Structure of Erythropoietin in African Clawed Frogs, Xenopus laevis, and Its Role in the Liver Erythropoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1148-1148
Author(s):  
Nami Nogawa ◽  
Nobuyoshi Kosaka ◽  
Youichi Aizawa ◽  
Hiroshi Miyazaki ◽  
Norio Komatsu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xenopus Laevis ◽  
Mammalian Species ◽  
Glycosylation Site ◽  
Dependent Manner ◽  
Erythropoietic Activity ◽  
Lectin Affinity Chromatography ◽  
Erythroid Progenitors ◽  
Peripheral Erythrocyte

Abstract Erythropoietin (EPO) is a main regulator of erythropoiesis ensuring oxygen supply in mammalian species. However the functions of EPO in nonmammalian vertebrates remain unclear. In this study, EPO was identified in Xenopus laevis (X. laevis), and its contribution to definitive erythropoiesis was studied. The X. laevis EPO (xlEpo) cDNA revealed that the deduced amino acid sequence had only 38% identity to human EPO (hEPO), while all four cysteine residues were conserved. xlEPO mRNA was expressed predominantly in the liver and lung. In order to assess the biological activity, recombinant xlEPO was produced by transfecting COS-1 with CMV promotor-driven vector. A mouse FDC/P2 cells stably expressing xlEPOR cDNA, that is a putative EPO receptor, showed proliferation in response to recombinant xlEPO in a dose dependent manner. This confirmed the ligand-receptor relationship of nonmammalian xlEPO and xlEPOR. To our surprise, xlEPO stimulated proliferation of EPO-dependent human cell line UT-7/EPO as well as murine EPOR expressing FDC/P2 cell lines. The cross-reactivity suggests the tertially structure is conserved through xlEPO to mammalian EPOs. In addition, the amino acid residues that are essential for hEPO binding to hEPOR are highly conserved in xlEPO. Since potent N-glycosylation site is absent in xlEPO, the glycosylation characteristics of recombinant xlEPO was studied by fractionation using wheat germ aggulutinin (WGA) and concanavalin A (ConA) lectin affinity chromatography. XlEPO activity was seen in flow-through fractions indicating the absence of O-glycosylation as well as N-glycosylation in xlEPO molecule. The absence of glycosylation suggests the high affinity of xlEPO to xlEPOR, and the shorter blood half-life. In order to investigate the biological function of xlEPO, in vitro colony forming assay of X. laevis erythroid progenitors was developed. Magnetic cell sorting analysis showed that xlEPOR-positive cells reside in the liver possessing typical erythroblastic morphology with high nucleus-to-cytoplasm ratio containing hemoglobin. The formation of erythroblast colonies from liver cells on addition of recombinant xlEPO was observed. The colonies formed were erythroblast colonies composed of hemoglobin-synthesising erythroblasts, confirming the erythropoietic function of xlEPO in X. laevis erythropoiesis. These results and the detection of xlEPO mRNA in liver hypothesized the paracrine regulation of xlEPO. In the colony assay, erythropoietic activity was observed in the serum of phenylhydrazine (PHZ) induced anemic X. laevis. The highest erythropoietic activity was observed 4 days after PHZ-administration, prior to the peripheral erythrocyte number reaches a nadir at day 8. These results proved that xlEPO is a functional ortholog of mammalian EPO and its role in vertebrate hematopoietic system, providing new insights into the basis of erythropoietic regulations.

scholarly journals Cloning, expression and map assignment of chicken prosaposin

1998 ◽  
Vol 330 (1) ◽  
pp. 321-327 ◽  
Author(s):  
Norihiro AZUMA ◽  
Hee-Chan SEO ◽  
Øystein LIE ◽  
Qiang FU ◽  
M. Robert GOULD ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Mammalian Species ◽  
Three Dimensional ◽  
Glycosylation Site ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Chicken Brain ◽  
Saposin C ◽  
Saposin B

Prosaposin is the precursor of four small glycoproteins, saposins A-D, that activate lysosomal sphingolipid hydrolysis. A full-length cDNA encoding prosaposin from chicken brain was isolated by PCR. The deduced amino acid sequence predicted that, similarly to human and other mammalian species studied, chicken prosaposin contains 518 residues, including four domains that correspond to saposins A-D. There was 59% identity and 76% similarity of human and chicken prosaposin amino acid sequences. The basic three-dimensional structures of these saposins is predicted to be similar on the basis of the conservation of six cysteine residues and an N-glycosylation site. Identity of amino acid sequences was higher among saposins A, B and D than in saposin C. The predicted amino acid sequence of saposin B matched exactly that of purified chicken saposin B protein. The chicken prosaposin gene was mapped to a single locus, PSAP, in chicken linkage group E11C10 and is closely linked to the ACTA2 locus. This confirms the homology between chicken and human prosaposins and defines a new conserved segment with human chromosome 10q21-q24.

scholarly journals Susceptibilities of Genotype 1a, 1b, and 3 Hepatitis C Virus Variants to the NS5A Inhibitor Elbasvir

2015 ◽  
Vol 59 (11) ◽  
pp. 6922-6929 ◽  
Author(s):  
Rong Liu ◽  
Stephanie Curry ◽  
Patricia McMonagle ◽  
Wendy W. Yeh ◽  
Steven W. Ludmerer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Genotype 1 ◽  
Genotype 3 ◽  
Ns5a Inhibitor ◽  
Genotype 1A ◽  
Genotype 1B ◽  
Ns5a Ravs

ABSTRACTElbasvir is an investigational NS5A inhibitor within vitroactivity against multiple HCV genotypes. Antiviral activity of elbasvir was measured in replicons derived from wild-type or resistant variants of genotypes 1a, 1b, and 3. The barrier to resistance was assessed by the number of resistant colonies selected by exposure to various elbasvir concentrations. In a phase 1b dose-escalating study, virologic responses were determined in 48 noncirrhotic adult men with chronic genotype 1 or 3 infections randomized to placebo or elbasvir from 5 to 50 mg (genotype 1) or 10 to 100 mg (genotype 3) once daily for 5 days. The NS5A gene was sequenced from plasma specimens obtained before, during, and after treatment. Elbasvir suppressed the emergence of resistance-associated variants (RAVs)in vitroin a dose-dependent manner. Variants selected by exposure to high elbasvir concentrations typically encoded multiple amino acid substitutions (most commonly involving loci 30, 31, and 93), conferring high-level elbasvir resistance. In the monotherapy study, patients with genotype 1b had greater reductions in HCV RNA levels than patients with genotype 1a at all elbasvir doses; responses in patients with genotype 3 were generally less pronounced than for genotype 1, particularly at lower elbasvir doses. M28T, Q30R, L31V, and Y93H in genotype 1a, L31V and Y93H in genotype 1b, and A30K, L31F, and Y93H in genotype 3 were the predominant RAVs selected by elbasvir monotherapy. Virologic findings in patients were consistent with the preclinical observations. NS5A-RAVs emerged most often at amino acid positions 28, 30, 31, and 93 in both the laboratory and clinical trial. (The MK-8742 P002 trial has been registered at ClinicalTrials.gov under identifier NCT01532973.)

Inhibition of Hemoglobin Degrading Protease Falcipain-2 as a Mechanism for Anti-Malarial Activity of Triazole-Amino Acid Hybrids

2020 ◽  
Vol 20 (5) ◽  
pp. 377-389 ◽  
Author(s):  
Vigyasa Singh ◽  
Rahul Singh Hada ◽  
Amad Uddin ◽  
Babita Aneja ◽  
Mohammad Abid ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cysteine Protease ◽  
Mammalian Cells ◽  
Antimalarial Activity ◽  
Docking Studies ◽  
Dependent Manner ◽  
Trophozoite Stage ◽  
Hemoglobin Degradation

Background: Novel drug development against malaria parasite over old conventional antimalarial drugs is essential due to rapid and indiscriminate use of drugs, which led to the emergence of resistant strains. Methods: In this study, previously reported triazole-amino acid hybrids (13-18) are explored against Plasmodium falciparum as antimalarial agents. Among six compounds, 15 and 18 exhibited antimalarial activity against P. falciparum with insignificant hemolytic activity and cytotoxicity towards HepG2 mammalian cells. In molecular docking studies, both compounds bind into the active site of PfFP-2 and block its accessibility to the substrate that leads to the inhibition of target protein further supported by in vitro analysis. Results: Antimalarial half-maximal inhibitory concentration (IC50) of 15 and 18 compounds were found to be 9.26 μM and 20.62 μM, respectively. Blood stage specific studies showed that compounds, 15 and 18 are effective at late trophozoite stage and block egress pathway of parasites. Decreased level of free monomeric heme was found in a dose dependent manner after the treatment with compounds 15 and 18, which was further evidenced by the reduction in percent of hemoglobin hydrolysis. Compounds 15 and 18 hindered hemoglobin degradation via intra- and extracellular cysteine protease falcipain-2 (PfFP-2) inhibitory activity both in in vitro and in vivo in P. falciparum. Conclusion: We report antimalarial potential of triazole-amino acid hybrids and their role in the inhibition of cysteine protease PfFP-2 as its mechanistic aspect.

Circulating Erythroid Progenitors and Predominant Contribution of Liver to Erythropoiesis in Adult Xenopus laevis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4213-4213
Author(s):  
Nami Nogawa ◽  
Youichi Aizawa ◽  
Nobuyoshi Kosaka ◽  
Takako Ishida ◽  
Takashi Kato
Keyword(s):  
Bone Marrow ◽  
Xenopus Laevis ◽  
Peripheral Blood ◽  
Polyclonal Antibodies ◽  
Erythropoietin Receptor ◽  
Assay System ◽  
Hematopoietic Progenitors ◽  
Erythroid Progenitors ◽  
Vitro Assay

Abstract Cross-species comparisons of hematopoietic systems will elucidate the conservation and diversity among species such as zebrafish, Xenopus, chick and mouse, which are not only of interest but different approaches would contribute to general hematology. To begin to understand their hematopoietic systems, particularly the whole animal-physiology, across non-mammalian vertebrates, we have focused on amphibian hematopoiesis. We tried to clarify the localization of definitive hematopoietic progenitors in adult Xenopus laevis, which is still to be determined. When Xenopus was induced acute hemolytic anemia by intraperitoneal phenylhydrazine (PHZ) administration, immature erythroblasts emerging in the circulation and notable increase in erythropoiesis within the liver were observed. We first screened putative hematopoietic tissues, liver, spleen, bone marrow and kidney, for erythroid progenitors using polyclonal antibodies to putative Xenopus erythropoietin receptor (xlEPOR) that we recently identified. MACS and FACS sorting and analysis revealed the existence of xlEPOR expressing cells in both liver and anemic peripheral blood. These xlEPOR positive cells were hemoglobin-positive with o-dianisidine staining, and had typical blastic morphology with high nucleus-to-cytoplasm ratio. We next developed and established an in vitro colony assay system to identify and score the hematopoietic progenitors retrospectively. The method enabled the identification and quantification of erythroid progenitors. Briefly, cells were prepared from liver, spleen, bone marrow and kidney followed by placing in semi-solid culture medium (α-MEM containing 0.8% methylcellulose, 20% FCS with appropriate hematopoietic stimulators), and cultured at 23°C with 5% CO2. The anemic serum exhibited the apparent erythropoietic stimulating activity toward the formation of remarkable number of colonies derived from anemic peripheral blood cells, resembling typical mammalian hematopoietic colony formation. Most of the colonies consisted of hemoglobin-expressing erythroids after two days culture, indicating that colony-forming units-erythroid (CFU-e) appeared in anemic blood. The normal and anemic liver also contained CFU-e, resulting in the formation of mixed and pure hematopoietic colonies. This also proved to be a useful in vitro assay system for identifying and quantifying various hematopoietic progenitors and activities of related cytokines. Figure shows the number of erythroid colonies derived from PHZ-induced anemic peripheral blood and liver stimulated with anemic serum. We furthermore examined spleen and bone marrow side-by-side, since amphibian hematopoietic system is known to unique as erythropoiesis, granulopoiesis, and thrombopoiesis occur at distinct organs. The results demonstrated the direct evidences of predominant contribution of adult liver to erythropoiesis rather than bone marrow or spleen. A new animal model developed here should provide new insights into the basis of hematopoietic regulations. Figure Figure

scholarly journals In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum- deprived cultures

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 248-256 ◽  
Author(s):  
G Migliaccio ◽  
AR Migliaccio ◽  
JW Adamson
Keyword(s):  
Recombinant Human Erythropoietin ◽  
Colony Growth ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Concentration Dependent Manner ◽  
Gm Csf ◽  
Human Erythropoietin ◽  
Normal Humans

Abstract The effects of recombinant human erythropoietin (Ep), granulocyte/macrophage (GM) and granulocyte (G) colony-stimulating factors (CSF), and interleukin-3 (IL-3) on erythroid burst and GM colony growth have been studied in fetal bovine serum (FBS)- supplemented and FBS-deprived culture. Sources of progenitor cells were nonadherent or nonadherent T-lymphocyte-depleted marrow or peripheral blood cells from normal humans. G-CSF, in concentrations up to 2.3 X 10(-10) mol/L, induced only the formation of neutrophil colonies. In contrast, GM-CSF and IL-3 both induced GM colonies and sustained the formation of erythroid bursts in the presence of Ep. However, the activities of these growth factors were affected by the culture conditions. IL-3 induction of GM colonies depended on the presence of FBS, whereas the degree of GM-CSF induction of GM colonies in FBS- deprived cultures depended on the method by which adherent cells were removed. GM-CSF increased colony numbers in a concentration-dependent manner only if the cells had been prepared by overnight adherence. Both GM-CSF and IL-3 exhibited erythroid burst-promoting activity in FBS- deprived cultures. However, some lineage restriction was evident because GM-CSF was two- to threefold more active than IL-3 in inducing GM colonies but IL-3 was two- to threefold more active in promoting erythroid burst growth. Furthermore, in FBS-deprived cultures, the number of both erythroid bursts and GM colonies reached the maximum only when Ep, GM-CSF, and IL-3 or GM-CSF, IL-3, and G-CSF, respectively, were added together. These results suggest that the colonies induced by IL-3, GM-CSF, and G-CSF are derived from different progenitors.

scholarly journals Deciphering Myostatin’s Regulatory, Metabolic, and Developmental Influence in Skeletal Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Catherine L. Omosule ◽  
Charlotte L. Phillips
Keyword(s):  
Muscle Fiber ◽  
Cell Biology ◽  
Mammalian Species ◽  
Real Life ◽  
Meat Production ◽  
Dependent Manner ◽  
Mineral Density ◽  
Bone Properties ◽  
Myostatin Inhibition

Current research findings in humans and other mammalian and non-mammalian species support the potent regulatory role of myostatin in the morphology and function of muscle as well as cellular differentiation and metabolism, with real-life implications in agricultural meat production and human disease. Myostatin null mice (mstn−/−) exhibit skeletal muscle fiber hyperplasia and hypertrophy whereas myostatin deficiency in larger mammals like sheep and pigs engender muscle fiber hyperplasia. Myostatin’s impact extends beyond muscles, with alterations in myostatin present in the pathophysiology of myocardial infarctions, inflammation, insulin resistance, diabetes, aging, cancer cachexia, and musculoskeletal disease. In this review, we explore myostatin’s role in skeletal integrity and bone cell biology either due to direct biochemical signaling or indirect mechanisms of mechanotransduction. In vitro, myostatin inhibits osteoblast differentiation and stimulates osteoclast activity in a dose-dependent manner. Mice deficient in myostatin also have decreased osteoclast numbers, increased cortical thickness, cortical tissue mineral density in the tibia, and increased vertebral bone mineral density. Further, we explore the implications of these biochemical and biomechanical influences of myostatin signaling in the pathophysiology of human disorders that involve musculoskeletal degeneration. The pharmacological inhibition of myostatin directly or via decoy receptors has revealed improvements in muscle and bone properties in mouse models of osteogenesis imperfecta, osteoporosis, osteoarthritis, Duchenne muscular dystrophy, and diabetes. However, recent disappointing clinical trial outcomes of induced myostatin inhibition in diseases with significant neuromuscular wasting and atrophy reiterate complexity and further need for exploration of the translational application of myostatin inhibition in humans.

The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome

2001 ◽  
Vol 114 (11) ◽  
pp. 2095-2104
Author(s):  
Régis Giet ◽  
Claude Prigent
Keyword(s):  
Xenopus Laevis ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Terminal Domain ◽  
Egg Extracts ◽  
Green Fluorescent ◽  
Xenopus Egg Extracts

Aurora kinases are involved in mitotic events that control chromosome segregation. All members of this kinase subfamily possess two distinct domains, a highly conserved catalytic domain and an N-terminal non-catalytic extension that varies in size and sequence. To investigate the role of this variable non-catalytic region we overexpressed and purified Xenopus laevis auroraA (pEg2) histidine-tagged N-terminal peptide from bacterial cells. The peptide has no effect on the in vitro auroraA kinase activity, but it inhibits both bipolar spindle assembly and stability in Xenopus egg extracts. Unlike the full-length protein, the N-terminal domain shows only low affinity for paclitaxel-stabilised microtubules in vitro, but localises to the centrosomes in a microtubule-dependent manner. When expressed in Xenopus XL2 cells, it is able to target the green fluorescent protein to centrosomes. Surprisingly, this is also true of the pEg2 catalytic domain, although to a lesser extent. The centrosome localisation of the N-terminal peptide was disrupted by nocodazole whereas localisation of the catalytic domain was not, suggesting that in order to efficiently localise to the centrosome, pEg2 kinase required the non-catalytic N-terminal domain and the presence of microtubules.

scholarly journals Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment.

1995 ◽  
Vol 131 (4) ◽  
pp. 913-927 ◽  
Author(s):  
M Lussier ◽  
A M Sdicu ◽  
T Ketela ◽  
H Bussey
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Dependent Manner ◽  
Reporter Protein ◽  
Golgi Localization ◽  
Golgi Compartment ◽  
Membrane Spanning

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.

Characteristics of N-Glycosylation-Modified Erythropoietin in Xenopus Laevis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4563-4563
Author(s):  
Kazumichi Nagasawa ◽  
Nami Kosaka-Nogawa ◽  
Youichi Aizawa ◽  
Takashi Kato
Keyword(s):  
Amino Acid ◽  
Xenopus Laevis ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Wild Type ◽  
Western Blots ◽  
Molecular Weights ◽  
Glycosylation Sites ◽  
Pngase F

Abstract Abstract 4563 Erythropoietin (EPO) and erythropoietin receptor (EPOR) are primary regulators of erythropoiesis in mammals. In human EPO (huEPO), for example, there are three well-conserved glycosylation sites for N-linked carbohydrates. It is reported that the N-glycosylation is not required for the in vitro activity but essential for the exhibition of the in vivo activity. The N-linked carbohydrates are also important for biosynthesis and/or secretion as well as its stability and biological function. Previously, we identified EPO cDNA in the South African clawed frog, Xenopus laevis but found no N-glycosylation sites in the deduced protein, xlEPO. To learn more about xlEPO lacking N-glycosylation, we constructed and characterized xlEPO mutants with N-linked carbohydrates. We altered xlEPO gene by site-directed mutagenesis to contain N-glycosylation consensus sequences (Asn-X-Ser/Thr, where X is any amino acid except proline) at the same positions as those in huEPO. These seven different xlEPO mutants contain one to three inserted N-glycosylation sites at Asn24, Asn38, and Asn83, respectively. Here, we describe xlEPO mutants as xlEPO-Y, where Y indicates N-glycosylation position 1 at Asn24, position 2 at Asn38, and position 3 at Asn83. Then, we isolated conditioned media from cultures of COS-1 cells transiently expressing wild-type and mutant xlEPOs. Western blots showed progressive increases in the molecular weights of xlEPO mutants from 18 kDa to 22, 26, and 30 kDa based on the number of additional N-linked carbohydrates. Non-glycosylated Asn83 was observed in xlEPO-3, xlEPO-13, xlEPO-23, and xlEPO-123. Treatment with peptide-N-glycosidase F (PNGase F) shifted the molecular weights of all mutants to 18 kDa, identical to wild-type. Thus we attributed the increased size of the mutants above that of wild-type xlEPO to increased N-linked carbohydrate content. Blots also revealed decreases in relative secretion levels that depended on addition of N-linked carbohydrates. The secretion levels of mutants with one and two additional N-linked carbohydrate(s) were approximately half and quarter that of wild-type xlEPO, respectively. The results suggested that in contrast to huEPO, N-glycosylation is not necessary for efficient secretion of xlEPO. To examine the effects of the added N-linked carbohydrates on in vitro activity of xlEPO, we used a proliferation assay with mouse FDC/P2 cells that express xlEPOR. xlEPO mutants showed dose-dependent stimulation and removal of N-linked carbohydrates from these mutants with PNGase F increased their activities. The maximum effects of xlEPO-1 (70%), xlEPO-12 (30%) were less than that of wild-type xlEPO, suggesting that carbohydrate at Asn24 was most effective. This is in accord with the observation that abolishing this N-glycosylation site in huEPO increases in vitro activity (Yamaguchi et al., J Biol Chem 266: 20434, 1991). Previously, we reported that xlEPO induced proliferation of both EPO-dependent human UT-7/EPO cells and murine EPOR expressing FDC/P2 cells though xlEPO has only 38% amino-acid sequence identity with human EPO (Nogawa et al., ASH 2006, Abstract 1148). Taken together, we consider that xlEPO retains an important aspect of the structural frame of EPO molecule required for the biological activity. The xlEPO and its receptor will be valuable tools to further study the structure-activity relationships of EPO molecule. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Candida albicans Induces Arginine Biosynthetic Genes in Response to Host-Derived Reactive Oxygen Species

2012 ◽  
Vol 12 (1) ◽  
pp. 91-100 ◽  
Author(s):  
Claudia Jiménez-López ◽  
John R. Collette ◽  
Kimberly M. Brothers ◽  
Kelly M. Shepardson ◽  
Robert A. Cramer ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Candida Albicans ◽  
Amino Acid ◽  
Reactive Oxygen ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Biosynthetic Genes ◽  
Content Type

ABSTRACTThe interaction ofCandida albicanswith phagocytes of the host's innate immune system is highly dynamic, and its outcome directly impacts the progression of infection. While the switch to hyphal growth within the macrophage is the most obvious physiological response, much of the genetic response reflects nutrient starvation: translational repression and induction of alternative carbon metabolism. Changes in amino acid metabolism are not seen, with the striking exception of arginine biosynthesis, which is upregulated in its entirety during coculture with macrophages. Using single-cell reporters, we showed here that arginine biosynthetic genes are induced specifically in phagocytosed cells. This induction is lower in magnitude than during arginine starvationin vitroand is driven not by an arginine deficiency within the phagocyte but instead by exposure to reactive oxygen species (ROS). Curiously, these genes are induced in a narrow window of sublethal ROS concentrations.C. albicanscells phagocytosed by primary macrophages deficient in thegp91phoxsubunit of the phagocyte oxidase do not express theARGpathway, indicating that the induction is dependent on the phagocyte oxidative burst.C. albicans argpathway mutants are retarded in germ tube and hypha formation within macrophages but are not notably more sensitive to ROS. We also find that theARGpathway is regulated not by the general amino acid control response but by transcriptional regulators similar to theSaccharomyces cerevisiaeArgR complex. In summary, phagocytosis induces this single amino acid biosynthetic pathway in an ROS-dependent manner.

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