Early changes in bacterial envelopes after inhibition of peptidoglycan synthesis, as shown by the use of a fluorescent probe

The probe 8-anilino-1-naphthalene sulphonate (ANS) showed increasing fluorescence with Bacillus subtilis metC lyt-2 cells taken from exponentially growing cultures treated with antibiotics that inhibit cell-wall peptidoglycan synthesis. This increase was due to the probe reaching hydrophobic cell constituents, probably membranes, and started within 30 min of the addition of the antibiotics. This corresponded to the time at which membrane function had been shown to be damaged. The increased fluorescence of the cells with ANS persisted after removal of the antibiotics.     

Effect of rubomycin, carminomycin and adriamycin on respiration in liver mitochondria in various metabolic states, respiratory control and ADP/O ratio

A comparative study on the effects of antitumour antibiotics of the anthracycline group (rubomycin, carminomycin and adriamycin) on respiration and oxidative phosphorylation in liver mitochondria in various metabolic states has been carried out for the first time. It was shown that the antibiotics under study cause partial inhibition of mitochondrial state 3 respiration, which is eliminated by an uncoupler. Treatment of liver mitochondria with the antibiotics decreases the ADP/O and respiratory control values and stimulates state 4 respiration. The latter is partly inhibited by oligomycin. The uncoupled respiration is decelerated in the presence of the antibiotics. Under these conditions the oxidation of succinate is inhibited by lower concentrations of the antibiotics than that of NAD+-dependent substrates. It was shown that the maximal activity is exerted by the most polar agent carminomycin, while the hydrophobic rubomycin is the least active. The experimental results are discussed in terms of the toxic effect of antitumour antibiotics.     

Hydrophobic interactions affect hydrogen bond strengths in complexes between peptides and vancomycin or ristocetin

A study on complexes of the glycopeptide antibiotics vancomycin and ristocetin with various dipeptides and tripeptides shows that the intermolecular hydrogen bond strengths of the complexes are reasonably well correlated with their free energy of formation. Such correlation is not anticipated on the basis of a purely hydrophobic interaction of the peptide side-chains with the antibiotics. It is also shown that the free energy changes observed are very different from those expected as a result of hydrophobic forces. These facts suggest that addition of a hydrophobic group not only allows hydrophobic bonding but also strengthens existing hydrogen bonds. The increased hydrogen bond strength can be an important factor in determining the overall binding energy.     

Antibiotic susceptibility, serum response and surface properties of Klebsiella species

Altogether 130 clinical isolates of five Klebsiella species (K. pneumoniae, K. planticola, K. oxytoca, K. ornithinolytica and K. terrigena) were characterized, for their susceptibility to five antibiotics, for susceptibility to serum bactericidal activity and for their hydrophobic properties. All strains exhibited ampicillin resistance. Ampicillin/sulbactam, gentamicin and ofloxacin showed effectiveness in 63.1, 67.7 and 71.5% of the Klebsiella isolates. K. planticola manifested the highest level of resistance to these antibiotics. The majority of Klebsiella strains (93.9%) were susceptible to cefuroxime. Sixty-four strains (49.2%) were serum resistant and intermediate serum sensitivity was shown by 57 strains (43.8%). A high percentage of serum resistant strains (65%) was found in K. planticola. Moderately hydrophobic properties determined by adherence of bacteria to xylene were demonstrated in 25 strains (19.2%).     

FbpA-Dependent biosynthesis of trehalose dimycolate is required for the intrinsic multidrug resistance, cell wall structure, and colonial morphology of Mycobacterium smegmatis

Ligation of mycolic acids to structural components of the mycobacterial cell wall generates a hydrophobic, impermeable barrier that provides resistance to toxic compounds such as antibiotics. Secreted proteins FbpA, FbpB, and FbpC attach mycolic acids to arabinogalactan, generating mycolic acid methyl esters (MAME) or trehalose, generating alpha,alpha'-trehalose dimycolate (TDM; also called cord factor). Our studies of Mycobacterium smegmatis showed that disruption of fbpA did not affect MAME levels but resulted in a 45% reduction of TDM. The fbpA mutant displayed increased sensitivity to both front-line tuberculosis-targeted drugs as well as other broad-spectrum antibiotics widely used for antibacterial chemotherapy. The irregular, hydrophobic surface of wild-type M. smegmatis colonies became hydrophilic and smooth in the mutant. While expression of M. smegmatis fbpA restored defects of the mutant, heterologous expression of the Mycobacterium tuberculosis fbpA gene was less effective. A single mutation in the M. smegmatis FbpA esterase domain inactivated its ability to provide antibiotic resistance. These data show that production of TDM by FbpA is essential for the intrinsic antibiotic resistance and normal colonial morphology of some mycobacteria and support the concept that FbpA-specific inhibitors, alone or in combination with other antibiotics, could provide an effective treatment to tuberculosis and other mycobacterial diseases.     

Outer membrane of Salmonella typhimurium: Transmembrane diffusion of some hydrophobic substances

The outer membrane, which is composed of lipopolysaccharide, phospholipids, and proteins, is a layer of the cell wall of Gram-negative bacteria, and apparently acts as a penetration barrier for various substances. It had been shown by other workers that “deep rough” mutants of Salmonella typhimurium, whose lipopolysaccharides lack most of the saccharide chains, were much more sensitive than the wild type strain to certain antibiotics and dyes, but not to others. We found that the former group of agents are usually hydrophobic and the latter group mostly hydrophilic. All hydrophilic antibiotics had molecular weights lower than 650, and one of them was shown to diffuse through the outer membrane at 0 °C. In contrast, some hydrophobic antibiotics had molecular weights in excess of 1200, and the rate of diffusion of one of them was shown to be extremely dependent both on temperature and on the structure of lipopolysaccharide present. These data and results presented elsewhere suggest, but do not necessarily prove, that most hydrophilic antibiotics diffuse through aqueous pores, whereas hydrophobic antibiotics and dyes mainly penetrate by dissolving into the hydrocarbon interior of the outer membrane. In contrast to the outer membrane of deep rough mutants, that of the wild type strain and less defective rough mutants was unusual among biological membranes in that it was practically impermeable to hydrophobic agents. It is proposed that the difference in hydrophobic permeability between the two types of strain is due to radical differences in the organization of the outer membrane, more specifically to the presence or absence of exposed phospholipid bilayer regions.     

Dependence of Ion Channel Properties Formed by Polyene Antibiotics Molecules on the Lactone Ring Structure

The properties of ion channels formed in membranes by polyene antibiotics of various chemical structure of hydrophilic and hydrophobic chains are investigated. Small differences in a hydrophylic chain with a changed number of hydroxyl and carbonyl groups significantly influence the values of conductivity and selectivity of the polyene channel. The greater number of double bonds in a hydrophobic part of polyene molecules leads to the higher biological activity of antibiotics. Measurement of anion–cationic selectivity of the channels formed by polyenes showed that anionic selectivity, as well as conductivity of channels, decreases among antibiotics: amphotericin B, nystatin, candidin, mycoheptin, and levorin. The study of physical and chemical properties of the single and hybrid ion channels on the bilayer lipid membranes in the presence of polyene antibiotics makes possible to create a theoretically reasonable recommendation for the targeted synthesis of new antibiotics with the desired properties.     

Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.     

Polyene antibiotics in the membrane environment.

The cytotoxic activity of the polyene antibiotics mainly depends on the appearance of the drug species which arises from drug-sterol complexation. The unsaturation and intact macrolide ring of the polyenes are the requirements for the biological activity. All the polyene antibiotics can form the complex with the sterol having 3 beta-OH group, and planar ring and a hydrophobic side chain. Aromatic polyene antibiotics with positively charged head group have been considered as most potential antifungal agents.     

Degradation of Antibiotics (Tetracycline,Sulfathiazole, Ampicillin) Using Enzymes of Glutathion S-Transferase

Swine wastewater is not easily treated in biological wastewater treatment plants. One reason is that some antibiotics are not easily degradable in a normal treatment system and inhibit the biological organisms in the treatment system. Specifically, tetracycline, sulfathiazole, and ampicillin are representative antibiotics found in poultry wastewater. To degrade these refractory and impediment antibiotics more easily, a special method is needed, such as an enzyme method. This research used a special enzyme in an experiment that tested feasibility with an enzyme assay of biological treatment in vitro. The Glutathion S-Transferases (GSTs) are a family of proteins that catalyze the conjugation of reduced glutathione with a variety of hydrophobic chemicals containing electrophilic centers. Using GSTs, these major antibiotics were transformed into components that were non-toxic to the microorganisms that treat manure wastewater. The initial concentration of tetracycline, sulfathiazole, and ampicillin were 100 mg/L, 100 mg/L, and 50 mg/L, respectively, and the concentration of pig feed was the same as usual. The GSTs have made the effect of biotransformation of antibiotics as their mode. They were 60–70% transformed by GSTs at the end of the degradation reaction. This lowered their inhibitory strength against microorganisms.     

双水相技术在抗生素分离中的应用

简单阐明了双水相技术在抗生素的分离中应用的发展状况,分析了影响双水相系统分离抗生素的各种因素,提出了导致双水相中抗生素非对称分配的主要作用力为疏水作用力,并对双水相技术在抗生素分离中应用的发展方向作了探讨。     

Supersusceptibility to hydrophobic antimicrobial agents and cell surface hydrophobicity in Branhamella catarrhalis

To clarify the cause of the supersusceptibility of Branhamella catarrhalis to macrolide antibiotics, which are well-known to be inactive to most Gram-negative bacteria, we determined its cell surface hydrophobicity by the partition experiment between water and hydrocarbons. Its cell surface was found to be markedly more hydrophobic than that of Escherichia coli or Pseudomonas aeruginosa cells. This suggested that the outer membrane of B. catarrhalis plays no role as a diffusion barrier towards hydrophobic agents.     

Molecular basis of NDM-1, a new antibiotic resistance determinant

The New Delhi Metallo-β-lactamase (NDM-1) was first reported in 2009 in a Swedish patient. A recent study reported that Klebsiella pneumonia NDM-1 positive strain or Escherichia coli NDM-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin. These can no longer be relied on to treat infections and therefore, NDM-1 now becomes potentially a major global health threat.In this study, we performed modeling studies to obtain its 3D structure and NDM-1/antibiotics complex. It revealed that the hydrolytic mechanisms are highly conserved. In addition, the detailed analysis indicates that the more flexible and hydrophobic loop1, together with the evolution of more positive-charged loop2 leads to NDM-1 positive strain more potent and extensive in antibiotics resistance compared with other MBLs. Furthermore, through biological experiments, we revealed the molecular basis for antibiotics catalysis of NDM-1 on the enzymatic level. We found that NDM-1 enzyme was highly potent to degrade carbapenem antibiotics, while mostly susceptible to tigecycline, which had the ability to slow down the hydrolysis velocity of meropenem by NDM-1. Meanwhile, the mutagenesis experiments, including D124A, C208A, K211A and K211E, which displayed down-regulation on meropenem catalysis, proved the accuracy of our model.At present, there are no effective antibiotics against NDM-1 positive pathogen. Our study will provide clues to investigate the molecular basis of extended antibiotics resistance of NDM-1 and then accelerate the search for new antibiotics against NDM-1 positive strain in clinical studies.     

Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H.marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.     

Factors affecting the entry of antibiotics into Escherichia coli

Factors affecting the entry into Escherichia coli of diverse antibacterial agents, especially beta-lactams were investigated. Agents of greater than a critical molecular weight (approximately 600 Daltons) penetrated extremely poorly. However, there was little correlation between penetrative ability and molecular weight for substances below the critical size. Within classes of related antibiotics (e.g. cephalosporins) penetrative ability was highly dependent on hydrophobicity. The relationship was parabolic rather than linear in nature. The proposal that the envelope of E. coli preferentially excludes hydrophobic molecules is to some extent an artefact arising from pre-selection of the agents used. For unrelated antibiotics hydrophobic nature was a poor guide to penetrative ability. A rather empirical property, diffusion ability through agar, was found to show good inverse correlation with penetrative ability for many unrelated antibiotics.     

Reduced expression of outer-membrane proteins in beta-lactam-resistant mutants of Enterobacter cloacae

Two antibiotic-resistant mutants of Enterobacter cloacae (AZT-R and AMA-R), obtained by selection with aztreonam and carumonam, were studied. Both mutants were resistant to beta-lactam antibiotics. In addition, AMA-R was also resistant to chloramphenicol, trimethoprim and brodimoprim, whereas AZT-R was hypersensitive to these compounds. Cytoplasmic and outer membranes of these bacteria were separated by sucrose density gradient centrifugation. Analysis of the outer membranes using SDS-PAGE showed marked changes in the bands corresponding to the porins (between 35 and 40 kDa). In the two mutants, the 39 kDa band was reduced to approximately 30% of the wild-type and the 36.5 kDa band was absent. Labelling of the outer membranes with the hydrophobic photolabel 3-(trifluoomethyl)- 3-(m-[125I]iodophenyl)diazirine ([125I]TID) enabled the above bands as well as a 28.8 kDa band to be identified as integral membrane proteins, thus supporting the suggestion that they correspond to porins and OmpA protein, respectively. Whereas the changes observed in outer-membrane proteins are assumed to be responsible for resistance to beta-lactam antibiotics, the basis of hypersensitivity of AZT-R to hydrophobic antibiotics remains to be more clearly defined.     

Discerning perturbed assembly of lipids in a model membrane in presence of violacein: Effects of membrane hydrophobicity

For the lack of effective antibiotics towards antibiotic resisting bacteria, it is required to discover new antibiotics and to understand their antimicrobial mechanism. Violacein is a violet pigment found in several gram-negative bacteria possessing antimicrobial properties to gram-positive bacteria. This present article investigates the insertion ability of this molecule into a model membrane composed of zwitterionic phospholipids. Thermodynamic characterization of lipid monolayers in the presence of violacein was carried out using a single lipid layer formed at air-water interface. The molecule inserts into the layer altering the area occupied by each lipid and the in-plane compressibility of the film. This insertion increases with the hydrophobic chain length of the lipid. The perturbed self-assembly of lipids in a bilayer is quantified using a lipid multilayer system applying the X-ray reflectivity technique. The electron density profile from the reflectivity data shows that the molecule inserts into the fluid phase creating a relatively ordered chain conformation. Further, the insertion into the gel phase is observed to increase with the increased thickness of the hydrophobic core of a bilayer.     

Structure-activity relationships in a series of semisynthetic polycyclic glycopeptide antibiotics

The main achievements in the development of methods for the design of semisynthetic antibiotics of a new generation belonging to the group of polycyclic glycopeptides directed against infections caused by multidrug-resistant bacteria and dangerous human and animal viruses are reviewed. The review is focused on the results obtained at the Gauze Institute in the area of chemical modification of natural antibiotics (eremomycin, vancomycin, teicoplanin, etc.) directed toward modification of their antibacterial and/or antiviral activity. A special emphasis is placed on the study of the mechanisms of action of these antibiotics, which could be the basis of a rational approach to their chemical modification involving the transformation of the inner binding pocket and the peripheral regions of the molecules that participate in the formation of their complexes with targets. The recently discovered antiviral activity of modified glycopeptides antibiotics is also discussed. A possibility of obtaining new highly active anti-HIV-1 and anti-HIV-2 preparations on the basis of hydrophobic derivatives of the aglycones of glycopeptide antibiotics was demonstrated. New semisynthetic derivatives of antibiotics that exhibit a high antibacterial activity in vivo, have good pharmacological characteristics, and are promising for practical use are described.     

Structure-activity relationships in a series of semisynthetic polycyclic glycopeptide antibiotics

The main achievements in the development of methods for the design of semisynthetic antibiotics of a new generation belonging to the group of polycyclic glycopeptides directed against infections caused by multidrug-resistant bacteria and dangerous human and animal viruses are reviewed. The review is focused on the results obtained at the Gauze Institute in the area of chemical modification of natural antibiotics (eremomycin, vancomycin, teicoplanin, etc.) directed toward modification of their antibacterial and/or antiviral activity. A special emphasis is placed on the study of the mechanisms of action of these antibiotics, which could be the basis of a rational approach to their chemical modification involving the transformation of the inner binding pocket and the peripheral regions of the molecules that participate in the formation of their complexes with targets. The study of the recently discovered antiviral activity of modified glycopeptide antibiotics is also discussed. A possibility of obtaining new highly active anti-HIV-1 and anti-HIV-2 preparations on the basis of hydrophobic derivatives of the aglycones of glycopeptide antibiotics was demonstrated. New semisynthetic derivatives of antibiotics that exhibit a high antibacterial activity in vivo, have good pharmacological characteristics, and are promising for practical use are described.     

Ribosomal crystallography: peptide bond formation, chaperone assistance and antibiotics activity

The peptidyl transferase center (PTC) is located in a protein free environment, thus confirming that the ribosome is a ribozyme. This arched void has dimensions suitable for accommodating the 3' ends of the A-and the P-site tRNAs, and is situated within a universal sizable symmetry-related region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between the elaborate PTC architecture and the A-site tRNA position revealed that the A- to P-site passage of the tRNA 3' end is performed by a rotatory motion, which leads to stereochemistry suitable for peptide bond formation and for substrate mediated catalysis, thus suggesting that the PTC evolved by gene-fusion. Adjacent to the PTC is the entrance of the protein exit tunnel, shown to play active roles in sequence-specific gating of nascent chains and in responding to cellular signals. This tunnel also provides a site that may be exploited for local co-translational folding and seems to assist in nascent chain trafficking into the hydrophobic space formed by the first bacterial chaperone, the trigger factor. Many antibiotics target ribosomes. Although the ribosome is highly conserved, subtle sequence and/or conformational variations enable drug selectivity, thus facilitating clinical usage. Comparisons of high-resolution structures of complexes of antibiotics bound to ribosomes from eubacteria resembling pathogens, to an archaeon that shares properties with eukaryotes and to its mutant that allows antibiotics binding, demonstrated the unambiguous difference between mere binding and therapeutical effectiveness. The observed variability in antibiotics inhibitory modes, accompanied by the elucidation of the structural basis to antibiotics mechanism justifies expectations for structural based improved properties of existing compounds as well as for the development of novel drugs.