scholarly journals Identification of Zinc-Dependent Mechanisms Used by Group B Streptococcus To Overcome Calprotectin-Mediated Stress

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Lindsey R. Burcham ◽  
Yoann Le Breton ◽  
Jana N. Radin ◽  
Brady L. Spencer ◽  
Liwen Deng ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Metal Ion ◽  
Invasive Disease ◽  
Female Reproductive Tract ◽  
Zinc Uptake ◽  
Wild Type ◽  
Metal Transporters ◽  
High Concentrations ◽  
Group B

ABSTRACT Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease. IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

scholarly journals Identification of zinc-dependent mechanisms used by Group B Streptococcus to overcome calprotectin-mediated stress

2020 ◽  
Author(s):  
Lindsey R. Burcham ◽  
Yoann Le Breton ◽  
Jana N. Radin ◽  
Brady L. Spencer ◽  
Liwen Deng ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Metal Binding ◽  
Metal Ion ◽  
Invasive Disease ◽  
Female Reproductive Tract ◽  
Zinc Uptake ◽  
Metal Transporters ◽  
High Concentrations ◽  
Group B

AbstractNutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Though GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin, are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative RT-PCR revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to the GBS WT strain. Further, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery are important for combatting zinc-chelation by calprotectin and establishing invasive disease.ImportanceGBS asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils, carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro, and in a mouse model of infection. We observed that a global zinc-uptake mutant was less virulent compared to the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by WT and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

scholarly journals The CiaR Response Regulator in Group B Streptococcus Promotes Intracellular Survival and Resistance to Innate Immune Defenses

2008 ◽  
Vol 191 (7) ◽  
pp. 2023-2032 ◽  
Author(s):  
Darin Quach ◽  
Nina M. van Sorge ◽  
Sascha A. Kristian ◽  
Joshua D. Bryan ◽  
Daniel W. Shelver ◽  
...  
Keyword(s):  
Response Regulator ◽  
Newborn Infants ◽  
Regulatory System ◽  
Intracellular Survival ◽  
Invasive Disease ◽  
Neonatal Meningitis ◽  
Host Defenses ◽  
Wild Type ◽  
Group B

ABSTRACT Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS ΔciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and ΔciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.

scholarly journals The sperm protein SPACA4 is required for efficient fertilization in mice

2021 ◽  
Author(s):  
Sarah Herberg ◽  
Yoshitaka Fujihara ◽  
Andreas Blaha ◽  
Karin Panser ◽  
Kiyonari Kobayashi ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Knockout Mice ◽  
Mammalian Species ◽  
Wild Type ◽  
Sperm Protein ◽  
Mammalian Sperm ◽  
Fundamental Process ◽  
Mammalian Fertilization

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncer's homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the testis. Male knockout mice are severely sub-fertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

scholarly journals Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits

2021 ◽  
Vol 17 (3) ◽  
pp. e1009116
Author(s):  
Allison N. Dammann ◽  
Anna B. Chamby ◽  
Andrew J. Catomeris ◽  
Kyle M. Davidson ◽  
Hervé Tettelin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amniotic Fluid ◽  
Deletion Strain ◽  
Growth Defect ◽  
Wild Type ◽  
Human Amniotic Fluid ◽  
Wide Range ◽  
Pregnant Mice ◽  
Group B

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

337 MITOCHONDRIA AND REACTIVE OXYGEN SPECIES IN PREBUPERTAL LAMB OOCYTES BEFORE AND AFTER IN VITRO MATURATION

2010 ◽  
Vol 22 (1) ◽  
pp. 325
Author(s):  
M. E. Dell'Aquila ◽  
B. Ambruosi ◽  
R. Guastamacchia ◽  
F. Binetti ◽  
E. Ciani ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Female Reproductive Tract ◽  
Nuclear Chromatin ◽  
Oxygen Species ◽  
Intracellular Ros ◽  
Before And After ◽  
Group A ◽  
Group B

Juvenile in vitro embryo transfer (JIVET) reduces the generation interval and increases the rate of genetic gain. The developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Oxidative stress in the oocyte is an emerging problem in reproductive in vitro technologies, due to the gas atmosphere used to incubate oocytes and the lack of physiological defense mechanisms available in the female reproductive tract. The major source of reactive oxygen species (ROS) is represented by mitochondria where ROS are produced during oxidative phosphorylation. The aim of the present study was to analyze mitochondria and ROS in ovine prepubertal oocytes before and after IVM in order to clarify their suitability in JIVET programs. Cumulus-oocyte complexes from the ovaries of 38 slaughtered prepubertal (less than 8 months of age) lambs of the Comisana breed were analyzed at retrieval (group A) or after IVM (group B; Ambruosi et al. 2009 Theriogenology 71, 1093-1104). After cumulus cell removal, all oocytes underwent nuclear chromatin, mitochondria and ROS evaluation by confocal analysis of fluorescence distribution and intensity. Hoechst 33258 and Mitotracker Orange CMTM Ros (Molecular Probes Inc., Eugene, OR) were used to label nuclear chromatin and mitochondria (Ambruosi et al. 2009) and 2′,7′-dichloro-dihydro-fluorescein diacetate was used for ROS labelling (Hashimoto et al. 2000 Mol. Reprod. Dev. 57, 353-360). Out of 65 oocytes from group A, 38 oocytes with regular size (>130 μm in diameter), morphology and nuclear chromatin at the GV stage were selected for analysis. One-hundred-thirty-eight oocytes underwent IVM (group B). Nuclear maturation rate (metaphase II with 1st polar body extruded) was 54%, 75/138. All MII oocytes were used for analysis. Significantly higher rate of oocytes from group B showed heterogeneous (large aggregates, clusters, pericortical, perinuclear) mitochondrial (mt) distribution pattern than oocytes from group A (55%, 41/75 v. 29%, 11/38, respectively; P < 0.05) which showed uniform distribution of small mt aggregates. Fluorescent intensity of mt labeling did not differ between groups (43.05 ± 16.15 v. 45.89 ± 10.36, for group A and B respectively; NS). In most of the oocytes from both groups, intracellular ROS were distributed in small or large aggregates (35/38, 92% and 62/75, 83%). No statistical difference was observed for intracellular ROS levels between oocytes from group A (66.36 ± 13.2) and group B (72.84 ± 20.63; NS). The culture conditions used in this study provided normal mt distribution and intracellular ROS levels. Qualitative and quantitative evaluation of mitochondria and intracellular ROS could be useful to improve in vitro culture methods in ovine prepubertal oocytes.

scholarly journals Mutations in PneumococcalcpsEGenerated viaIn VitroSerial Passaging Reveal a Potential Mechanism of Reduced Encapsulation Utilized by a Conjunctival Isolate

2015 ◽  
Vol 197 (10) ◽  
pp. 1781-1791 ◽  
Author(s):  
Mara G. Shainheit ◽  
Michael D. Valentino ◽  
Michael S. Gilmore ◽  
Andrew Camilli
Keyword(s):  
Streptococcus Pneumoniae ◽  
Invasive Disease ◽  
Adaptive Mutation ◽  
Specific Gene ◽  
High Dose ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Sepsis Model

ABSTRACTThe polysaccharide capsule ofStreptococcus pneumoniaeis required for nasopharyngeal colonization and for invasive disease in the lungs, blood, and meninges. In contrast, the vast majority of conjunctival isolates are acapsular. The first serotype-specific gene in the capsule operon,cpsE, encodes the initiating glycosyltransferase and is one of the few serotype-specific genes that can tolerate null mutations. This report characterizes a spontaneously arising TIGR4 mutant exhibiting a reduced capsule, caused by a 6-nucleotide duplication incpsEwhich results in duplication of Ala and Ile at positions 45 and 46. This strain (AI45dup) possessed more exposed phosphorylcholine and was hypersusceptible to C3 complement deposition compared to the wild type. Accordingly, the mutant was significantly better at forming abiotic biofilms and binding epithelial cellsin vitrobut was avirulent in a sepsis model.In vitroserial passaging of the wild-type strain failed to reproduce the AI45dup mutation but instead led to a variety of mutants with reduced capsule harboring single nucleotide polymorphisms (SNPs) incpsE. A single passage in the sepsis model after high-dose inoculation readily yielded revertants of AI45dup with restored wild-type capsule level, but the majority of SNP alleles ofcpsEcould not revert, suppress, or bypass. Analysis ofcpsEin conjunctival isolates revealed a strain with a single missense mutation at amino acid position 377, which was responsible for reduced encapsulation. This study supports the hypothesis that spontaneous, nonreverting mutations incpsEserve as a form of adaptive mutation by providing a selective advantage toS. pneumoniaein niches where expression of capsule is detrimental.IMPORTANCEWhile the capsule ofStreptococcus pneumoniaeis required for colonization and invasive disease, most conjunctival isolates are acapsular by virtue of deletion of the entire capsular operon. We show that spontaneous acapsular mutants isolatedin vitroharbor mostly nonrevertible single nucleotide polymorphism (SNP) null mutations incpsE, encoding the initiating glycosyltransferase. From a small collection of acapsular conjunctival isolates, we identified one strain with a complete capsular operon but containing a SNP incpsEthat we show is responsible for the acapsular phenotype. We propose that acapsular conjunctival isolates may arise initially from such nonreverting SNP null mutations incpsE, which can be followed later by deletion of portions or all of thecpsoperon.

scholarly journals Characterization of a Novel Leucine-Rich Repeat Protein Antigen from Group B Streptococci That Elicits Protective Immunity

2005 ◽  
Vol 73 (3) ◽  
pp. 1671-1683 ◽  
Author(s):  
Ravin Seepersaud ◽  
Sean B. Hanniffy ◽  
Peter Mayne ◽  
Phil Sizer ◽  
Richard Le Page ◽  
...  
Keyword(s):  
Significant Risk ◽  
Invasive Disease ◽  
Invasive Infection ◽  
Dependent Manner ◽  
Leucine Rich Repeat ◽  
Group B Streptococci ◽  
Lethal Challenge ◽  
Life Threatening ◽  
Group B

ABSTRACT Group B streptococci (GBS) usually behave as commensal organisms that asymptomatically colonize the gastrointestinal and urogenital tracts of adults. However, GBS are also pathogens and the leading bacterial cause of life-threatening invasive disease in neonates. While the events leading to transmission and disease in neonates remain unclear, GBS carriage and level of colonization in the mother have been shown to be significant risk factors associated with invasive infection. Surface antigens represent ideal vaccine targets for eliciting antibodies that can act as opsonins and/or inhibit colonization and invasion. Using a genetic screen for exported proteins in GBS, we identified a gene, designated lrrG, that encodes a novel LPXTG anchored surface antigen containing leucine-rich repeat (LRR) motifs found in bacterial invasins and other members of the LRR protein family. Southern blotting showed that lrrG was present in all GBS strains tested, representing the nine serotypes, and revealed the presence of an lrrG homologue in Streptococcus pyogenes. Recombinant LrrG protein was shown in vitro to adhere to epithelial cells in a dose-dependent manner, suggesting that it may function as an adhesion factor in GBS. More importantly, immunization with recombinant LrrG elicited a strong immunoglobulin G response in CBA/ca mice and protected against lethal challenge with virulent GBS. The data presented in this report suggest that this conserved protein is a highly promising candidate antigen for use in a GBS vaccine.

scholarly journals Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

2002 ◽  
Vol 70 (6) ◽  
pp. 3080-3084 ◽  
Author(s):  
Bhavna G. Gordhan ◽  
Debbie A. Smith ◽  
Heidi Alderton ◽  
Ruth A. McAdam ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Auxotrophic Mutant ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

scholarly journals Availability of Zinc Impacts Interactions Between Streptococcus sanguinis and Pseudomonas aeruginosa in Co-culture

2019 ◽  
Author(s):  
Kewei Li ◽  
Alex Gifford ◽  
Thomas Hampton ◽  
George A. O’Toole
Keyword(s):  
Relative Abundance ◽  
Zinc Uptake ◽  
Microbial Interactions ◽  
Growth Defect ◽  
Zinc Toxicity ◽  
Wild Type ◽  
Airway Infections ◽  
Mechanistic Basis ◽  
Zinc Levels

ABSTRACTAirway infections associated with cystic fibrosis (CF) are polymicrobial. We reported previously that clinical isolates of P. aeruginosa promote the growth of a variety of streptococcal species. To explore the mechanistic basis of this interaction, we performed a genetic screen to identify mutants of Streptococcus sanginuis SK36 whose growth was no longer enhanced by P. aeruginosa PAO1. Mutations in zinc uptake systems of S. sanginuis SK36 reduced growth of these strains by 1-3 log compared to wild-type S. sanginuis SK36 when grown in coculture with P. aeruginosa PA01, while exogenous zinc (0.1-10 μm) rescued the coculture defect of zinc uptake mutants of S. sanginuis SK36. Zinc uptake mutants of S. sanginuis SK36 had no obvious growth defect in monoculture. Consistent with a competition for zinc driving coculture dynamics, S. sanginuis SK36 grown in coculture with P. aeruginosa showed increased expression of zinc uptake genes compared to S. sanginuis grown alone. Strains of P. aeruginosa PAO1 defective in zinc transport also supported more robust growth by S. sanginuis compared to coculture with wild-type P. aeruginosa PAO1. An analysis of 118 CF sputum samples revealed that total zinc levels varied from ~5-145 μM. At relatively low zinc levels, Pseudomonas and Streptococcus were found in approximately equal abundance; at higher zinc levels, we observed an increasing relative abundance of Pseudomonas and decline of Streptococcus, perhaps as a result of increasing zinc toxicity. Together, our data indicate that the relative abundance of these microbes in the CF airway may be impacted by zinc levels.IMPORTANCEPolymicrobial infections in CF likely impact patient health, but the mechanism(s) underlying such interactions are poorly understood. Here we show that interactions between Pseudomonas and Streptococcus are modulated by zinc availability using an in vitro model system, and clinical data are consistent with this model. Together with previous studies, our work supports a role for metal homeostasis as a key factor driving microbial interactions.

scholarly journals The Vacuolar Zinc Transporter TgZnT Protects Toxoplasma gondii from Zinc Toxicity

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Nathan M. Chasen ◽  
Andrew J. Stasic ◽  
Beejan Asady ◽  
Isabelle Coppens ◽  
Silvia N. J. Moreno
Keyword(s):  
Toxoplasma Gondii ◽  
Metal Ion ◽  
Essential Element ◽  
Lytic Cycle ◽  
Zinc Toxicity ◽  
Content Type ◽  
Zinc Concentrations ◽  
High Concentrations ◽  
Yeast Mutants

ABSTRACT Zinc (Zn2+) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory proteins. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii. TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in in vitro assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+ tolerance of T. gondii extracellular tachyzoites. IMPORTANCE Toxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+. We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.

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