小学校教科書語彙の研究

<div><p>Abstract</p><p><i>Leishmania</i> parasites replicate within the phagolysosome compartment of mammalian macrophages. Although <i>Leishmania</i> depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient <i>Leishmania major</i> mutant (<i>∆gnat</i>) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in <i>ex vivo</i> infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the <i>L</i>. <i>major ∆gnat</i> mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in <i>ex vivo</i> infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in <i>Leishmania</i>-induced skin lesions and is efficiently internalized into <i>Leishmania</i> containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides <i>Leishmania</i> with essential carbon sources, creating a uniquely favorable niche for these parasites.</p></div

Identification and characterization of T47.

<p>A) Second-round screening of cherry-picked members of combinatorial library as inhibitors of extension of octyl α-d-mannopyranoside by <i>L. mexicana Δgmp</i> cell membranes containing β-1,2-mannosyltransferase. Assay conditions: 40 µM octyl α-D-mannopyranoside, 50 µM GDP-[³H]mannose, inhibitors at 1.27 mM, 15 min, 27°C. TLCs were developed in “solvent A”. “-ve” indicates reaction mixture that does not contain any inhibitor. M2,3 denote the oligomer length of oligomannosides formed from the substrate. B) Comparative IC₅₀ values of GMP and T47. Open squares denote GMP, closed triangles denote <b>T47</b>.</p

Characterization of β-1,2-mannosyltransferase activity and inhibition. A) Extension of octyl α-d-mannoside by <i>L. mexicana Δgmp</i> β-1,2-mannosyltransferases.

<p>B) Fluorogram of HPTLC of 0.05 mM octyl α-d-mannopyranoside incubated with GDP-[³H]mannose in a membrane preparation of <i>L. mexicana Δgmp</i> with GDP-[³H]mannose; C) Michaelis-Menten plot (inset: Lineweaver-Burke replot) of rate versus [GDP-mannose] at saturating (50 mM) octyl α-d-mannopyranoside; D) Michaelis-Menten plot (inset: Lineweaver-Burke replot) of rate versus [octyl α-d-mannopyranoside] at saturating (10 mM) GDP-mannose; E) Fluorogram of HPTLC showing inhibition of β-1,2-mannosyltransferase-catalyzed extension of octyl α-d-mannopyranoside by GMP and GDP. M2–5 denote the oligomer length of oligomannosides formed from the substrate.</p

Synthesis of GMP-alkyne.

<p>a) (ⁱPr₂N)₂POCH₂CH₂CN, diisopropylammonium tetrazolide, CH₂Cl₂, 0°C→rt, 75 min, 46%; b) <i>i</i> 1<i>H</i>-tetrazole, propargyl alcohol, CH₂Cl₂, 0°C→rt, 15 min; <i>ii</i> 0.1 M I₂ in H₂O/pyridine/THF (2∶20∶80), 10 min, 57%; c) 1.0 M TBAF/THF, 45 min, Dowex 50W-X4 (Na⁺ form), 37%; d) Cu⁰, CuSO₄, DMSO/H₂O (9∶1), 50%.</p

Structures of biphenyl-based mono/diphosphonucleotide triazole adducts.

<p>Structures of biphenyl-based mono/diphosphonucleotide triazole adducts.</p

Characterization of Metabolically Quiescent <i>Leishmania</i> Parasites in Murine Lesions Using Heavy Water Labeling

<div><p>Information on the growth rate and metabolism of microbial pathogens that cause long-term chronic infections is limited, reflecting the absence of suitable tools for measuring these parameters <i>in vivo</i>. Here, we have measured the replication and physiological state of <i>Leishmania mexicana</i> parasites in murine inflammatory lesions using ²H₂O labeling. Infected BALB/c mice were labeled with ²H₂O for up to 4 months, and the turnover of parasite DNA, RNA, protein and membrane lipids estimated from the rate of deuterium enrichment in constituent pentose sugars, amino acids, and fatty acids, respectively. We show that the replication rate of parasite stages in these tissues is very slow (doubling time of ~12 days), but remarkably constant throughout lesion development. Lesion parasites also exhibit markedly lower rates of RNA synthesis, protein turnover and membrane lipid synthesis than parasite stages isolated from <i>ex vivo</i> infected macrophages or cultured <i>in vitro</i>, suggesting that formation of lesions induces parasites to enter a semi-quiescent physiological state. Significantly, the determined parasite growth rate accounts for the overall increase in parasite burden indicating that parasite death and turnover of infected host cells in these lesions is minimal. We propose that the <i>Leishmania</i> response to lesion formation is an important adaptive strategy that minimizes macrophage activation, providing a permissive environment that supports progressive expansion of parasite burden. This labeling approach can be used to measure the dynamics of other host-microbe interactions <i>in situ</i>.</p></div

First-round screening of combinatorial library as inhibitors of extension of octyl α-D-mannopyranoside by <i>L. mexicana Δgmp</i> cell membranes containing β-1,2-mannosyltransferase.

<p>Assay conditions: 40 µM octyl α-d-mannopyranoside, 50 µM GDP-[³H]mannose, inhibitors at 4 mM, 15 min, 27°C. TLCs were developed in “solvent A”. “-ve” indicates reaction mixture that does not contain any inhibitor. M2–5 denote the oligomer length of oligomannosides formed from the substrate.</p

Stage-specific changes in <i>Leishmania</i> growth rates.

<p><b>A</b>. Schematic overview of ²H₂O labeling protocol. Parasite stages were cultivated axenically in the presence 5% ²H₂O, or isolated from infected macrophages or BALB/c lesion incubated or infused with ²H₂O (final concentration 5%). Parasite stages were harvested at multiple time points and extracts containing total DNA/RNA, or total proteins and lipids generated from purified parasite fraction. Levels of deuterium enrichment in constituent dRib/Rib, amino acids and fatty acids were subsequently quantitated by GC-MS. <b>B</b>. Kinetics of ²H-labeling of DNA dRib in cultured promastigotes (Pro^log, Pro^stat) and amastigotes (Ama^axenic), and in amastigotes isolated from macrophages (Ama^Mø) and murine lesions (Ama^lesion). The fraction of new cells (Y-axis) was calculated from the level of ²H-enrichment in dRib relative to maximum labeling observed in each parasite stage after long term (equilibrium) labeling. Calculated doubling times for each stage are shown in inset boxes. <b>C</b>. Comparative growth rates of different <i>Leishmania</i> stages, calculated from ²H-enrichment in dRib. <b>D</b>. Section of stained cutaneous lesion (with detail in insert) and calculated range of parasite numbers/phagolysosome. Abbreviations: dRib; deoxyribose, Rib; ribose.</p

Rates of protein turnover in cultured and intracellular <i>Leishmania</i> stages.

<p>Parasite stages were ²H₂O-labeled in culture or <i>in situ</i> in infected BALB/c mice and harvested at the indicated time points. Kinetics of ²H-labeling of proteinogenic alanine in (<b>A</b>) cultured parasite stages (Pro^log, Pro^stat, Ama^axenic) and (<b>B</b>) amastigotes isolated from BALB/c lesion (Ama^lesion). The fraction of new molecules (Y-axis) was calculated from the level of ²H-enrichment in alanine relative to maximum labeling observed in each parasite stage after long term labeling. Inset boxes in A and B show turnover (t_1/2) in days. <b>C</b>. Comparative rates of protein turnover in different <i>Leishmania</i> developmental stages. Note that similar estimates of protein turnover were obtained by measuring deuterium incorporation into other proteinogenic amino acids (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004683#ppat.1004683.s006" target="_blank">S6 Fig</a>.).</p

Rates of RNA turnover in cultured and intracellular <i>Leishmania</i> stages.

<p>Kinetics of ²H-labeling of RNA ribose in (<b>A</b>) cultured parasite stages (Pro^log, Pro^stat, Ama^axenic) (<b>B</b>) amastigotes isolated from infected J774 macrophages (Ama^Mø) and (<b>C</b>) amastigotes isolated from BALB/c lesions (Ama^lesion). The fraction of new molecules (Y-axis) was calculated from the level of ²H-enrichment in Rib relative to maximum labeling observed in each parasite stage after long term labeling. Inset boxes shows estimated RNA turnover (t_1/2 in days) in each stage. <b>D</b>. Comparative rates of RNA turnover in different <i>Leishmania</i> developmental stages.</p