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 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 A role for glutathione in buffering excess intracellular copper in Streptococcus pyogenes

2020 ◽  
Author(s):  
Louisa J. Stewart ◽  
Cheryl-lynn Y. Ong ◽  
May M. Zhang ◽  
Stephan Brouwer ◽  
Liam McIntyre ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Metal Binding ◽  
Metal Ion ◽  
Invasive Disease ◽  
Metal Availability ◽  
Bacterial Physiology ◽  
Fundamental Understanding ◽  
Group A

ABSTRACTCopper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally-responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (Group A Streptococcus, GAS), this system is encoded by the copYAZ operon. In this study, we demonstrate that the site of GAS infection in vivo represents a Cu-rich environment but inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde 3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of non-cognate metal-binding sites. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. We further show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. Our results indicate that GSH contributes to buffering of excess intracellular Cu when the transcriptionally-responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance that allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. This study advances fundamental understanding of Cu handling in the bacterial cytoplasm.IMPORTANCEThe control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it is established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and is long assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this role of glutathione beyond measurement of the effect of Cu on growth of glutathione-deficient mutants. Our work with Group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.

scholarly journals A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

2018 ◽  
Vol 293 (21) ◽  
pp. 7993-8008 ◽  
Author(s):  
Subrata Debnath ◽  
Dalibor Kosek ◽  
Harichandra D. Tagad ◽  
Stewart R. Durell ◽  
Daniel H. Appella ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Metal Ions ◽  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Protein Phosphatases ◽  
Random Order ◽  
Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

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 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 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.

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 Defining the Role of theStreptococcus agalactiaeSht-Family Proteins in Zinc Acquisition and Complement Evasion

2019 ◽  
Vol 201 (8) ◽  
Author(s):  
P. Moulin ◽  
V. Rong ◽  
A. Ribeiro E Silva ◽  
V. G. Pederick ◽  
E. Camiade ◽  
...  
Keyword(s):  
Human Blood ◽  
Streptococcus Agalactiae ◽  
Metal Ion ◽  
Binding Proteins ◽  
Factor H ◽  
Zinc Uptake ◽  
Zinc Homeostasis ◽  
Content Type

ABSTRACTStreptococcus agalactiaeis not only part of the human intestinal and urogenital microbiota but is also a leading cause of septicemia and meningitis in neonates. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including the transition metal ion zinc. The primary zinc acquisition system of the pathogen is the Adc/Lmb ABC permease, which is essential for viability in zinc-restricted environments. Here, we show that in addition to the AdcCB transporter and the three zinc-binding proteins, Lmb, AdcA, and AdcAII,S. agalactiaezinc homeostasis also involves two streptococcal histidine triad (Sht) proteins. Sht and ShtII are required for zinc uptake via the Lmb and AdcAII proteins with apparent overlapping functionality and specificity. Both Sht-family proteins possess five-histidine triad motifs with similar hierarchies of importance for Zn homeostasis. Independent of its contribution to zinc homeostasis, Sht has previously been reported to bind factor H leading to predictions of a contribution to complement evasion. Here, we investigated ShtII to ascertain whether it had similar properties. Analysis of recombinant Sht and ShtII reveals that both proteins have similar affinities for factor H binding. However, neither protein aided in resistance to complement in human blood. These findings challenge prior inferences regarding thein vivorole of the Sht proteins in resisting complement‐mediated clearance.IMPORTANCEThis study examined the role of the two streptococcal histidine triad (Sht) proteins ofStreptococcus agalactiaein zinc homeostasis and complement resistance. We showed that Sht and ShtII facilitate zinc homeostasis in conjunction with the metal-binding proteins Lmb and AdcAII. Here, we show that the Sht-family proteins are functionally redundant with overlapping roles in zinc uptake. Further, this work reveals that although the Sht-family proteins bind to factor Hin vitrothis did not influence survival in human blood.

Production and secretion of progesterone in vitro and presence of platelet activating factor (PAF) in early pregnancy of the marsupial, Macropus eugenii

1993 ◽  
Vol 5 (1) ◽  
pp. 15 ◽  
Author(s):  
T Kojima ◽  
LA Hinds ◽  
WJ Muller ◽  
C O'Neill ◽  
CH Tyndale-Biscoe
Keyword(s):  
Platelet Activating Factor ◽  
Explant Culture ◽  
Ovarian Vein ◽  
Progesterone Concentration ◽  
Plasma Progesterone ◽  
Blood Samples ◽  
Basal Plasma ◽  
High Concentrations ◽  
Group B

The corpus luteum (CL) of the tammar is suppressed by lactation. Removal of the sucking pouch young induces reactivation of the CL which produces a peak in plasma progesterone concentration on Day 5, 6 or 7; reactivation of the embryo after diapause precedes by one day this transient peak of progesterone. This study examines progesterone production and secretion in vitro by the CL and the production of platelet activating factor (PAF) by the endometrium during this stage of pregnancy (Days 0 and 3-8). Blood samples were collected twice daily to determine for each animal (n = 28) at autopsy the relationship of PAF production to the day of the progesterone peak. Significant changes in basal plasma progesterone concentrations were used to define four groups according to the time the animals were killed: Group A, Day 0; Group B, other animals showing no significant change; Group C, peripheral progesterone concentration increasing; Group D, peripheral progesterone concentration decreasing after the peak. At autopsy on successive days, blood samples were taken from the heart, caudal vein and uterine branch of the ovarian vein, and CL and endometria were prepared for explant culture. Progesterone contents of pre- and post-incubation luteal tissues and of medium were determined at 4, 12 and 24 h to estimate production and secretion rates. The values for all progesterone parameters from animals in Group C were significantly higher than in the other groups. It is concluded that the progesterone peak results from a change in rate of production and secretion of progesterone by the CL, one day before the peak in peripheral plasma, and that this change could provide the first signal to the uterus. Endometrium was incubated for 24 h and the medium assayed for PAF. High concentrations of PAF were detected in half the animals in Groups B and C, that is, before or at the time progesterone was increasing. This is the first measurement of PAF in any marsupial and the first demonstration of the release of PAF by the endometrium. The detection of PAF provides a candidate for an endometrial signal that is responsive to the rising progesterone concentration in the utero-ovarian vein draining the CL-bearing ovary and that may stimulate reactivation of the blastocyst.

scholarly journals Unraveling the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2)

2019 ◽  
Vol 294 (20) ◽  
pp. 8046-8063 ◽  
Author(s):  
Gergely Gyimesi ◽  
Giuseppe Albano ◽  
Daniel G. Fuster ◽  
Matthias A. Hediger ◽  
Jonai Pujol-Giménez
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
In Silico ◽  
Metal Ion ◽  
Substrate Binding ◽  
Functional Characterization ◽  
Ph Sensitivity ◽  
Hek293 Cells ◽  
Substrate Binding Site

The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–translocation pore. Interestingly, the H63A substitution completely abrogated pH sensitivity, and substitutions of Glu-67 and Phe-269 altered the pH and voltage modulation of transport. In contrast, substitution of Glu-106, which might be part of a dimerization interface, altered pH but not voltage modulation. Substitution of Phe-269, located close to the substrate-binding site, also affected substrate selectivity. These findings were supported by an additional model of ZIP2 that was based on the structure of a prokaryotic homolog, Bordetella bronchiseptica ZrT/Irt-like protein (bbZIP), and in silico pKa calculations. We also found that residues Glu-179, His-175, His-202, and Glu-276 are directly involved in the coordination of the substrate metal ion. We noted that, unlike bbZIP, human ZIP2 is predicted to harbor a single divalent metal-binding site, with the charged side chain of Lys-203 replacing the second bound ion. Our results provide the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport, identify the substrate-binding site, and suggest a structure-based transport mechanism for the ZIP2 transporter.

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