Cytosolic clearance of replication-deficient mutants reveals Francisella tularensis interactions with the autophagic pathway

Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ∆dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival and proliferation.     

Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3⁺ bacteria were present within single-membrane phagosomes that are LAMP1⁺. Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3⁺ bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.     

Morphometric analysis of autophagy-related structures in Saccharomyces cerevisiae

When macroautophagy (autophagy) is induced by nutrient starvation or rapamycin treatment, Atg (autophagy-related) proteins are assembled at a restricted region close to the vacuole. Subsequently, the phagophore expands to form a closed autophagosome. In Saccharomyces cerevisiae cells overexpressing precursor Ape1 (prApe1), a specific autophagosome cargo protein, the phagophore can be visualized as a cup-shaped structure labeled with green fluorescent protein (GFP)-tagged Atg8. Previously, our group has shown that the maximum length of GFP-Atg8-labeled structures reflects the magnitude of bulk autophagy. In that study, the morphological parameters of the autophagy-related structures were extracted manually, requiring a great deal of time. Moreover, only well-expanded phagophores were subjected to further analysis. Here we report Qautas (Quantitative autophagy-related structure analysis system), a high-throughput and comprehensive system for morphological analysis of autophagy-related structures using a combination of image processing and machine learning. We describe both the manual method and Qautas in detail.     

Synthesis of Some Novel Acyclolumazine N-1 Nucleosides

Abstract

Reactjon of (2-acetoxyethoxy)methyl bromide with the silylated lumazine bases (1-6) in the presence of n-Bu₄NI leads to the formation of the nucleosides 8, 10, 12, 14, 16 and 18 respectively. Deacetylation with methanolic ammonia afforded the free nucleosides 9, 11, 13, 15, 17 and 19, respectively, in good yields. Structural proofs of the newly synthesized compounds are based on elemental analyses, UV and ¹H-NMR spactra. None of the acyclic nucleosides exhibited antiviral activity against HSV-1 in vitro.     

Synthesis of Certain 5′-Substituted Derivatives of Ribavirin and Tiazofurin

Abstract

The synthesis of several 5′-substituted derivatives of ribavirin (1) and tiazofurin (3) are described. Direct acylation of 1 with the appropriate acyl chloride in pyridine-DMF gave the corresponding 5′-O-acyl derivatives (4a-h). Tosylation of the 2′, 3′-O-isopropylidene-ribavirin (6) and tiazofurin (11) with p-toluenesulfonyl chloride gave the respective 5′-O-p-tolylsulfonyl derivatives (7a and 12a), which were converted to 5′-azido-5′-deoxy derivatives (7b and 12b) by reacting with sodium/lithium azide. Deisopropylidenation of 7b and 12b, followed by catalytic hydrogenation afforded 1-(5-amino-5-deoxy-β-D)-ribofuranosyl)-1, 2, 4-triazole-3-carboxamide (10b) and 2 - (5 -amino- 5-deoxy- β-D-ribofuranosyl) thiazole-4-carboxamide (16), respectively. Treatment of 6 with phthalimide in the presence of triphenylphosphine and diethyl azodicarboxylate furnished the corresponding 5′-deoxy-5′-phthaloylamino derivative (9). Reaction of 9 with n-butylamine and subsequent deisopropylidenation provided yet another route to 10b. Selective 5′-thioacetylation of 6 and 11 with thiolacetic acid, followed by saponification and deisopropylidenation afforded 5′-deoxy-5′-thio derivatives of 1-β-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide (8a) and 2-β-D-ribofuranosylthiazole-4-carboxamide (15), respectively.     

The Discovery of Intramolecular Stereoelectronic Forces That Drive The Sugar Conformation in Nucleosides and Nucleotides

Abstract

This report summarizes our results⁸ on how the determination of the thermodynamics of the two-state North (N, C2′-exo-C3′-endo) ? South (S,C2′-endo-C3′-exo) pseudorotational equilibrium in aqueous solution (pD 0.6 - 12.0) basing on vicinal ³J_HH extracted from ¹H-NMR spectra measured at 500 MHz from 278K to 358K yields an experimental energy inventory of the unique stereoelectronic forces that dictate the conformation of the sugar moiety in β-D-ribonucleosides (rNs), β-D-nucleotides, in the mirror-image β-D- versus β-L-2′-deoxynucleosides (dNs) as well as in α-D- or L- versus β-D- or L-2′-dNs. Our work shows for the first time that the free-energies of the inherent internal flexibilities of β-D- versus β-L-2′-dNs and α-D- versus α-L-2′-dNs are identical, whereas the aglycone promoted tunability of the constituent sugar conformation is grossly affected in the α-nucleosides compared to the β-counterparts.     

Synthetic Nucleosides and Nucleotides. XX1. Synthesis of Various 1-β-D-Xylofuranosyl-5-Alkyluracils and Related Nucleosides

Abstract

Treatment of D-xylose (1) with 0.5% methanolic hydrogen chloride under controlled conditions followed by benzoylation and acetolysis afforded crystalline 1-O-acetyl-2, 3, 5-tri-O-benzoyl-α-D-xylofuranose (4) in good yield. Coupling of 4 with 2, 4-bis-trimethylsilyl derivatives of 5-alkyluracils (methyl, ethyl, propyl and butyl) (5a-5d), 5-fluorouracil (5e) and uracil (5f) in acetonitrile in the presence of stannic chloride gave 1-(2,3,5-tri-O-benzoyl-β-D-xylofuranosyl)-nucleosides (6a-6f). Saponification of 6 with sodium methoxide afforded 1-β-D-xylofuranosyl-5-substituted uracils (7a-7f). Condensation of 4 with free adenine in similar fashion and deblocking gave carcinostatic 9-β-D-xylofuranosyladenine (7g).     

Synthesis and Antitumour Properties of 2-Thio-5-chloro-nucleosides

Abstract

Four methods are described for the synthesis of 2-thio-5-chlorouracil (1). β- and α-5-Chloro-2-thio-2′-deoxyuridines (12 and 13) were obtained by Lewis acid catalysed condensation of TMS derivative of 1 with 2-deoxy-3,5-di-O-p-toluyl-α-D-ribosyl chloride and deblocking of toluylated derivatives with methanolic ammonia. Selective enzymatic phosphorylation of 12 led to its 5′-monophosphate, the latter being a moderate inhibitor of thymidylate synthase, while 12 showed moderate cytotoxicity in vitro against mouse leukemic cells L15178Y.     

Identification of a novel MTOR activator and discovery of a competing endogenous RNA regulating autophagy in vascular endothelial cells

MTOR, a central regulator of autophagy, is involved in cancer and cardiovascular and neurological diseases. Modulating the MTOR signaling balance could be of great significance for numerous diseases. No chemical activators of MTOR have been found, and the urgent challenge is to find novel MTOR downstream components. In previous studies, we found a chemical small molecule, 3-benzyl-5-((2-nitrophenoxy) methyl)–dihydrofuran-2(3H)-one (3BDO), that inhibited autophagy in human umbilical vein endothelial cells (HUVECs) and neuronal cells. Here, we found that 3BDO activated MTOR by targeting FKBP1A (FK506-binding protein 1A, 12 kDa). We next used 3BDO to detect novel factors downstream of the MTOR signaling pathway. Activation of MTOR by 3BDO increased the phosphorylation of TIA1 (TIA1 cytotoxic granule-associated RNA binding protein/T-cell-restricted intracellular antigen-1). Finally, we used gene microarray, RNA interference, RNA-ChIP assay, bioinformatics, luciferase reporter assay, and other assays and found that 3BDO greatly decreased the level of a long noncoding RNA (lncRNA) derived from the 3′ untranslated region (3′UTR) of TGFB2, known as FLJ11812. TIA1 was responsible for processing FLJ11812. Further experiments results showed that FLJ11812 could bind with MIR4459 targeting ATG13 (autophagy-related 13), and ATG13 protein level was decreased along with 3BDO-decreased FLJ11812 level. Here, we provide a new activator of MTOR, and our findings highlight the role of the lncRNA in autophagy.     

The tRNA “WOBBLE POSITION” Uridines. III.1 the Synthesis of 5-[S-Methoxycarbonyl (Hydroxy)Methyl] Uridine and its 2-Thio Analogue

Abstract

The diastereoisomers 2a, 2b and their 2-thio analogues 4a and 4b were obtained by three-step transformation of uridine and 2-thiouridine, respectively. The absolute configuration at C-5¹ in 2a and 2b was established by CD, while for 4a and 4b the configurational assignment was based on the chemical correlation. The acids 1 and 3 were obtained by alkaline hydrolysis of 2a and 4a, respectively.     

Conformation of Diastereomers of Adenosine Cyclic 3′, 5′-Phosphorothioate

Abstract

¹H and ³¹P NMR spectra of cAMP (1) and both diastereomers of cAMPS (2 and 3) were compared with these of structurally related bicyclic phosphate 4 and phosphorothioates 5 and 6. Conformational analysis was also performed by NMR techniques for bicyclic phosphoranilidates 7 and 8 and (Rp)-cdAMP anilidate (9). Chair conformation is predominant for all investigated compounds 1–8, while the phosphoranilidate 9 exists in solution in chair-twist equilibrium. Thus, antagonistic properties of (Rp)-cAMPS with respect to cAMP are inferred by the change in the overall molecular shape caused by the presence of the bulky sulfur atom in the equatorial position of the cAMPS molecule.     

Nucleosides,I Ribosylation of 8-Substituted Theophylline Derivatives

Abstract

The attempted ribosylation reaction of 8-nitro-theophylline (2) with 1-o-acetyl-2, 3, 5-tri-o-benzoyl-D-ribo-furanose (5) failed to give any nucleoside product, whereas the reaction of 8-chlorotheophylline (3) with 5 afforded the 8-chloro-7-(2,3,5-tri-o-benzoyl) β-D-ribofuranosyltheophylline (6) in good yield. The product 6 reacted with benzylamine producing the 8-benzylamino-7-(2, 3, 5-tri-O-benzoyl) β-D-ribo-furanosyltheophylline (10), which could also be synthesised by ribosylation of 8-benzylaminotheophylline (8) with 5. Debenzoylation of 6 and 10 gave the corresponding 7-β-D-ribofuranosyltheophylline nucleosides (7) and (11), respectively. Compound 7 could be converted into 11 by reaction with benzylamine. The newly synthesised compounds have been characterised by elemental analysis, ¹H-NMR and UV spectra.     

A Facile Synthesis of the Phosphoramidites of 2-N-Methyl-2′-deoxy (or 2′-O-allyl)-ψ-isocytidine, 1, 3-Dimethyl-2′-deoxy-ψ-uridine and N1-Methyl-2′-O-allyl-ψ-uridine as Synthons Suitable for Oligonucleotide Synthesis

Abstract

An efficient and facile syntheses of 5′-O-(4, 4′-dimethoxytrityl)-3′-[2-cyanoethyl bis(1-methylethyl)]phosphoramidites of 2-N-methyl-2′-deoxy-ψ-isocytidine (6), 2-N-methyl-2′-deoxy-α-ψ-isocytidine (13), 2-N-methyl-2′-O-allyl-ψ-isocytidine (11), 1, 3-dimethyl-2′-deoxy-ψ-uridine (4) and N1-methyl-2′-O-allyl-ψ-uridine (19) have been accomplished in good overall yields. The pyrimidine-pyrimidine transformation reaction was found to be useful for the preparation of 2-N-methyl-2′-O-allyl-ψ-isocytidine (10). The utility of these novel phosphoramidites is demonstrated by their incorporation into oligonucleotides via solid-support, oligonucleotide methodology.     

Synthesis of Some Novel 1-(5-Thio-ß-D-glucopyranosyl)-6-azauracil Derivatives - Thiosugar Nucleosides

Abstract

The chemical syntheses of 1-(2,3,4,6-tetra-0-acety]-5-thio-β-D-glucopyranosyl)-6-azauracil (4) and the 5-bromo analogue 6 are described. Deblocking of 4 and 6 with sodium methoxide afforded the free nucleosides 5 and 7, respetively. Treatment of 6 with benzylmercaptan in basic medium led to the formation of 6-benzylthio-1-((2,3,4,6-tetra-0-acetyl-5-thio-β-D-glucopyranosyl)-6-azauracil (8), in good yield, which was deblocked to 9 on treatment with sodium methoxide. Reaction of 6 with benzlamine gave 5-benzylamino-1-(5-thio-β-D-glucopyranosyl)-6-azauracil (10).     

Studies on the Dehydration of New 2- (Pentitol-1-YL) Pyridines Having D-Gulo,D-IDO,and L-Manno Configurations

Abstract

Fusion of 2-trimethylsilylpyridine and tetra-O-acetyl-aldehydo-D-xylose or 2,3:4,5-di-O-isopropylidene-aldehydo-L-arabinose led, after removing of the protecting groups, to 2-(pentitol-1-yl)pyridines of D-gulo and D-ido or L-manno configurations. Dehydration of the sugar-chain with D-gulo and D-ido configurations gave the corresponding 2′,5′-anhydro derivatives, whereas 2-(5-O-isopropyl-L-manno-pentitol-1-yl)-pyridine was the only compound formed by dehydration of the sugar-chain with L-manno configuration. Structural proofs are based on ¹H and ¹³C NMR spectra.     

Synthetic Studies on the Isomeric N -Methyl Derivatives of C-Ribavirin

Abstract

The syntheses of all three of the mono-N-methy1 derivatives of C-ribavirin (3-β-D-ribofuranosyl-1, 2, 4-triazole-5-carboxamide, 2) have been accomplished. Reaction of 1-(β-D-ribofuranosyliminomethyl)-2-methyl-hydrazine ( 7 ) with ethyl oxamate (8) in boiling ethanol gave the N′-methyl-C-ribavirin ( 3 ). A similar treatment of β-D-ribofuranosyl-1-carboximidic acid methyl ester ( 6 ) with N′-methyloxamic hydrazide ( 10 ) furnished the N²-methyl-C-ribavirin ( 4 ). Direct methylation of unprotected 2 with methyl iodide in the presence of potassium carbonate in dimethyl sulfoxide gave N ⁴-methyl isomer ( 5 ) as the major product. Structural assignments of 3 , 4 , and 5 were based on the unequivocal synthetic sequences, ¹H and ¹³C NMR data and confirmed by single crystal X-ray diffraction analysis.     

Improved and Reliable Synthesis of 3′‐Azido‐2′,3′‐dideoxyguanosine Derivatives

An improved synthesis of N²‐protected‐3′‐azido‐2′,3′‐dideoxyguanosine 20 and 23 is described. Deoxygenation of 2′‐O‐alkyl (and/or aryl) sulfonyl‐5′‐dimethoxytritylguanosine coupled with [1,2]‐hydride shift rearrangement gave protected 9‐(2‐deoxy‐threo‐pentofuranosyl)guanines ( 10 , 12 and 16 ). This rearrangement was accomplished in high yield with a high degree of stereoselectivity using lithium triisobutylborohydride (l‐Selectride®). Compounds 10 , 12 and 16 were transformed into 3′‐O‐mesylates ( 18 and 21 ), which can be used for 3′‐substitution. The 3′‐azido nucleosides were obtained by treatment of 18 and 21 with lithium azide. This procedure is reproducible with a good overall yield.     

5′-O-[N-(Amnoacyl)Sulfamoyl]Nucleosides. Synthesis and Antiviral and Cytostatic Activities

Abstract

5′-O-[N-(Aminoacyl)sulfamoyl]-uridines and -thymidines 4a-12a and 4b-12b have been synthesized and tested against Herpes Simplex virus type 2 (HSV-2) and as cytostatics. Condensation of 2′,3′-O-isopropylidene-5′-O-sulfamoyluridine and 3′-O-acetyl-5′-O-sulfamoylthymidine with the N-hydroxysuccinimide esters of Boc-L-Ser(Bzl), (2R, 3S)-3-benzyloxycarbonylamino-2-hydroxy-4-phenylbuta-noic acid [(2R, 3S-N-Z-AHPBA], (2R, 3S) and (2S, 3R)-N-Boc-AHPBA gave 4a,b-7a,b, which after removal of the protecting groups provided 1Oa,b-12a,b. A study of the selective removal of the O-Bzl protecting group from the L-Ser derivatives 4a,b, without hydrogenation of the pyrimidine ring, has been carried out. Only the fully protected uridine derivatives 4a-7a did exhibit high anti-HSV-2 activity, and none of the synthesized compounds showed significant cytostatic activity against HeLa cells cultures.     

Synthesis of Brunfelsamidine Ribonucleoside and Certain Related Compounds by the Stereospecific Sodium Salt Glycosylation Procedure

Abstract

A synthesis of 2,4-dideazaribavirin ( 2 ), brunfelsamidine ribonucleoside ( 8c ) and certain related derivatives are described for the first time using the stereospecific sodium salt glycosylation procedure. Glycosylation of the sodium salt of pyrrole-3-carbonitrile ( 4 ) with 1-chloro-2, 3-O-t-isopropylidene-5-O-t-butyldimethylsilyl-α-D-ribofuranose ( 5 ) gave exclusively the corresponding blocked nucleoside ( 6 ) with β-anomeric configuration, which on deprotection provided 1-β-D-ribofuranosylpyrrole-3-carbonitrile ( 7 ). Functional group tranformation of 7 gave 2 , 8c and related 3-substituted pyrrole ribonucleosides. These compounds are devoid of any significant antiviral/antitumor activity invitro.