Pristinamycin accumulation by Staphylococcus aureus.

Pristinamycins IA and IIA (PIA and PIIA) accumulation by Staphylococcus aureus has been studied with two hydrogenated analogs, (H2)PIA and (H2)PIIA. Rapid accumulation of both antibiotics at 37 degrees C is observed and internal concentrations can reach up to 58-fold the external concentration; this accumulation cannot be reduced by either metabolic inhibitors or tetracycline. The synergistic activity of pristinamycins IA and IIA is not observed at the bacterial accumulation level. We propose that pristinamycins enter into bacteria by a passive diffusion process and that the internal concentration is maintained by binding of the antibiotic to the bacterial ribosomes.

Have deoxystreptamine aminoglycoside antibiotics the same binding site on bacterial ribosomes?

(3H) Tobramycin was used as a probe to determine the relationship between the structure of aminoglycoside antibiotics and their ability to remove this drug from its higher affinity binding site on the ribosome. The dissacharide moieties (neamine, tobramine, gentamine) appeared to have a common binding site, whereas the kanosamine, garosamine and ribose moieties determined the specificity of this binding. Amikacin and butikacin behaved in an anomalous manner in spite of their close structural relationship to tobramycin.

Binding of tobramycin to Escherichia coli ribosomes: characteristics and equilibrium of the reaction.

A sample of [3H] tobramycin (5,000 Ci/Mole) has been synthetized and incubated with the bacterial ribosome and its subunits. The results obtained show that this antibiotic has two types of binding sites. The primary one is probably responsible for the inhibition of protein synthesis whereas the secondary one is probably related to the misreading and reading through of the messenger RNA.

Studies on pristinamycin synergism in Staphylococcus aureus.

Binding experiments were performed with both components of the pristinamycin complex (pristinamycin IA (PIA) and pristinamycin IIA (PIIA] using ribosomes from sensitive and resistant Staphylococcus aureus. Fluorescence polarization was used to measure PIA binding. The results obtained show a direct correlation between inhibition, synergy and the enhancement of the affinity of PIA for its receptor in the presence of PIIA. The uptake of PIA by intact cells seems to be directly correlated with affinity between PIA and ribosomes, a phenomenon which is probably shared with the macrolide antibiotics.

Irreversible binding of pristinamycin IIA (streptogramin A) to ribosomes explains its "lasting damage" effect.

In vitro and in vivo studies are presented to test the hypothesis that the synergistic action of the pristinamycins is not due to a catalytic effect of pristinamycin IIA (PIIA) on the bacterial ribosome. We demonstrate that there is a proportionality between the quantity of PIIA bound on the ribosome and pristinamycin IA (PIA) retained by it. Moreover in vitro and in vivo experiments correlated to biological effects (growth and protein synthesis) demonstrate that pristinamycin IIA is tightly bound on 70S ribosome, which satisfactory explains the so called "lasting damage effect".

Effects of cations, polyamines and other aminoglycosides on gentamicin C2. Binding to ribosomes from sensitive and resistant Escherichia coli strains.

Gentamicin C2 interacts cooperatively with ribosomes from a sensitive Escherichia coli strain in a multiphasic way with several classes of sites. It is shown that this binding is highly-dependent on Mg++ and natural endogenous polyamine concentrations. The differences observed between ribosomes from sensitive and resistant strains may be explained by the absence of specific cooperative gentamicin interactions with resistant ribosomes. The effects of other aminoglycoside antibiotics are discussed in terms of structure-activity relationships.

Plasmid-mediated pristinamycin resistance. PAC IIA: a new enzyme which modifies pristinamycin IIA.

A wild strain of Staphylococcus aureus which inactivates a wide variety of antibiotics has been found to inactivate pristinamycin IIA, an antistaphylococcal antibiotic. This phenomenon has been demonstrated to be plasmid mediated. The plasmid directs the biosynthesis of an acetyltransferase which is able to O-acetylate the drug. We propose to call the new enzyme PAC (IIA): Pristinamycin acetyltransferase.

Photo-induced labelling of Escherichia coli ribosomes by a tobramycin analog.

An [3H]azidobenzyl derivative of tobramycin, a 4,6-disubstituted 2-deoxystreptamine aminoglycoside, has been synthesized, and its ability to label Escherichia coli 70-S ribosomes under photoactivation has been studied. Two concentrations of the photolabel, corresponding to the saturation of the two classes of tobramycin sites on the ribosomes, were used. The results show that, at high antibiotic concentrations which induce maximal misreading during protein synthesis, most of the ribosomal proteins are labelled. At low antibiotic concentration, which results in the saturation of the first-class sites, a few proteins of both subunits are labelled, including L6, S4, S5, and, to a lesser extent, L2, L13 and S18. The 30-S subunit is, on the whole, labelled more efficiently than the 50-S subunit.

Overexpression, purification and photoaffinity labeling with a 3H-analogue of norfloxacin, of the GyrA and GyrB subunits of the DNA gyrase.

In spite of much work on DNA gyrase and quinolones for many years, our knowledge of the molecular basis of quinolone-gyrase action is still incomplete. We designed a photoaffinity labeling reagent for the quinolone target, and synthesized a norfloxacin analogue with an azide function which, under UV irradiation, becomes covalently linked to its target. For that, a large amount of purified gyrase was needed. Both subunits were purified using exclusion and affinity chromatography. A plasmid was used that allowed the overproduction of GyrA as a fusion-protein with six histidine residues at its carboxy-terminal domain. GyrA-(His)6 was purified after chromatography on a nickel-containing column, and native GyrB after chromatography on immobilized novobiocin. Reconstituted DNA gyrase (A2B2) had supercoiling activity. Photoaffinity labeling showed covalent binding of the 3H-photoaffinity analogue of norfloxacin to the gyrase-DNA complex, and mainly to the GyrA. The specific binding site remains to be explored.

[Analysis of biospecific complexes by electrophoresis: antibiotics specific to bacterial ribosomes]. / Etude de complexes biospécifiques par électrophorèse: application aux antibiotiques spécifiques du ribosome bactérien

Electrophoresis is a widespread method in biochemistry. Apparently it has never been used in the study of interactions found in enzyme-substrates or drug-receptor complexes. The aim of this Note is to describe some possibilities given by this simple technique especially in the case of interactions found between some antibiotics and the bacterial ribosome. Chloramphenicol, pristinamycin are located in 50 S subunit whereas dihydrostreptomycin is found in 30 S particle (fig). Such results have been previously obtained by other methods. The distribution of the gentamicin and kanamycin components is quite different. This suggests that the mechanism of action of these molecules may be different from that of streptomycin group antibiotics.

RP 59500: a proposed mechanism for its bactericidal activity.

RP 59500 is a combination of RP 57669 and RP 54476, which are semisynthetic water soluble derivatives of pristinamycin IA (PIA) and pristinamycin IIA (PIIA), respectively. Like their precursors, these molecules are bacteristatic in their own right. In association, they exert bactericidal activity against a variety of Gram-positive bacteria. Experiments involving the binding of these antibiotics to the target bacterial ribosome showed that both the binding sites and the mechanism of action of the components of RP 59500 are identical to those of the parent molecules. By affinity-labelling with a structural analogue of RP 57669, it was demonstrated that L24, a protein of the 70S ribosomal subunit, was specifically labelled. Experiments using radioactive N-ethylmaleimide to label proteins possessing a thiol residue, indicated that proteins L24, L10 and L11 are not only close to each other in the ribosomal structure, but are also adjacent (if not actually part of) the channel through which newly synthesized proteins are extruded. We propose that the mechanism of action of these compounds is to close or narrow the extrusion channel of these proteins, which could lead to their accumulation on the ribosome. We cannot exclude, of course, the possibility that this accumulation disturbs peptidyl-tRNA hydrolase (PHT) activity, thereby depleting free tRNAs within the cell and inhibiting protein synthesis.

Photo-induced affinity labeling of Escherichia coli ribosomes by chloramphenicol.

In order to obtain more information about the binding site for chloramphenicol (D-threo diastereoisomer) on the bacterial ribosome, photo-affinity labeling experiments of this receptor have been performed with [3H]chloramphenicol itself. Control experiments show that this drug can be split photochemically by ultraviolet irradiation, whereas the ribosome is not modified structurally or functionally by such a treatment. When photolysis of a mixture of chloramphenicol and ribosomes is performed under critical conditions, some proteins like L1, L11, S3 and S4 are radiolabeled. L11, S3 and S4 are radiolabeled specifically as demonstrated by photo-incorporation experiments with isotopically diluted [3H]chloramphenicol or by comparison of the results obtained here with reversible experiments performed by the isotopic dilution method. When the D-erythro diastereoisomer of chloramphenicol is photo-incorporated into the bacterial ribosome, proteins are radiolabeled only in a non-specific way. These results show that this material could be used as an efficient scavenger. When finally D-threo [3H]chloramphenicol is photo-incorporated in the presence of a large amount of the D-erythro diastereoisomer, the radiolabeling pattern obtained for the proteins is quite different from that expected: while L11 is still labeled fairly extensively, L27 is the most radiolabeled protein found.

Mechanism of action of aminoglycoside antibiotics. Binding studies of tobramycin and its 6'-N-acetyl derivative to the bacterial ribosome and its subunits.

6'-N-[14C]Acetyl-tobramycin and [3H]tobramycin were synthesized and their binding to Escherichia coli ribosomes and ribosomal subunits studied using equilibrium dialysis. THE 70-S ribosome, as well as its 50-S and 30-S subunits, bound tightly to 6'-N-acetyl-tobramycin. The binding of [3H]tobramycin to ribosomes was quite different. The 70-S ribosome was observed to possess several classes of binding sites; of these, one was determined to be of higher affinity and lower capacity, the 6'-N-[14C]acetyl-tobramycin site. The isotopic dilution method was used to define the specificity of the interaction. The selective binding of 6'-N-[14C]acetyl-tobramycin was highly reversible by tobramycin, kanamycins A, B, C and neomycin, but not by streptomycin or erythromycin. Gentamicin C1a was a poor inhibitor. This suggested that either the kanosamin or garosamin rings might be determinant in the binding of these molecules, as well as the 6'-amino group.

[Water-soluble derivatives of factor IA of pristinamycins. Interaction with the bacterial ribosome]. / Dérivés hydrosolubles du facteur IA des pristinamycines. Interaction avec le ribosome bactérien.

The IA component of pristinamycins is a depsipeptide with a bacteriostatic effect on Gram positive bacteria. IA is made bactericidal by association with the IIA component of pristinamycins. Use of IA is limited because of its poor solubility in water. For this reason several water soluble IA derivatives have been synthesized. As for IA, these derivatives are fluorescent, a property used to study the binding of each compound to bacterial ribosomes of Escherichia coli (G-) and Staphylococcus aureus (G+). Two different techniques were used: direct study of fluorescence of the ribosome-antibiotic complex, and study of polarization of the fluorescence of the antibiotic bound to its receptor site. In addition to determination of binding parameters, these techniques can evaluate molecular synergy between pristinamycin IA (PIA) derivatives and pristinamycin IIA (PIIA) derivatives. MICs for the tested molecules correlate strongly with their binding parameters.

Autoradiography of tissue distribution of the IIA constituent of the pristinamycins.

The tissue distribution of a radioactive analogue of the IIA constituent of the pristinamycins was studied in female mice by autoradiography. Examination of slides and photographs discloses the presence of the antibiotic on the skin and in the bone marrow only a short time after injection. Elimination of the antibiotic is quick and is mainly through the digestive tract.

Mechanism of action of gentamicin components. Characteristics of their binding to Escherichia coli ribosomes.

The binding of gentamicin (Gm) to Escherichia coli ribosomes and ribosomal subunits has been studied. By means of equilibrium dialysis and of statistical interpretation of the data it was found that [3H]gentamicin C2 and 6'-N-[3H]methylgentamicin C1a interact with three classes of sites on tight-coupled 70-S species: a first class concerning the tight and non-cooperative interaction with one drug molecule (Kd = 0.6 microM), a second class in which about five Gm molecules bind cooperatively (mean Kd = 10 microM), and a third class of very high capacity in which up to 70 drug molecules may interact. The extreme cooperativity of the third class of sites induces such an increase in the affinity for Gm that it may allow the shift of molecules already bound from high-affinity sites towards lower-affinity sites. The alteration of a ribosomal protein, L6, in a gentamicin-resistant mutant of E. coli abolished the multiclass and the cooperative aspects of ribosomes--gentamicin interaction. The large ribosomal subunits from E. coli MRE 600 strain interact cooperatively with Gm, whereas 50-S particles from the resistant mutant bind the drug in a diffuse way with high capacity and low affinity. The small subunits from both strains behave identically towards Gm. A good correlation is observed in comparing the gentamicin concentrations capable of saturating the different ribosomal classes of sites with concentrations inducing its multiphasic effects on protein synthesis.

Antibacterial action of amphipathic derivatives of isoniazid against the Mycobacterium avium complex.

The antibacterial action of amphipathic derivatives of isoniazid (INH) as compared to the parent hydrophilic molecule was determined against the bacteria of the Mycobacterium avium complex (MAC) using a 7H11 agar-dilution method. The results obtained showed a higher activity of 1-isonicotinyl-2-palmitoyl hydrazine and 1-isonicotinyl-2-(12 hydroxy dodecanoyl) hydrazine as compared to INH. However, when one mannose residue was terminally attached to the fatty acid chain of the latter, it lost its anti-MAC activity. 1-isonicotinyl-2-D-galacturonic acid hydrazone (but not hydrazine) also showed increased activity against MAC. Although pristinamycin was shown to bind to M. avium surface lipids, the INH-pristinamycin derivative was not more active than INH alone. These findings are discussed in respect to a proposed mechanism of diffusion across a lipid barrier.

2"-O-phosphorylation of gentamicin components by a Staphylococcus aureus strain carrying a plasmid.

A wild-type strain of Staphylococcus aureus that inactivates the 4,6-glycosidically linked deoxystreptamine aminoglycoside antibiotics by a plasmid-mediated process was found to harbor two enzymes: an acetyltransferase of the AAC(6') type and a new phosphotranferase specific to the gentamicin components. The target of this last enzyme is the 2''-hydroxyl function of these antibiotics, since one inactivated compound is 2''-(O)-phosphorylsisomicin.