticus’ (CLas). HLB plants commonly present zinc deficiency chlorosis, which is evidenced with decreased zinc content and upregulation of genes encoding zinc transport proteins (ZIPs). However, the mechanism of how ZIPs are activated in response to CLas infection is unknown. Herein, the phylogenetic analysis was done for ZIPs and its potential upstream regulator bZIPs on the genome-wide level. Fifty-six bZIP transcription factor members classified into thirteen groups and twelve ZIP members were identified from C. sinensis. CsbZIP23 and CsbZIP24 harboring Zinc-sensor motif (ZSM) were characterized as the homologues of Arabidopsis thaliana bZIP19/23 in regulation of zinc homeostasis, and both were highly expressed in leaves compared to stems and roots. CsbZIP24 was further demonstrated in activation of CsZIP3 gene expression by binding to its promoter containing Zinc Deficiency Response Elements (ZDRE) rather than CsbZIP23. Additionally, the induction of both CsbZIP24 and CsZIP3 was observed in HLB diseased plants. Taken together, zinc sensor members were firstly identified in citrus and CsbZIP24 was proposed as the key factor in regulation of CsZIP3 gene expression to accommodate zinc-deficient conditions that caused by CLas colonization.

LncRNA cis- and trans-regulation provides new insight into drought stress responses in wild barley.

Drought is one of the most common abiotic stresses that affect barley productivity. Long noncoding RNA (lncRNA) has been reported to be widely involved in abiotic stress, however, its function in the drought stress response in wild barley remains unclear. In this study, RNA sequencing was performed to identify differentially expressed lncRNAs (DElncRNA) among two wild barley and two cultivated barley genotypes. Then, the cis-regulatory networks were according to the chromosome position and the expression level correction. The GO annotation indicates that these cis-target genes are mainly involved in "ion transport transporter activity" and "metal ion transport transporter activity". Through weighted gene co-expression network analysis (WGCNA), 10 drought-related modules were identified to contract trans-regulatory networks. The KEGG annotation demonstrated that these trans-target genes were enriched for photosynthetic physiology, brassinosteroid biosynthesis, and flavonoid metabolism. In addition, we constructed the lncRNA-mediated ceRNA regulatory network by predicting the microRNA response elements (MREs). Furthermore, the expressions of lncRNAs were verified by RT-qPCR. Functional verification of a candidate lncRNA, MSTRG.32128, demonstrated its positive role in drought response and root growth and development regulation. Hormone content analysis provided insights into the regulatory mechanisms of MSTRG.32128 in root development, revealing its involvement in auxin and ethylene signal transduction pathways. These findings advance our understanding of lncRNA-mediated regulatory mechanisms in barley under drought stress. Our results will provide new insights into the functions of lncRNAs in barley responding to drought stress.

Genetic analyses of truncated variant rs200185429 in ZNT8 encoding SLC30A8 gene with respect to prediabetes and type 2 diabetes in Bangladeshi population

Zinc transporter ZnT8, encoded by SLC30A8, is expressed highly in pancreatic β-cells that effluxes Zn2+ into insulin granules which is required to secret insulin from the granules. Genome-wide association study identified twelve loss of function mutations in SLC30A8 that play protective role against type 2 diabetes (T2D). This study aimed to find genetic association of a protein truncating variant rs200185429 in Bangladeshi healthy individuals (n = 184), patients with prediabetes (n = 130) and patients with T2D (n = 179). Genetic association study with respect to rs200185429 was performed using TaqMan® probe followed by allelic discrimination plots. Wild type CC genotype was found to be evenly distributed in healthy individuals (96.2 %), patients with prediabetes (95.38 %) and patients with T2D (94.41 %). CT genotype was more prevalent in T2D (5.59 %), less in healthy individuals (3.38 %). However, TT genotype was absent in the study participants. Mutant T allele was neither associated with prediabetes (OR = 1.22, χ2 = 0.12, p = 0.72) nor with T2D (OR = 1.42, χ2 = 0.52, p = 0.47). Similarly, none of the genetic inheritance models showed statistically significant association with T2D. Thus, a large-scale study is warranted to establish our finding regarding the association of rs200185429 with prediabetes and T2D in Bangladeshi population.

Islet autoantibody frequency in relatives of children with type 1 diabetes who have a type 2 diabetes diagnosis.

This study aimed to evaluate characteristics of autoimmunity in individuals who have a type 2 diagnosis and are relatives of children with type 1 diabetes. Pre-diagnosis samples (median 17 months before onset) from relatives who were later diagnosed with type 2 diabetes were measured for autoantibodies to glutamate decarboxylase 65 (GADA), islet antigen-2 (IA-2A), zinc transporter 8 (ZnT8A) and insulin (IAA) as well as the type 1 diabetes genetic risk score (GRS2). Associations between islet autoantibodies, insulin treatment and GRS2 were analysed using Fisher's exact and t-tests. Among 226 relatives (64% men; mean age at sampling 41 years; mean age 54 years at diagnosis), 32 (14%) were islet autoantibody-positive for at least one autoantibody more than a decade before diagnosis. Approximately half of these (n = 15) were treated with insulin. GADA-positivity was higher in insulin-treated relatives than in non-insulin-treated relatives (12/18 [67%] vs. 6/18 [33%], p < 0.001). IAA-positivity was observed in 13/32 (41%) of relatives with autoantibodies. GRS2 scores were increased in autoantibody-positive relatives (p = 0.032), but there was no clear evidence for a difference according to treatment (p = 0.072). This study highlights the importance of measuring islet autoantibodies, including IAA, in relatives of people with type 1 diabetes to avoid misdiagnosis.

The role of zinc and matrix metalloproteinases in myofibrillar protein degradation in critical illness myopathy

Due to an unexpected activation of different zinc (Zn) transporters in a recent prospective clinical study, we have revisited the role of Zn homeostasis and the activation of matrix metalloproteinases (MMPs) in skeletal muscle exposed to the intensive care unit (ICU) condition (immobilization and mechanical ventilation). ICU patients exposed to 12 days ICU condition were followed longitudinally with six repeated muscle biopsies while they showed a progressive preferential myosin loss, i.e., the hallmark of Critical Illness Myopathy (CIM), in parallel with the activation of Zn-transporters. In this study, we have revisited the expression of Zn-transporters and the activation of MMPs in clinical as well as in experimental studies using an established ICU model. MMPs are a group Zn-dependent endopeptidases which do not only target and cleave extracellular proteins but also intracellular proteins including multiple sarcomeric proteins. MMP-9 is of specific interest since the hallmark of CIM, the preferential myosin loss, has also been reported in dilated cardiomyopathy and coupled to MMP-9 activation. Transcriptional activation of Zn-transporters was observed in both clinical and experimental studies as well as the activation of MMPs, in particular MMP-9, in various limb and respiratory muscles in response to long-term exposure to the ICU condition. The activation of Zn-transporters was paralleled by increased Zn levels in skeletal muscle which in turn showed a negative linear correlation with the preferential myosin loss associated with CIM, offering a potential intervention strategy. Thus, activation of Zn-transporters, increased intramuscular Zn levels, and activation of the Zn-dependent MMPs are forwarded as a probable mechanism involved in CIM pathophysiology. These effects were confirmed in different rat strains subjected to a model of CIM and exacerbated by old age. This is of specific interest since old age and muscle wasting are the two factors most strongly associated with ICU mortality.

Emerging Perspectives in Zinc Transporter Research in Prostate Cancer: An Updated Review.

Dysregulation of zinc and zinc transporters families has been associated with the genesis and progression of prostate cancer. The prostate epithelium utilizes two types of zinc transporters, the ZIP (Zrt-, Irt-related Protein) and the ZnTs (Zinc Transporter), to transport zinc from the blood plasma to the gland lumen. ZIP transporters uptake zinc from extracellular space and organelle lumen, while ZnT transporters release zinc outside the cells or to organelle lumen. In prostate cancer, a commonly observed low zinc concentration in prostate tissue has been correlated with downregulations of certain ZIPs (e.g., ZIP1, ZIP2, ZIP3, ZIP14) and upregulations of specific ZnTs (e.g., ZnT1, ZnT9, ZnT10). These alterations may enable cancer cells to adapt to toxic high zinc levels. While zinc supplementation has been suggested as a potential therapy for this type of cancer, studies have yielded inconsistent results because some trials have indicated that zinc supplementation could exacerbate cancer risk. The reason for this discrepancy remains unclear, but given the high molecular and genetic variability present in prostate tumors, it is plausible that some zinc transporters-comprising 14 ZIP and 10 ZnT members-could be dysregulated in others patterns that promote cancer. From this perspective, this review highlights novel dysregulation, such as ZIP-Up/ZnT-Down, observed in prostate cancer cell lines for ZIP4, ZIP8, ZnT2, ZnT4, ZnT5, etc. Additionally, an in silico analysis of an available microarray from mouse models of prostate cancer (Nkx3.1;Pten) predicts similar dysregulation pattern for ZIP4, ZIP8, and ZnT2, which appear in early stages of prostate cancer progression. Furthermore, similar dysregulation patterns are supported by an in silico analysis of RNA-seq data from human cancer tumors available in cBioPortal. We discuss how these dysregulations of zinc transporters could impact zinc supplementation trials, particularly focusing on how the ZIP-Up/ZnT-Down dysregulation through various mechanisms might promote prostate cancer progression.

General population screening for type 1 diabetes using islet autoantibodies at the preschool vaccination visit: a proof-of-concept study (the T1Early study)

ObjectiveType 1 diabetes (T1D) screening programmes testing islet autoantibodies (IAbs) in childhood can reduce life-threatening diabetic ketoacidosis. General population screening is required to detect the majority of children with T1D,...

Trace metals in the teleost fish gill: biological roles, uptake regulation, and detoxification mechanisms.

In fish, the gill plays a vital role in regulating the absorption of trace metals and is also highly susceptible to metal toxicity. Trace metals such as iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn) are involved in various catalytic activities and molecular binding within the gill, thereby supporting a range of physiological processes in this organ. While beneficial at normal levels, these metals can become toxic when present in excess. Conversely, nonessential metals like cadmium (Cd) and lead (Pb) can gain entry into gill cells through similar metal transport pathways, potentially interfering with various cellular processes. The transepithelial transport of these metals across the gill epithelium is governed by a variety of metal transport and metal binding proteins. These include the Cu transporter 1 (CTR1), divalent metal transporter 1 (DMT1), and members of the Zrt-/Irt-like protein (ZIP) and zinc transport (ZnT) families. Additionally, some of these metals can compete with major ions (e.g., calcium, sodium) for absorption sites in the gill. This complex crosstalk suggests an interdependent mechanism that balances metal uptake to meet physiological needs while preventing excessive accumulation. In this article, I review the roles of trace metals in proteins/enzymes that support the different functions in the gill of teleost fish. I also discuss current understanding of the pathways involved in regulating the branchial uptake of metals and their influence on ionic regulation, and the potential detoxification mechanisms in the gill. Finally, I summarize knowledge gaps and potential areas for further investigation.

Transcriptome-wide m6A methylation profile reveals tissue specific regulatory networks in switchgrass (Panicum virgatum L.) under cadmium stress

The heavy metal cadmium (Cd), known for its high toxicity, poses a grave threat to human health through the food chain. N6-methyladenosine (m6A), the most abundant internal modification, regulates plant adaptation to various adversities, yet the panorama of m6A modifications in switchgrass under cadmium stress remains elusive. This study examines the physiological responses of switchgrass roots and shoots exposed to 50 μM CdCl2, alongside an overview of transcriptome-wide m6A methylation patterns. After cadmium treatment, methylation modifications are primarily enriched near stop codons and the 3′UTR region, with a negative correlation between m6A modification and gene expression levels. In shoots, approximately 58 % of DEGs with m6A modifications show upregulation in expression and decrease in m6A peaks, including zinc transporter 4-like (ZIP4). In roots, about 43 % of DEGs with m6A modifications exhibit downregulation in expression and increase in m6A peaks, such as the ABC transporter family member (ABCG25). We further validate the m6A enrichment, gene expression and mRNA stability of ZIP4 in response to Cd treatment. The results suggest that the negative correlation of m6A enrichment and gene expression is due to altered mRNA stability. Our study establishes an m6A regulatory network governing cadmium transport in switchgrass roots and shoots, offering new avenues for candidate gene manipulation in phytoremediation applications of heavy metal pollution.

Placental Expression of Glucose and Zinc Transporters in Women with Gestational Diabetes

Background/Objectives: Gestational diabetes (GDM) is a metabolic disorder with altered glucose levels diagnosed in pregnant women. The pathogenesis of GDM is not fully known, but it is thought to be caused by impaired insulin production and insulin resistance induced by diabetogenic factors. The placenta may play an important role in the development of GDM. Glucose transporters (GLUTs) are responsible for the delivery of glucose into the foetal circulation. Placental zinc transporters regulate insulin and glucagon secretion, as well as gluconeogenesis and glycolysis. The aim of this study was to investigate the placental expression of GLUT3, GLUT4, GLUT7 and SLC30A8 in women with GDM. Furthermore, we evaluated whether the expression profiles of these transporters were correlated with clinical parameters. Methods: This study included 26 patients with GDM and 28 patients with normal glucose tolerance (NGT). Results: The placental expression of GLUT3 was significantly reduced in the GDM group, while the placental expression of GLUT4, GLUT7 and SLC30A8 was significantly upregulated in the GDM group. GLUT3 expression correlated significantly with body mass index (BMI) increase during pregnancy and body mass increase during pregnancy, while GLUT4 expression correlated negatively with BMI at birth. Conclusions: These results suggest the involvement of GLUT3 and GLUT4, GLUT7 and SLC30A8 in the pathogenesis of GDM.

Digestion and absorption characteristics of iron-chelating silver carp scale collagen peptide and insights into their chelation mechanism

Iron deficiency is widespread throughout the world, supplementing sufficient iron or improving the bioavailability of iron is the fundamental strategy to solve the problem of iron scarcity. Herein, we explored a new form of iron supplement, iron chelates of silver carp scales (SCSCP-Fe) were prepared from collagen peptide of silver carp scales (SCSCP) and FeCl2·4H2O, the effects of external environment and simulated gastrointestinal digestive environment on the stability of SCSCP-Fe and the structural changes of peptide iron chelates during digestion were investigated. The results of in vitro iron absorption promotion showed that the iron bioavailability of SCSCP-Fe was higher than that of FeSO4. Two potential high iron chelating peptides DTSGGYDEY (DY) and LQGSNEIEIR (LR) were screened and synthesized from the SCSCP sequence by molecular dynamics and LC-MS/MS techniques. The FTIR results displayed that the binding sites of DY and LR for Fe2+ were the carboxyl group, the amino group, and the nitrogen atom on the amide group on the peptide. ITC results indicated that the chelation reactions of DY and LR with Fe2+ were mainly dominated by electrostatic interactions, forming chelates in stoichiometric ratios of 1:2 and 1:1, respectively. Both DY and LR had a certain ability to promote iron absorption. The transport of DY-Fe chelate may be a combination of the three pathways: PepT1 vector pathway, cell bypass, and endocytosis, while LR-Fe chelate was dominated by bivalent metal ion transporters. This study is expected to provide theoretical reference and technical support for the high-value utilization of silver carp scales and the development of novel iron supplements.

Marginal Zinc Deficiency Promotes Pancreatic Islet Enlargement While Zinc Supplementation Improves the Pancreatic Insulin Response in Zucker Diabetic Fatty Rats.

Zinc deficiency has been associated with the worsening of diabetes while zinc supplementation has been proposed to ameliorate diabetes. This study examined the effects of marginal zinc deficiency (MZD) and zinc supplementation (ZS) on obesity, glycemic control, pancreatic islets, hepatic steatosis and renal function of Zucker diabetic fatty (ZDF) rats. Male ZDF rats were fed an MZD, zinc control (ZC) or ZS diet (4, 30 and 300 mg Zn/kg diet, respectively), and lean Zucker rats were fed a ZC diet for 8 weeks. MZD and ZS did not alter body weight or whole-body composition in ZDF rats. MZD ZDF rats had reduced zinc concentrations in the femur and pancreas, a greater number of enlarged pancreatic islets and a diminished response to an oral glucose load based on a 1.8-fold greater incremental area-under-the-curve (AUC) for glucose compared to ZC ZDF. ZS ZDF rats had elevated serum, femur and pancreatic zinc concentrations, unchanged pancreatic parameters and a 50% reduction in the AUC for insulin compared to ZC ZDF rats, suggesting greater insulin sensitivity. Dietary zinc intake did not alter hepatic steatosis, creatinine clearance, or levels of proteins that contribute to insulin signaling, inflammation or zinc transport in epididymal fat. Potential adverse effects of ZS were suggested by reduced hepatic copper concentrations and elevated serum urea compared to ZC ZDF rats. In summary, ZS improved the pancreatic insulin response but not the glucose handling. In contrast, reduced zinc status in ZDF rats led to impaired glucose tolerance and a compensatory increase in the number and size of pancreatic islets which could lead to β-cell exhaustion.

Development of a live cell assay for the zinc transporter ZnT8

Zinc is an essential trace element that is involved in many biological processes and in cellular homeostasis. In pancreatic β-cells, zinc is crucial for the synthesis, processing, and secretion of insulin, which plays a key role in glucose homeostasis and which deficiency is the cause of diabetes. The accumulation of zinc in pancreatic cells is regulated by the solute carrier transporter SLC30A8 (or Zinc Transporter 8, ZnT8), which transports zinc from cytoplasm in intracellular vesicles. Allelic variants of SLC30A8 gene have been linked to diabetes. Given the physiological intracellular localization of SLC30A8 in pancreatic β-cells and the ubiquitous endogenous expression of other Zinc transporters in different cell lines that could be used as cellular model for SLC30A8 recombinant over-expression, it is challenging to develop a functional assay to measure SLC30A8 activity. To achieve this goal, we have firstly generated a HEK293 cell line stably overexpressing SLC30A8, where the over-expression favors the partial localization of SLC30A8 on the plasma membrane. Then, we used the combination of this cell model, commercial FluoZin-3 cell permeant zinc dye and live cell imaging approach to follow zinc flux across SLC30A8 over-expressed on plasma membrane, thus developing a novel functional imaging- based assay specific for SLC30A8. Our novel approach can be further explored and optimized, paving the way for future small molecule medium-throughput screening.

The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

The metal ion transporter SLC39A8 is associated with physiological traits and diseases, including blood manganese (Mn) levels and inflammatory bowel diseases (IBD). The mechanisms by which SLC39A8 controls Mn homeostasis and epithelial integrity remain elusive. Here, we generate Slc39a8 intestinal epithelial cell-specific-knockout (Slc39a8-IEC KO) mice, which display markedly decreased Mn levels in blood and most organs. Radiotracer studies reveal impaired intestinal absorption of dietary Mn in Slc39a8-IEC KO mice. SLC39A8 is localized to the apical membrane and mediates 54Mn uptake in intestinal organoid monolayer cultures. Unbiased transcriptomic analysis identifies alkaline ceramidase 1 (ACER1), a key enzyme in sphingolipid metabolism, as a potential therapeutic target for SLC39A8-associated IBDs. Importantly, treatment with an ACER1 inhibitor attenuates colitis in Slc39a8-IEC KO mice by remedying barrier dysfunction. Our results highlight the essential roles of SLC39A8 in intestinal Mn absorption and epithelial integrity and offer a therapeutic target for IBD associated with impaired Mn homeostasis.

Physiological and molecular responses of strawberry plants to Cd stress

Cadmium (Cd), a toxic metal element, can be absorbed by plants via divalent metal ion transporters, thereby retarding plant growth and posing a threat to human health. Strawberries are popular and economically valuable berry species that are sensitive to soil pollutants, especially Cd. However, the mechanisms underlying Cd stress responses in strawberry plants remain largely unclear. Here, we investigated the physiological and molecular basis of Cd stress responses in strawberry plants using the diploid strawberry ‘Yellow Wonder’ as a material. The results indicated that Cd stress induced oxidative damage, repressed photosynthetic efficiency, and interfered with the accumulation and redistribution of trace elements. Furthermore, Cd stress reduced the concentrations of indoleacetic acid, trans-zeatin riboside and gibberellic acid while increasing the concentration of abscisic acid, thus altering the phytohormone signaling pathway in strawberry plants. Cd stress also inhibited the expression of genes involved in nitrogen uptake and assimilation while promoting the energy supply for plant survival under Cd toxicity. Moreover, the flavonoid biosynthesis pathway was induced, and the anthocyanin concentration increased, thereby improving the free radical scavenging capacity of strawberry plants under Cd toxicity. Additionally, we identified several transcription factors and functional genes as hub genes based on a weighted gene coexpression network analysis. These results collectively provide a theoretical foundation for strawberry breeding and ensuring agriculture and food safety.

Trans-synaptic Association of Vesicular Zinc Transporter 3 and Shank3 Supports Synapse-Specific Dendritic Spine Structure and Function in the Mouse Auditory Cortex.

Shank3 is a synaptic scaffolding protein that assists in tethering and organizing structural proteins and glutamatergic receptors in the postsynaptic density of excitatory synapses. The localization of Shank3 at excitatory synapses and the formation of stable Shank3 complexes is regulated by the binding of zinc to the C-terminal sterile-alpha-motif (SAM) domain of Shank3. Mutations in the SAM domain of Shank3 result in altered synaptic function and morphology, and disruption of zinc in synapses that express Shank3 leads to a reduction of postsynaptic proteins important for synaptic structure and function. This suggests that zinc supports the localization of postsynaptic proteins via Shank3. Many regions of the brain are highly enriched with free zinc inside glutamatergic vesicles at presynaptic terminals. At these synapses, zinc transporter 3 (ZnT3) moves zinc into vesicles where it is co-released with glutamate. Alterations in ZnT3 are implicated in multiple neurodevelopmental disorders, and ZnT3 knock-out (KO) mice-which lack synaptic zinc-show behavioral deficits associated with autism spectrum disorder and schizophrenia. Here we show that male and female ZnT3 KO mice have smaller dendritic spines and miniature excitatory postsynaptic current amplitudes than wildtype (WT) mice in the auditory cortex. Additionally, spine size deficits in ZnT3 KO mice are restricted to synapses that express Shank3. In WT mice, synapses that express both Shank3 and ZnT3 have larger spines compared to synapses that express Shank3 but not ZnT3. Together these findings suggest a mechanism whereby presynaptic ZnT3-dependent zinc supports postsynaptic structure and function via Shank3 in a synapse-specific manner.

Intergenerational Impact of Parental Zinc Deficiency on Metabolic and Redox Outcomes in Drosophila melanogaster.

Zinc deficiency is a common nutritional disorder with detrimental health consequences. Whether parental zinc deficiency induces intergenerational effects remains largely unknown. We investigated the effects of a combined maternal and paternal zinc deficiency on offspring's metabolic outcomes and gene expression changes in Drosophila melanogaster. The parent flies were raised on zinc-deficient diets throughout development, and their progeny were assessed. Offspring from zinc-deprived parents exhibited a significant (p < 0.05) increase in body weight and whole-body zinc levels. They also displayed disrupted glucose metabolism, altered lipid homeostasis, and diminished activity of antioxidant enzymes. Gene expression analysis revealed significant (p < 0.05) alterations in zinc transport genes, with increases in mRNA levels of dZIP1 and dZnT1 for female and male offspring, respectively. Both sexes exhibited reduced dZnT35C mRNA levels and significant (p < 0.05) increases in the mRNA levels of DILP2 and proinflammatory markers, Eiger and UPD2. Overall, female offspring showed higher sensitivity to parental zinc deficiency. Our findings underscore zinc's crucial role in maintaining health and the gender-specific responses to zinc deficiency. There is the need for further exploration of the underlying mechanisms behind these intergenerational effects.

Reduced Mn uptake of pleiotropic ZIP8 SNP is caused by its loss of Mn-responsive accumulation on the cell-surface.

Zrt/Irt-like protein 8 (ZIP8), which is a Zn transporter, plays a pivotal role as a Mn transporter. Recent studies have shown that a ZIP8 SNP (rs13107325 C→T, A391T) is associated with multiple diseases, likely by causing systemic Mn deficiency. However, the underlying molecular mechanisms remain unclear. We attempted to address this issue in cell-based experiments using Madin-Darby canine kidney cells stably expressing ZIP8 WT or the A391T SNP mutant under the control of the Tet-regulatable promoter. We showed that the A391T mutant lost the property of Mn-responsive accumulation on the cell surface, which was observed in WT ZIP8. We also showed that the loss of Mn-responsive accumulation of A391T mutant was associated with its reduced Mn uptake, compared with WT ZIP8, in the Mn uptake assay using the radioisotope 54Mn. Our results potentially explain how the ZIP8 A391T substitution is associated with disease pathogenesis caused by Mn deficiency.

FUNCTIONAL METATRANSCRIPTOMICS REVEALS A NOVEL MULTI METAL TOLERANT GENE OF NRAMP-FAMILY, ISOLATED FROM METAL-CONTAMINATED SOIL

The continuous release of heavy metal pollutants into soil through human activities posesthreat to both macro- and micro-organisms within the ecosystem. Functional metatranscriptomics is a robust method for discovering novel genes associated with a particular biological process. In this study, functional meta transcriptomic approach was employed to isolate a gene from size-fractionated cDNA libraries of micro-eukaryotes. The cDNA libraries were raised using high-quality RNA from heavy metal-contaminated soil. The identified gene aligned with Nramp (Natural resistance associated macrophage protein) family. The gene, JMC-10, was submitted to GenBank under Accession No. OR882126, and it encodes a protein with high sequence similarity to other Nramp family members, including Smf1p, Smf2p, and Smf3p in Saccharomyces cerevisiae. To study the function of JMC-10 gene, a series of growth assays were conducted in yeast cells that either overexpressed JMC-10 or carried a deletion of gene. The study revealed the putative role of JMC-10 in the transportation of divalent metal ions, including iron, manganese, zinc and cadmium. Notably, the overexpression of JMC-10 was induced by exposure to toxic levels of cadmium, suggesting it’s potential role in metal detoxification. The study provides new intuitions into the roles of Nramp family in metal homeostasis and highlights the potentiality of functional metatranscriptomics for identifying new genes having crucial roles in complex biological processes. Further, it may serve as a biomarker for identifying metal pollution.

Identification and characterization of zinc importers in Corynebacterium diphtheriae.

Corynebacterium diphtheriae is the causative agent of diphtheria, a severe respiratory disease in humans. C. diphtheriae colonizes the human upper respiratory tract, where it acquires zinc, an essential metal required for survival in the host. While the mechanisms for zinc transport by C. diphtheriae are not well characterized, four putative zinc ABC-type transporter loci were recently identified in strain 1737: iutABCD/E (iut), znuACB (znu), nikABCD1 (nik1), and nikABCD2 (nik2). A mutant deleted for all four loci (Δ4) exhibited similar growth to that of the wild-type strain in a zinc-limited medium, suggesting there are additional zinc transporters. Two additional gene loci predicted to be associated with metal import, mntABCD (mnt) and sidAB (sid), were deleted in the Δ4 mutant to construct a new mutant designated Δ6. The C. diphtheriae Δ6 mutant exhibited significantly reduced growth under zinc limitation relative to the wild type, suggesting a deficiency in zinc acquisition. Strains retaining the iut, znu, mnt, or sid loci grew to near-wild-type levels in the absence of the other five loci, indicating that each of these transporters may be involved in zinc uptake. Plasmid complementation with cloned iut, znu, mnt, or nik1 loci also enhanced the growth of the Δ6 mutant. Quantification of intracellular zinc content by inductively coupled plasma mass spectrometry was consistent with reduced zinc uptake by Δ6 relative to the wild type and further supports a zinc uptake function for the transporters encoded by iut, znu, and mnt. This study demonstrates that C. diphtheriae zinc transport is complex and involves multiple zinc uptake systems.IMPORTANCEZinc is a critical nutrient for all forms of life, including human bacterial pathogens. Thus, the tools that bacteria use to acquire zinc from host sources are crucial for pathogenesis. While potential candidates for zinc importers have been identified in Corynebacterium diphtheriae from gene expression studies, to date, no study has clearly demonstrated this function for any of the putative transporters. We show that C. diphtheriae encodes at least six loci associated with zinc import, underscoring the extent of redundancy for zinc acquisition. Furthermore, we provide evidence that a previously studied manganese-regulated importer can also function in zinc import. This study builds upon our knowledge of bacterial zinc transport mechanisms and identifies potential targets for future diphtheria vaccine candidates.