48 research outputs found
REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant
AbstractThe peptidoglycan of Gram-positive bacteria consists of glycan chains with attached short peptide stems cross-linked to one another by glycyl bridges. The bridge of Staphylococcus aureus has five glycyl units and that of its FemA mutant has one. These long- and short-bridge cross-links create totally different cell-wall architectures. S. aureus and its FemA mutant grown in the presence of an alanine-racemase inhibitor were labeled with d-[1-13C]alanine, l-[3-13C]alanine, [2-13C]glycine, and l-[5-19F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between 13C-labeled alanines and 19F-labeled lysine incorporated in the peptidoglycan. The alanyl 13C labels in the parent strain were preselected for C{F} and C{P} REDOR measurement by their proximity to the glycine label using 13C13C spin diffusion. The observed 13C13C and 13C31P distances are consistent with a tightly packed architecture containing only parallel stems in a repeating structural motif within the peptidoglycan. Dante selection of d-alanine and l-alanine frequencies followed by 13C13C spin diffusion rules out scrambling of carbon labels. Cell walls of FemA were also labeled by a combination of d-[1-13C]alanine and l-[15N]alanine. Proximity of chains was measured by C{N} and N{C} REDOR distances and asymptotic plateaus, and both were consistent with a mixed-geometry model. Binding of an 19F-labeled eremomycin analog in the FemA cell wall matches that of binding to the parent-strain cell wall and reveals the proximity of parallel stems in the alternating parallel–perpendicular mixed-geometry model for the FemA peptidoglycan lattice. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky
An analogue of the antibiotic teicoplanin prevents flavivirus entry in vitro.
There is an urgent need for potent inhibitors of dengue virus (DENV) replication for the treatment and/or prophylaxis of infections with this virus. We here report on an aglycon analogue of the antibiotic teicoplanin (code name LCTA-949) that inhibits DENV-induced cytopathic effect (CPE) in a dose-dependent manner. Virus infection was completely inhibited at concentrations that had no adverse effect on the host cells. These findings were corroborated by quantification of viral RNA levels in culture supernatant. Antiviral activity was also observed against other flaviviruses such as the yellow fever virus and the tick-borne encephalitis virus (TBEV). In particular, potent antiviral activity was observed against TBEV. Time-of-drug-addition experiments indicated that LCTA-949 inhibits an early stage in the DENV replication cycle; however, a virucidal effect was excluded. This observation was corroborated by the fact that LCTA-949 lacks activity on DENV subgenomic replicon (that does not encode structural proteins) replication. Using a microsopy-based binding and fusion assay employing DiD-labeled viruses, it was shown that LCTA-949 targets the early stage (binding/entry) of the infection. Moreover, LCTA-949 efficiently inhibits infectivity of DENV particles pre-opsonized with antibodies, thus potentially also inhibiting antibody-dependent enhancement (ADE). In conclusion, LCTA-949 exerts in vitro activity against several flaviviruses and does so (as shown for DENV) by interfering with an early step in the viral replication cycle.Fil: De Burghgraeve, Tine. Katholikie Universiteit Leuven; BélgicaFil: Kaptein, Suzanne J. F.. Katholikie Universiteit Leuven; BélgicaFil: Ayala Nunez, Nilda V.. University of Groningen; PaÃses BajosFil: Mondotte, Juan Alberto. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones BioquÃmicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones BioquÃmicas de Buenos Aires; ArgentinaFil: Pastorino, Boris. Université de la Méditerranée; FranciaFil: Printsevskaya, Svetlana S.. Russian Academy of Medical Sciences; RusiaFil: de Lamballerie, Xavier. Université de la Méditerranée; FranciaFil: Jacobs, Michael. Royal Free & University College Medical School; Reino UnidoFil: Preobrazhenskaya, Maria. Russian Academy of Medical Sciences; RusiaFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones BioquÃmicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones BioquÃmicas de Buenos Aires; ArgentinaFil: Smit, Jolanda M.. University of Groningen; PaÃses BajosFil: Neyts, Johan. Katholikie Universiteit Leuven; Bélgic
Fucans, but Not Fucomannoglucuronans, Determine the Biological Activities of Sulfated Polysaccharides from Laminaria saccharina Brown Seaweed
Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed
Two approaches to the use of benzo[c][1,2]oxaboroles as active fragments for synthetic transformation of clarithromycin
Clarithromycin (active against Gram positive infections) and 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole derivatives (effective for Gram negative microbes) are the ligands of bacterial RNA. The antimicrobial activities of these benzoxaboroles linked with clarithromycin at 9 or 4″ position were compared. Two synthetic pathways for these conjugates were elaborated. First pathway explored the substitution of the C-9 carbonyl group of macrolactone’s cycle via oxime linker, the second direction used the modification of the 4″-O-group of cladinose via the formation of carbamates of benzoxaboroles. 4″-O-(3-S-(1-Hydroxy-1,3-dihydro-benzo[c][1,2]oxaborole)-methyl-carbamoyl-clarithromycin showed twofold decrease in MICs for S. epidermidis and S. pneumoniae than clarithromycin. 4″-O-Modified clarithromycin demonstrated an efficacy against Gram positive strains only. Compounds with C-9 substitution were more active than 4″-O-substituted antibiotics for susceptible strains E. coli tolC and did not exceed the activity of initial antibiotics
<i>Staphylococcus aureus</i> Peptidoglycan Stem Packing by Rotational-Echo Double Resonance NMR Spectroscopy
<i>Staphylococcus aureus</i> grown in the presence of an alanine-racemase
inhibitor was labeled with d-[1-<sup>13</sup>C]Âalanine and l-[<sup>15</sup>N]Âalanine to characterize some details of the
peptidoglycan tertiary structure. Rotational-echo double-resonance
NMR of intact whole cells was used to measure internuclear distances
between <sup>13</sup>C and <sup>15</sup>N of labeled amino acids incorporated
in the peptidoglycan, and from those labels to <sup>19</sup>F of a
glycopeptide drug specifically bound to the peptidoglycan. The observed <sup>13</sup>C–<sup>15</sup>N average distance of 4.1–4.4
Ã… between d- and l-alanines in nearest-neighbor
peptide stems is consistent with a local, tightly packed, parallel-stem
architecture for a repeating structural motif within the peptidoglycan
of <i>S. aureus</i>
Role of the Glycopeptide Framework in the Antibacterial Activity of Hydrophobic Derivatives of Glycopeptide Antibiotics
The Carboxyl Terminus of Eremomycin Facilitates Binding to the Non‑d‑Ala‑d‑Ala Segment of the Peptidoglycan Pentapeptide Stem
Glycopeptide
antibiotics inhibit cell wall biosynthesis in Gram-positive
bacteria by targeting the peptidoglycan (PG) pentapeptide stem structure
(l-Ala-d-iso-Gln-l-Lys-d-Ala-d-Ala). Structures
of the glycopeptide complexed with a PG stem mimic have shown that
the d-Ala-d-Ala segment is the primary drug binding
site; however, biochemical evidence suggests that the glycopeptide–PG
interaction involves more than d-Ala-d-Ala binding.
Interactions of the glycopeptide with the non-d-Ala-d-Ala segment of the PG stem were investigated using solid-state nuclear
magnetic resonance (NMR). LCTA-1421, a double <sup>15</sup>N-enriched
eremomycin derivative with a C-terminal [<sup>15</sup>N]Âamide and
[<sup>15</sup>N]ÂAsn amide, was complexed with whole cells
of <i>Staphylococcus aureus</i> grown in a defined medium
containing l-[3-<sup>13</sup>C]ÂAla and d-[1-<sup>13</sup>C]ÂAla in the presence of alanine racemase inhibitor alaphosphin. <sup>13</sup>CÂ{<sup>15</sup>N} and <sup>15</sup>NÂ{<sup>13</sup>C} rotational-echo
double-resonance (REDOR) NMR measurements determined the <sup>13</sup>C–<sup>15</sup>N internuclear distances between the [<sup>15</sup>N]ÂAsn
amide of LCTA-1421 and the <sup>13</sup>C atoms of the bound d-[1-<sup>13</sup>C]ÂAla-d-[1-<sup>13</sup>C]ÂAla to be 5.1
and 4.8 Ã…, respectively. These measurements also determined the
distance from
the C-terminal [<sup>15</sup>N]Âamide of LCTA-1421 to the l-[3-<sup>13</sup>C]ÂAla of PG to be 3.5 Ã…. The measured REDOR
distance constraints position the C-terminus
of the glycopeptide in the proximity of the l-Ala
of the PG, suggesting that the C-terminus of the glycopeptide interacts
near the l-Ala segment of the PG stem. <i>In vivo</i> REDOR measurements provided structural insight into
how C-terminally modified glycopeptide antibiotics operate