Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter.

MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram-positive and Gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB-TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA-MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex.

The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter.

Escherichia coli MacAB-TolC is a tripartite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins.

Impact of stereochemistry on the biological activity of novel oleandomycin derivatives.

A set of 8-methylene-, 8-methyl-, and 8-methyl-9-dihydro-oleandomycin derivatives having different combinations of stereochemistries at positions C-8 and/or C-9 have been prepared in a chemoselective and stereoselective manner and tested in vitro for antibacterial activity and inhibition of IL-6 production. Configurations of the stereocenters at C-8 and C-9 were determined using 2D NMR techniques. We have shown that change of stereochemistry at these positions can exert a major influence on antibacterial activity as well as IL-6 inhibition, providing novel macrolide derivatives with diminished antibacterial and potent anti-inflammatory activity. In addition, the anti-inflammatory activity observed in vitro was confirmed in an in vivo model of lipopolysaccharide-induced inflammation.

[Evolution of antibiotic-resistance Staphylococcus aureus in Saint Camille Medical Centre in Ouagadougou]. / Evolution de la résistance de Staphylococcus aureus aux antibiotiques au Centre Médical Saint Camille de Ouagadougou.

BACKGROUND: Monitoring the antibiotic resistance of microorganisms in a specific geographic area can be useful in developing new approaches to first-intention antibiotherapy. OBJECTIVE: The purpose of this study was to describe the evolution of resistance of Staphylococcus aureus to antibiotics routinely used at Saint Camille Medical Centre in Ouagadougou, Burkina Faso from 1996 to 2006. METHOD: Strains of S. aureus, isolated from various pathologic sources were tested to determine their susceptibility to antibiotics. Sensitivity tests were performed in accordance with the guidelines of the Antibiogram Committee of the French Society for Microbiology (version 2007). RESULTS: During the study period, 1160 staphylococci strains were isolated including 73.45% identified as S. aureus. Susceptibility tests demonstrated a significant increase in resistance to beta-lactam antibiotics. The proportion of strains showing resistance to ampicillin reached 58.29% in 2000. Resistance to these antibiotics regressed significantly from 2000 to 2006. Resistance to pristinamycin and erythromycin showed a tendency to increase while resistance to gentamicin and oleandomycin showed no statistically significant change. CONCLUSION: This study demonstrated that S. aureus was the most common Staphylococcus genus present at the center and that it was resistant to several antibiotics. Reducing use of beta-lactam probably accounted for the significant decline in resistance to this type of antibiotic. Care should also be given to the use of other antibiotics such as pristinamycin and erythromycin since resistance appears to be increasing.

Differential activity profiles of translation inhibitors in whole-cell and cell-free approaches.

AIMS: Evaluation of the activity profiles of standard prokaryotic translation inhibitors with different physicochemical properties under whole-cell and cell-free conditions. METHODS AND RESULTS: The minimal inhibitory concentration values (cell-free/whole-cell microg ml(-1)) for three aminoglycosides (neomycin, 0.01/6.92; paromomycin, 0.7/1.96; streptomycin 1.45/1.57), three macrolides (erythromycin, 1.53/56.9; josamycin, 1.61/87.7; oleandomycin, 5.12/565.9), chloramphenicol (11.9/3.04), and two tetracyclines (tetracycline hydrochloride, not determined/0.63; minocycline hydrochloride, 2.53/1.09), towards Escherichia coli A19 cells were determined with a microtitre plate-based broth dilution method and compared with values determined in a coupled transcription/translation system based on a S30 extract of the same E. coli strain (cell-free) for the production of the green fluorescent protein. CONCLUSIONS: The analysed prokaryotic translation inhibitors showed substance-specific activity profiles under cell-free vs whole-cell conditions that are explainable by the physicochemical properties of the molecules. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the advantages and limits of cell- free transcription/translation (CFTT) experiments for the discovery of novel antimicrobials. The main advantage is the direct access of the target structures (ribosomes) for the inhibitors, and our results provide an estimation of the concentration necessary to detect new agents. The main limitations are that the inhibitory properties of different agents in CFTT experiments do not necessarily reflect their growth inhibition activity in cell cultures.

Identification and antibacterial activity of a new oleandomycin derivative from Streptomyces antibioticus.

During the study on the oleandomycin production, we purified a new oleandomycin derivative having a macrolactone of which biosynthesis does not follow the genetic architecture of the oleandomycin PKS. The molecular formula for the compound was suggested as C35H59NO11 on the basis of the analysis of NMR and HRMS data (m/z 670.4185, Delta-1.9mmu, calcd for C35H60NO11). 13C NMR assignments and analysis of COSY, HMBC and HMQC data suggested that the compound differs from oleandomycin by formation of the olefinic functionality resulting from the dehydration of a hydroxy group in oleandomycin. The new oleandomycin derivative has antibacterial activities similar to those of oleandomycin agaisnt Enterococcus faecalis, Bacillus subtilis and Staphylococcus aureus.

Ring contraction of oleandrose on the macrolide antibiotic oleandomycin with [(Methoxycarbonyl)sulfamoyl]triethylammonium hydroxide inner salt.

Ring contraction of the neutral oleandrose sugar in the 14-membered-ring macrolide antibiotic oleandomycin (2) has been accomplished using [(methoxycarbonyl)sulfamoyl]triethylammonium hydroxide inner salt (1). The product of this interesting rearrangement, after methanolic hydrolysis of the 2'-acetate, is the 11-acetyl-3-O-(3"-methoxy-4"-vinylfuranosyl)oleandomycin (12). The in vitro activity of furanoside 12 is only moderately less than that of 11-acetyloleandomycin (13).

In vivo effects of erythromycin, oleandomycin and erythralosamine derivatives on hepatic cytochrome P-450.

Rats have been treated with several derivatives of the erythromycin, erythralosamine or oleandomycin series, in order to compare their ability to induce cytochrome P-450 and to form stable 456 nm-absorbing cytochrome P-450 metabolite complexes. The data obtained confirm that the cytochromes P-450 induced in rats by various macrolides are similar to that induced by pregnenolone 16 alpha-carbonitrile: the cytochrome P-450 IIIA1 isozyme. It showed that: (i) formation of a stable inhibitory 456 nm-absorbing cytochrome P-450 complex is not a prerequisite for cytochrome P-450 induction but enhances induction by stabilization of the IIIA isozyme. Therefore, the best inducers lead also to the maximal in vivo amounts of cytochrome P-450 metabolite complex (except for 2'MBEM); (ii) affinity for cytochrome P-450 IIIA1 is not directly involved for induction; and (iii) hydrophobicity favors induction and formation of complexes. Structural factors are also involved.

Characterization of the mac-1 gene encoding a putative ABC transporter from Myxococcus xanthus.

The mac-1 gene of Myxococcus xanthus TA, an antibiotic TA producer, encoded a protein with strong sequence similarity to the antibiotic ATP-binding cassette (ABC) transporter for macrolide antibiotics. The mac-1 gene encoding protein (Mac-1) had two ATP-binding domains containing Walker A and B motifs, and no hydrophobic transmembrane regions. Insertional inactivation of mac-1 caused enhanced sensitivity to oleandomycin, a macrolide antibiotic, while the mac-1 mutant showed normal export of antibiotic TA into the extracellular fluid. The mac-1 mutant could form mounds, but was unable to form fruiting bodies or sporulate under nutrient starvation. A primary role for Mac-1 in M. xanthus may be as a transporter which exports or imports a molecule required for the sporulation process.

Purification and characterization of macrolide 2'-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics.

The resistance mechanism of Escherichia coli BM2506 to macrolides was found to be due to inactivation. Inactivated oleandomycin was identified as oleandomycin 2'-phosphate by thin-layer chromatography. A new type of macrolide-phosphorylating enzyme, macrolide 2'-phosphotransferase type II (MPH(2')II), was detected, purified 95-fold and its enzymological properties investigated. MPH(2')II was a constitutive intracellular enzyme which showed high levels of activity with both 14-member-ring and 16-member-ring macrolides. The optimum pH for the inactivation of oleandomycin was 8.2 and the optimum temperature of the reaction was 40 degrees C. Enzyme activity was lost by heat treatment at 60 degrees C for 1 min. The isoelectric point and M(r) of the enzyme were 5.3 and 48,000, respectively. Purine nucleotides, such as ITP, GTP and ATP, were effective as cofactors in the inactivation of macrolides. An inhibitory effect of iodine, EDTA, or divalent cations on MPH(2')II activity was observed.

Topological studies of the membrane component of the OleC ABC transporter involved in oleandomycin resistance in Streptomyces antibioticus.

The OleC ABC transporter of Streptomyces antibioticus is constituted by an ATP-binding protein (OleC) and a hydrophobic protein (OleC5). Here we present experimental evidence demonstrating that the OleC5 protein is an integral membrane protein and we propose a topological model for its integration into the membrane. This model is based on the generation of hybrid proteins between different regions of OleC5 and a Escherichia coli beta-lactamase (BlaM) and the determination of the minimal inhibitory concentrations to ampicillin in these constructions. Fusions were generated both by cloning specific fragments of oleC5 and by creating ExoIII nested deletions of the gene. In the topological model proposed there will be six alpha-helix transmembrane regions, two cytoplasmic and four periplasmic loops and a hydrophobic linker domain.

A dyadic plasmid that shows MLS and PMS resistance in Staphylococcus aureus.

Out of a collection of 56 Staphylococcus aureus clinical strains from 1971 to 1990 in Japan, we found one 1971 isolate, strain MS8968, harboring plasmid pMS97. A transductant strain, MS15009(pMS97), showed inducible resistance to a group of drugs, the so-called MLS antibiotics in the presence of a low concentration of erythromycin (EM). However, in the case of oleandomycin (OL), the strain showed resistance to another group of antibiotics: 14-membered macrolides (EM and OL), a 16-membered macrolide (mycinamicin I), and type B streptogramin, the so-called PMS antibiotics. Moreover, plasmid pMS97 contained an erm gene with universal primers specific for erm A, AM, B, BC, C, C', and G and an msrA gene with primers specific for msrA. The first finding suggests that two genes encoding functionally different mechanisms for MLS and PMS resistance, erm and msrA, are present together within plasmid pMS97 originating from S. aureus.

Identification of Escherichia coli clinical isolates producing macrolide 2'-phosphotransferase by a highly sensitive detection method.

Macrolide is inactivated with ATP plus crude extract of Escherichia coli producing macrolide 2'-phosphotransferase (MPH(2')), but not by living cells. Therefore, a convenient method for detection of MPH(2') using intact cells is needed. In this report, we determine that the modified lysozyme-DNase-RNase (LDR) method (named ELDR method) is at least one hundred times more sensitive for the detection of MPH(2') activity than the LDR method and, in addition, highly sensitive for the detection of aminoglycoside-modifying enzymes. Therefore, three new MPH(2')-producing strains were found in clinically isolated E. coli in Japan in 1997 by this method. It suggests that MPH(2')-producing E. coli have been spread in Japanese clinical fields.

Synergy of four macrolide antibiotics with chloroquine against chloroquine-resistant Plasmodium falciparum in vitro.

The antimalarial activity of four macrolide antibiotics was investigated against the multidrug resistant K1 strain of Plasmodium falciparum in vitro. ID50 (50% inhibitory concentration) values for erythromycin, spiramycin, tylosin tartrate and oleandomycin phosphate in 48-hour assays were 1.6 X 10(-4)M, 2.5 X 10(-5)M, 1.2 X 10(-5)M and 9 X 10(-6)M respectively, and in 96 hour assays were 10(-5)M, 2.6 X 10(-6)M, 2.6 X 10(-6) and 3 X 10(-6)M, respectively. Comparable values were obtained in assays in which drug effect was quantified from either parasite counts or 14C isoleucine incorporation. Each of the four macrolides displayed synergy with chloroquine at the IC90 (90% inhibitory concentration) level, but at the IC50 level synergy was either less pronounced or absent. For each combination this difference in the degree of synergy was significant at the 95% level of confidence. In replicate assays in which 3H hypoxanthine was the marker of drug effect, synergy between chloroquine and either erythromycin or spiramycin could not be detected.

Mutagenicity of antibiotics in microbial assays. Problems of evaluation.

5 antibiotics, 4 of which inhibit protein synthesis in different ways, and 1 of which inhibits bacterial cell-wall synthesis, were tested in a battery of microbial assays for possible genetic effects. All the antibiotics, chloramphenicol, tetracycline, gentamicin, oleandomycin and phosphonomycin induced forward mutation to L-azetidine-2-carboxylic acid resistance in Escherichia coli WP2. This response was closely correlated with the toxic effects and was inferred to be deletion mutation. In addition, chloramphenicol was weakly active in reversion of the frame-shift mutation in Salmonella typhimurium TA98, gentamicin caused petite induction in S. cerevisiae at pH 4.4--4.7 and tetracycline gave a significant reponse with gene conversion and petite induction also in S. cerevisiae but at pH 7.2. The results, particularly those with E. coli, cast doubts on the validity of testing specifically designed antibacterial agents in bacteria, and raise serious problems in the evaluation of such data in terms to human populations.

New oleandomycin 9-oximes. Synthesis, characterization and biological activity.

A series of the novel oleandomycin 9-oximes has been prepared and characterized by spectroscopic data and X-ray analysis. The antibacterial in vitro activities of the oximes (6-10) were compared with that of oleandomycin (1). Among the novel derivatives the most active compound was 8(R)-methyloleandomycin-9-oxime (9) in contrast ot its 8(S)-isomer (10) which possessed only low potency. Some preliminary pharmacokinetic data of 9 confirmed its activity. Compound 9 has been advanced to further biological study.

A new semisynthetic macrolide antibiotic 3-O-oleandrosyl-5-O-desosaminylerythronolide A oxime.

A new antibiotic, 3-O-oleandrosyl-5-O-desosaminylerythronolide A oxime (3) was produced from erythronolide A oxime (1) by the oleandomycin-producing culture, Streptomyces antibioticus ATCC 11891. The structure of 3 was determined by degradative studies and confirmed by X-ray analysis. Compound 3 was found to be less active, but more stable to acid, then erythromycin A oxime.

Effect of various feed additives on immune response of turkeys vaccinated with live Pasteurella multocida in drinking water.

Various feed additives were included in diets of young turkeys being administered live Pasteurella multocida, Clemson University (CU) strain, in drinking water. Erythromycin, Neomycin, and Oleandomycin reduced immune response the most. Most medications that are not effective against P. multocida can be included in turkey feed during CU vaccination.

Resistance to macrolide-lincosamide-streptogramin antibiotics in enterococci from the intestines of animals.

Macrolide-lincosamide-streptogramin group B (MLS) resistant strains were found among enterococci isolated from caeca of poultry, pigs and cattle. The percentage of MLS resistance among the poultry strains was 70 per cent. The Streptococcus faecium strains were more susceptible than the other enterococcal strains to virginiamycin, a member of the streptogramin class of antibiotics. This was due to higher susceptibility of the S faecium strains to the virginiamycin component M which belongs to the streptogramin group A antibiotics. Ability to inactivate clindamycin, an antibiotic of the lincosamide class of antibiotics, was noted in an unclassified group D streptococcus strain isolated from a pig.

Induction of mutants resistant to antibiotics in Brevibacterium flavum.

The optimum conditions for the induction of mutants resistant to antibiotics in Brevibacterium flavum ATCC 14067 were determined. UV irradiation at the energy fluence of 6.5 kJ/m2 and N-methyl-N'-nitro-N-nitrosoguanidine (1 mg/mL) at pH 6.0 were used for the induction of mutants. Mutant strains resistant to rifampicin, oleandomycin, streptomycin and erythromycin were prepared.