scholarly journals The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii

2022 ◽  
Author(s):  
Dana Aghabi ◽  
Megan Sloan ◽  
Zhicheng Dou ◽  
Olga Antipova ◽  
Alfredo Guerra ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Oxygen Radicals ◽  
Critical Role ◽  
Growth Defect ◽  
Iron Storage ◽  
Iron Transporter ◽  
Expression Of Genes ◽  
Veterinary Importance ◽  
Slight Growth

Abstract Iron is essential to living cells, acting as a cofactor in a number of important enzymes in metabolism; however in the absence of correct storage iron forms dangerous oxygen radicals. In both yeast and plants, iron is stored in a membrane-bound vacuole through the action of a vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii, a pathogen of medical and veterinary importance. Here, we assess the role of VIT, and iron storage, in T. gondii. We show that iron is restricted to a compartment in the parasite that does not overlap with zinc. By deleting VIT we find a slight growth defect in vitro, however the absence of VIT leads to hypersensitivity to iron, confirming its essential role in iron detoxification in the parasite. This hypersensitivity can be rescued by scavenging of oxygen radicals. In the absence of VIT, parasites store less iron and are at a growth disadvantage when moving into an iron-depleted environment. We show parasite VIT expression is regulated by iron levels at both the transcript and protein level, and by altering the distribution of VIT within the cell. In the absence of VIT, we find that T. gondii responds by altering expression of genes with a role in iron metabolism and by increasing the activity of the antioxidant protein catalase. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

scholarly journals The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii

2021 ◽  
Author(s):  
Dana Aghabi ◽  
Megan Sloan ◽  
Zhicheng Dou ◽  
Alfredo J. Guerra ◽  
Clare R. Harding
Keyword(s):  
Toxoplasma Gondii ◽  
Critical Role ◽  
Growth Defect ◽  
Iron Storage ◽  
Excess Iron ◽  
Iron Transporter ◽  
Expression Of Genes ◽  
Veterinary Importance ◽  
Slight Growth

AbstractIron is essential to living cells, acting as a cofactor in a number of essential enzymes in metabolism; however, iron requires proper storage or it can be dangerous to the cell. In both yeast and plants, iron is stored in a vacuole through the action of a vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii, a pathogen of medical and veterinary importance. Here, we assess the role of VIT in T. gondii. We show that deletion of VIT causes a slight growth defect in vitro, however leads to hypersensitivity in the presence of excess iron, confirming its essential role in iron detoxification in the parasite. In the absence of VIT, parasites contain less iron and are at a growth disadvantage when moving into an iron-depleted environment. We show parasite VIT expression is regulated by environmental iron levels at both the transcript and protein level, and by altering the distribution of VIT within the cell. In the absence of VIT, we find that the T. gondii responds by altering expression of genes with a role in iron metabolism and by increasing the activity of the antioxidant protein catalase. We also show that iron detoxification has an important role both in parasite survival within macrophages and in pathogenesis in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

scholarly journals Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

2020 ◽  
Author(s):  
Joshua A. Mayoral ◽  
Tadakimi Tomita ◽  
Vincent Tu ◽  
Jennifer T. Aguilan ◽  
Simone Sidoli ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Critical Role ◽  
Cell Types ◽  
Effector Proteins ◽  
Secreted Proteins ◽  
Growth Defect ◽  
Label Free

ABSTRACTToxoplasma gondii is a highly successful parasite that infects a significant portion of the human population. As an intracellular parasite, T. gondii thrives within many different cell types due to its residence in the parasitophorous vacuole, a specialized and heavily modified compartment in which parasites divide. Within this vacuole, numerous secreted proteins facilitate functions that optimize intracellular survival. We characterized one such protein, TgPPM3C, which is predicted to contain a domain belonging to the PP2C class of serine/threonine phosphatases and is secreted by both tachyzoites and differentiating bradyzoites into the vacuolar lumen. Genetic deletion of TgPPM3C established that parasites lacking this predicted phosphatase exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. A label-free phosphoproteomic approach was utilized to identify putative TgPPM3C substrates and demonstrated several secreted proteins with altered phosphorylation status in the absence of TgPPM3C. Altered phosphorylation status was seen in MYR1, a protein essential to the process of protein effector export from the parasitophorous vacuole into the host cell, and in GRA16 and GRA28, two exported effector proteins. Defects were seen in the export of GRA16 and GRA28, but not the effector TgIST, in the TgPPM3C knockout strain. Parasites lacking TgPPM3C also exhibited defects in host c-Myc induction, a process influenced by effector export. Phosphomimetic mutations of GRA16 serine residues recapitulated export defects, implicating de-phosphorylation as an important process in facilitating the export of GRA16. These findings provide an example of the emerging critical role that phosphatases play in regulating the complex environment of the T. gondii parasitophorous vacuole.

scholarly journals Energy metabolism adaptations and gene expression reprogramming in a cellular MAFLD model

2021 ◽  
Author(s):  
Tian-Ran Zhou ◽  
Cagla Cömert ◽  
Xiaoyu Zhou ◽  
Lin Lin ◽  
Lars Bolund ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Energy Metabolism ◽  
Cell Model ◽  
Critical Role ◽  
Huh7 Cell ◽  
Mitochondrial Superoxide ◽  
Expression Of Genes ◽  
Bioenergetic Analysis ◽  
Vitro Model

Mitochondrial dysfunction plays a critical role in metabolic associated fatty liver disease (MAFLD). This study aims to characterize mitochondrial dysfunctions in a human MAFLD Huh7 cell model triggered by free fatty acid (FFA) (palmitate and oleate) overload for 24 hours. We investigate its impact on cellular energy metabolism and identify potential targets for MAFLD treatment. FFA-treated cells displayed an accumulation of lipid droplets and slightly decreased viability but no significant changes in mitochondrial superoxide levels. Bioenergetic analysis showed a shift to more respiration and less glycolytic fermentation. Comprehensive transcriptomics and proteomics analyses identified changes in the expression of genes prominently involved in fatty acid handling and metabolism. The expressions of seven genes were consistently and significantly (p<0.05) altered (4 upregulated and 3 downregulated genes) in both proteomics and transcriptomics. The FFA-treated Huh7 cell model is an appropriate in vitro model to study fatty acid metabolism and suitable to investigate the role of mitochondria, glycolysis, and multiple metabolic pathways in MAFLD. Our comprehensive analyses form a basis for drug discovery and screening using this model.

scholarly journals Microneme Rhomboid Protease TgROM1 Is Required for Efficient Intracellular Growth of Toxoplasma gondii

2008 ◽  
Vol 7 (4) ◽  
pp. 664-674 ◽  
Author(s):  
Fabien Brossier ◽  
G. Lucas Starnes ◽  
Wandy L. Beatty ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Cell Invasion ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Host Cells ◽  
Wild Type ◽  
Intracellular Growth ◽  
Adhesive Proteins

ABSTRACT Rhomboids are serine proteases that cleave their substrates within the transmembrane domain. Toxoplasma gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments. Toxoplasma rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in Plasmodium falciparum processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of T. gondii. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of TgROM1 using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type T. gondii. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by T. gondii.

scholarly journals Inactivation of NMB0419, Encoding a Sel1-Like Repeat (SLR) Protein, in Neisseria meningitidis Is Associated with Differential Expression of Genes Belonging to the Fur Regulon and Reduced Intraepithelial Replication

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Ming-Shi Li ◽  
Paul R. Langford ◽  
J. Simon Kroll
Keyword(s):  
Epithelial Cells ◽  
Neisseria Meningitidis ◽  
Iron Acquisition ◽  
Regulatory Function ◽  
Growth Defect ◽  
Wild Type ◽  
Content Type ◽  
Expression Of Genes ◽  
New Perspective

ABSTRACT Neisseria meningitidis is a commensal microbe that colonizes the human nasopharynx but occasionally invades the bloodstream to cause life-threatening infection. N. meningitidis MC58 NMB0419 encodes a Sel1-like repeat (SLR)-containing protein, previously implicated in invasion of epithelial cells. A gene-regulatory function was revealed in Escherichia coli expressing plasmid-borne NMB0419 and showing significantly increased epithelial adherence compared to the wild type, due to increased expression of mannose-sensitive type 1 pili. While a meningococcal NMB0419 mutant did not have altered epithelial adherence, in a transcriptome-wide comparison of the wild type and an NMB0419 mutant, a large proportion of genes differentially regulated in the mutant were involved in iron acquisition and metabolism. Fifty-one percent and 38% of genes, respectively, up- and downregulated in the NMB0419 mutant had previously been identified as being induced and repressed by meningococcal Fur. An in vitro growth defect of the NMB0419 mutant under iron restriction was consistent with the downregulation of tbpAB and hmbR, while an intraepithelial replication defect was consistent with the downregulation of tonB, exbB, and exbD, based on a known phenotype of a meningococcal tonB mutant. Disruption of the N-terminal NMB0419 signal peptide, predicted to export the protein beyond the cytoplasmic membrane, resulted in loss of functional traits in N. meningitidis and E. coli. Our study indicates that the expression of NMB0419 is associated with transcriptional changes counterbalancing the regulatory function of Fur, offering a new perspective on regulatory mechanisms involved in meningococcal interaction with epithelial cells, and suggests new insights into the roles of SLR-containing genes in other bacteria.

scholarly journals Critical role of miR-10b in BRafV600E dependent anchorage-independent growth and invasion of melanoma cells

2018 ◽  
Author(s):  
Ila Datar ◽  
Gardiyawasam Kalpana ◽  
Ivana de la Serna ◽  
Robert Trumbly ◽  
Jungmin Choi ◽  
...  
Keyword(s):  
Cell Invasion ◽  
High Throughput Sequencing ◽  
Genetic Locus ◽  
Critical Role ◽  
Melanoma Cells ◽  
Growth And Survival ◽  
Anchorage Independent Growth ◽  
Expression Of Genes ◽  
Kinase Cascade

AbstractRecent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the BRaf genetic locus as one of the critical events in melanomagenesis. BRaf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of BRaf is tightly regulated and is required for cell growth and survival. BRaf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of BRaf mutated melanoma cells are stringently dependent on BRaf-MEK-ERK activation, the downstream effector targets that are required for oncogenic BRaf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic BRaf (BRafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of BRaf wild-type melanoma cells, miR-10b silencing decreases BRafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for BRafV600E-mediated anchorage-independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic BRafV600E activity in melanoma.

scholarly journals Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae

2015 ◽  
Vol 84 (2) ◽  
pp. 511-523 ◽  
Author(s):  
Eric D. Peng ◽  
Elizabeth E. Wyckoff ◽  
Alexandra R. Mey ◽  
Carolyn R. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Iron Acquisition ◽  
Growth Conditions ◽  
Growth Defect ◽  
Null Mutant ◽  
Content Type ◽  
Infant Mouse ◽  
Iron Transporter

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number ofV. choleraeiron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter inV. cholerae. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron sourcein vitro, we demonstrate that heme is not available toV. choleraein the infant mouse intestine.

scholarly journals Krüppel-like factor 6–mediated loss of BCAA catabolism contributes to kidney injury in mice and humans

2021 ◽  
Vol 118 (23) ◽  
pp. e2024414118
Author(s):  
Sian E. Piret ◽  
Yiqing Guo ◽  
Ahmed A. Attallah ◽  
Sylvia J. Horne ◽  
Amy Zollman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kidney Function ◽  
Critical Role ◽  
Kidney Injury ◽  
Sequencing Analysis ◽  
Kidney Fibrosis ◽  
Atp Production ◽  
Expression Of Genes ◽  
Genes Encoding

Altered cellular metabolism in kidney proximal tubule (PT) cells plays a critical role in acute kidney injury (AKI). The transcription factor Krüppel-like factor 6 (KLF6) is rapidly and robustly induced early in the PT after AKI. We found that PT-specific Klf6 knockdown (Klf6PTKD) is protective against AKI and kidney fibrosis in mice. Combined RNA and chromatin immunoprecipitation sequencing analysis demonstrated that expression of genes encoding branched-chain amino acid (BCAA) catabolic enzymes was preserved in Klf6PTKD mice, with KLF6 occupying the promoter region of these genes. Conversely, inducible KLF6 overexpression suppressed expression of BCAA genes and exacerbated kidney injury and fibrosis in mice. In vitro, injured cells overexpressing KLF6 had similar decreases in BCAA catabolic gene expression and were less able to utilize BCAA. Furthermore, knockdown of BCKDHB, which encodes one subunit of the rate-limiting enzyme in BCAA catabolism, resulted in reduced ATP production, while treatment with BCAA catabolism enhancer BT2 increased metabolism. Analysis of kidney function, KLF6, and BCAA gene expression in human chronic kidney disease patients showed significant inverse correlations between KLF6 and both kidney function and BCAA expression. Thus, targeting KLF6-mediated suppression of BCAA catabolism may serve as a key therapeutic target in AKI and kidney fibrosis.

scholarly journals Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

2020 ◽  
Vol 16 (12) ◽  
pp. e1008771
Author(s):  
Joshua Mayoral ◽  
Tadakimi Tomita ◽  
Vincent Tu ◽  
Jennifer T. Aguilan ◽  
Simone Sidoli ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Cell Types ◽  
Effector Proteins ◽  
Growth Defect ◽  
Phosphatase Domain ◽  
A Minor

The intracellular parasite Toxoplasma gondii infects a large proportion of humans worldwide and can cause adverse complications in the settings of immune-compromise and pregnancy. T. gondii thrives within many different cell types due in part to its residence within a specialized and heavily modified compartment in which the parasite divides, termed the parasitophorous vacuole. Within this vacuole, numerous proteins optimize intracellular survival following their secretion by the parasite. We investigated the contribution of one of these proteins, TgPPM3C, predicted to contain a PP2C-class serine/threonine phosphatase domain and previously shown to interact with the protein MYR1, an essential component of a putative vacuolar translocon that mediates effector export into the host cell. Parasites lacking the TgPPM3C gene exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. Phosphoproteomic assessment of TgPPM3C deleted parasite cultures demonstrated alterations in the phosphorylation status of many secreted vacuolar proteins including two exported effector proteins, GRA16 and GRA28, as well as MYR1. Parasites lacking TgPPM3C are defective in GRA16 and GRA28 export, but not in the export of other MYR1-dependant effectors. Phosphomimetic mutation of two GRA16 serine residues results in export defects, suggesting that de-phosphorylation is a critical step in the process of GRA16 export. These findings provide another example of the emerging role of phosphatases in regulating the complex environment of the T. gondii parasitophorous vacuole and influencing the export of specific effector proteins from the vacuolar lumen into the host cell.

scholarly journals CcpA Is Important for Growth and Virulence of Enterococcus faecium

2014 ◽  
Vol 82 (9) ◽  
pp. 3580-3587 ◽  
Author(s):  
Sudha R. Somarajan ◽  
Jung H. Roh ◽  
Kavindra V. Singh ◽  
George M. Weinstock ◽  
Barbara E. Murray
Keyword(s):  
Enterococcus Faecium ◽  
Deletion Mutant ◽  
Growth Delay ◽  
Growth Defect ◽  
Single Mutation ◽  
Competitive Growth ◽  
Content Type ◽  
Mixed Inoculum ◽  
Slight Growth

ABSTRACTThe collagen adhesin Acm was the first virulence determinant reported to be important for the pathogenesis ofEnterococcus faeciumin a rat infective endocarditis model. We had previously reported that there was a slight growth delay associated withacmallelic replacement (cat) mutant strain TX6051 used in that study. Recently, we generated a nonpolar markerlessacmdeletion mutant and did not observe a delay in growth. We therefore performed comparative genome sequence analysis of wild-type strain TX82 and TX6051 and found a single mutation, a nonsense mutation in theccpAgene of TX6051. After correcting this mutation, the growth defect of TX6051 was abolished, implicating a role for CcpA in the growth ofE. faecium. To confirm this, we created accpAdeletion mutant of TX82, which also exhibited a slight delay in growth. Furthermore, theccpAdeletion mutant was attenuated (P= 0.0024) in a mixed-inoculum (TX82 plus TX82 ΔccpA) rat endocarditis model and also in anin vitrocompetitive growth assay; accpA-complemented strain showed neither reduced growth nor reduced virulence. We also found attenuation in the endocarditis model with the newacmdeletion mutant although not as great as that previously observed with TX6051 carrying theccpAmutation. Taken together, our data confirm the role of Acm in the pathogenesis of endocarditis. We also show that CcpA affects the growth ofE. faecium, that an intactccpAgene is important for full virulence, and that accpAmutation was partly responsible for the highly attenuated phenotype of TX6051.

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