In vitro selection of RNA aptamers that can bind specifically to Tat protein of HIV-1.

The Tat protein of human immunodeficiency virus interacts specifically with its target RNA sequence, TAR, and activates viral gene expression at the early stage of infection. Here, we have used in vitro genetic selection strategy to determine what sequence or structural motifs might exist between RNA's that interact specifically with the Tat protein. Starting from a RNA pool that has a 120 base random sequence core, aptamers that bind specifically to the Tat protein were selected by repeating 11 rounds of selection and amplification. A comparative analysis of 64 aptamers that were isolated from the 11th generation revealed two main sequence classes. Interestingly, one of these two classes of aptamers had minimum of one U residues in bulge loop and 2 specific adjacent base pairs. This region is very much homologous to the core sequence of TAR RNA that is essential for the specific Tat-TAR interactions. Further analyses of the sequences from the 11th generation should reveal what kind of RNA structures are required in order to show a high affinity for the Tat protein.

The susceptibility of conjugative resistance transfer in gram-negative bacteria to physicochemical and biochemical agents.

Over thirty years of studies have established that conjugative transfer of plasmid-encoded resistance to drugs and heavy metals can take place at high frequency between various organisms under laboratory conditions. The detected transfer frequencies in soil, in aquatic environments, and in the urogenital and respiratory tracts of healthy animals and man have generally been low. However, the conversion of bacteria from susceptible to resistant to antibiotics has been observed often during antimicrobial therapy. This has formed a challenge for the antibacterial treatment of pathogenic bacteria and called for the evaluation of the extent of conjugative transfer in various environments. Several biochemical and physicochemical factors inhibit conjugation, show preferential toxicity against plasmid-bearing cells, or stimulate plasmid curing. These factors include various agents such as detergents, anesthetics, mutagens and antibiotics which affect membrane potential, membrane permeability, protein synthesis and the processing of DNA. The application of the data on these agents, summarized in this review, might be helpful in preventing drug multi-resistance from spreading. Also these data might be valuable in studies which use conjugation as a tool or which treat the molecular mechanisms involved in conjugation.

The outer membrane permeability-increasing action of deacylpolymyxins.

The outer membrane permeability-increasing action of deacylpolymyxins was compared to the well-known potent action of polymyxin B nonapeptide (PMBN). Deacylpolymyxin B (DAPB), prepared by treating polymyxin B with polymyxin acylase, was found to be a slightly more effective permeabilizer than PMBN. As low a DAPB concentration as 1 microgram/ml sensitized Escherichia coli to the probe antibiotics (rifampin, fusidic acid, erythromycin, clindamycin, novobiocin) by factors 30-100 and Salmonella typhimurium by factors 10-100. A higher concentration (3 micrograms/ml) of DAPB elicited further sensitization. Also deacylcolistin (DAC) was found to be an effective permeabilizer.

Effect of small cationic leukocyte peptides (defensins) on the permeability barrier of the outer membrane.

Defensins are small cationic antibacterial peptides that are abundant in polymorphonuclear leukocytes from human and other sources (T. Ganz, M. Selsted, D. Szklarek, S. Harwig, K. Daher, D. F. Bainton, and R. J. Lehrer, J. Clin. Invest. 76:1427-1435, 1985). We studied whether subinhibitory concentrations of defensins increase the outer membrane (OM) permeability of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to hydrophobic probes, as do many other polycations that have been studied previously. Throughout the study, we used polymyxin B nonapeptide (PMBN) as a reference peptide. PMBN has a known potent OM permeability-increasing action. As a sharp contrast to PMBN, subinhibitory concentrations of defensins did not permeabilize (or, under some test conditions, permeabilized very slightly) the OM to the probes that were used (rifampin and Triton X-100). At bacteriostatic or bactericidal defensin concentrations, some degree of synergism with rifampin was seen.

Polycations which disorganize the outer membrane inhibit conjugation in Escherichia coli.

Outer membrane disorganizing polycation, polymyxin B nonapeptide (PMBN), reduced drastically the production of recombinants when present at sub-MIC concentrations during F'-mediated Escherichia coli conjugation. The decrease of recombinants was accompanied by a less marked decrease of viability in the recipient population in a manner resembling lethal zygosis. No reduction was seen when either donor or recipient was grown in the same PMBN concentration, washed and resuspended to PMBN-free medium before mating. The same concentration of outer membrane disorganizing polycations of higher bactericidal activity (protamine and polylysine) caused only a moderate reduction in transconjugant frequency when present during mating. Spermine and tetralysine, which are not effective disorganizers of the outer membrane, did not reduce the recombinant frequency or the viability of the recipients.

The effect of polymyxin B nonapeptide (PMBN) on transformation.

The effect of the outer membrane permeabilizing polycation, polymyxin B nonapeptide (PMBN) on the transformation of E. coli HB101 with pBR322 plasmid DNA was investigated. Pretreatment of cells with PMBN (followed by suspending the cells in PMBN-free medium) did not stimulate the development of competence induced by the calcium heat pulse. In the absence of calcium-ions, a high PMBN concentration (1 mM) was able to induce a low transformation frequency provided that PMBN was not removed before the addition of DNA.

Fimbrial phase variation and systemic E. coli infection studied in the mouse peritonitis model.

Mouse peritonitis induced by intraperitoneal injection of a virulent (LD50 4 x 10(5) E. coli 018:K1:H7 strain isolated from neonatal meningitis was studied. These bacteria are capable of producing both type 1 and S fimbriae, binding to mannose or sialic acid containing glycoconjugates, respectively; the production of both fimbrial types is subject to phase variation. A broth culture of the bacteria was fractionated into subpopulations containing either type 1 or S fimbriae or neither (nonfimbriated cells), and each fraction, grown in broth to logarithmic growth phase, was used to infect groups of mice. The type 1 fraction was associated with decreased virulence as the fraction was eliminated rapidly without causing a progressive infection even at 10(6) bacteria/mouse, whereas both S and nonfimbriated cells started rapid multiplication in the peritoneal cavity and spread to the blood. In nonfibriated cells, however, S fimbriae production was induced at the same time so that at 1 h after injection, 60-70% of the bacteria in the peritoneal cavity and in the blood of the mice had S fimbriae. The injected S-fimbriated fraction remained completely S-fimbriated. Rapid induction of S fimbriae also took place in vitro when the nonfimbriated bacteria were grown in mouse serum or peritoneal fluid. Anti-S serum protected the mice from a lethal dose of S-fimbriated bacteria.

Susceptibility of gram-negative bacteria to the synergistic bactericidal action of serum and polymyxin B nonapeptide.

Polymyxin B nonapeptide was able to sensitize Escherichia coli strains and strains of Salmonella typhimurium, Klebsiella spp., Enterobacter cloacae, Pseudomonas aeruginosa, and Haemophilus influenzae to the bactericidal action of fresh normal human serum. The degree of sensitization varied significantly within the strains. Strains of Proteus mirabilis, Neisseria gonorrhoeae, and N. meningitidis remained resistant.

Binding of polymyxin B nonapeptide to gram-negative bacteria.

The binding of the outer membrane-disorganizing peptide polymyxin B nonapeptide (PMBN) to gram-negative bacteria was studied by using tritium-labeled PMBN. Smooth Salmonella typhimurium had a binding capacity of ca. 6 nmol of PMBN per mg (dry weight) of bacteria, which corresponds to ca. 1 X 10(6) to 2 X 10(6) molecules of PMBN per single cell. The binding was of relatively high affinity (Kd, 1.3 microM). The isolated outer membrane of S. typhimurium bound ca. 100 nmol of PMBN per mg of outer membrane protein (Kd, 1.1 microM), whereas the cytoplasmic membrane bound 9 to 10 times less. Other bacteria which are susceptible to the action of PMBN (Escherichia coli strains, Pseudomonas aeruginosa, Haemophilus influenzae) also bound large amounts of PMBN. The S. typhimurium pmrA mutant, Neisseria gonorrhoeae, and Proteus mirabilis (all known as resistant to polymyxin and PMBN) bound 3.3, 4, and 12 times less than S. typhimurium, respectively. The binding of PMBN to S. typhimurium was effectively inhibited by low concentrations of polymyxin B, compound EM49 (octapeptin), polylysine, and protamine. Spermine, Ca2+, and Mg2+ also inhibited the PMBN binding although they were ca. 160, 700, and 2,400 times less active (based on molarity) than polymyxin B, respectively. No binding inhibition was found at the tested concentrations of streptomycin, tetralysine, spermidine, or cadaverine.

New Salmonella typhimurium mutants with altered outer membrane permeability.

We describe three new classes of Salmonella typhimurium mutants with increased sensitivity to hydrophobic agents. In contrast to many previously described mutants, the phage sensitivity pattern of these mutants did not give any indication of defective lipopolysaccharide. Furthermore, they had no detectable changes in their phospholipid or outer membrane protein composition, and their growth rate and cell morphology were normal. Class B mutants were nearly as sensitive to novobiocin, fusidic acid, erythromycin, rifampin, and clindamycin as are deep rough (heptoseless) mutants; in addition they were sensitive to methicillin, penicillin (to which heptoseless mutants are resistant), gentian violet, and anionic and cationic detergents. Class A and C mutants had less sensitive, but characteristic phenotypes. None of the three classes were sensitive to serum bactericidal action. The class B mutation mapped between map positions 7 and 11 on the S. typhimurium chromosome, and the class C mutation mapped between positions 5 and 7. The map position for the class A mutation remained undefined, but it was separate from the class B and C mutations and, like those, did not correspond to any gene loci known to participate in the synthesis of major outer membrane constituents.

Susceptibility of gram-negative bacteria to polymyxin B nonapeptide.

Subinhibitory concentrations of polymyxin B nonapeptide sensitized all 21 polymyxin-susceptible gram-negative bacterial strains studied to hydrophobic antibiotics such as fusidic acid, novobiocin, and erythromycin. The susceptibility increases were usually 30- to 300-fold. The strains included representatives of Escherichia coli with different O- and K-antigens, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter agglomerans, Salmonella typhimurium, Acinetobacter calcoaceticus, Pseudomonas aeruginosa, and Pseudomonas maltophilia. In contrast, polymyxin-resistant strains (Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia stuartii, and Serratia marcescens) were resistant to the action of polymyxin B nonapeptide.

An outer membrane-disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action.

The small cationic outer membrane-disorganizing peptide PMBN sensitized four smooth, encapsulated strains of Escherichia coli (serotypes 02:K1, 04:K12, 018:K1, and 018:K5) to the lethal action of serum. The concentrations of PMBN required were low (0.3 to 1.0 microgram/ml). One E. coli strain (IH 11030; 075:K5) remained virtually resistant to serum and also to anti-075 hyperimmune serum plus complement (C) even in the presence of PMBN. This strain was nevertheless sensitive to the outer membrane permeability-increasing action of PMBN. In the bactericidal system, PMBN could be replaced by high concentrations of lysine20 or protamine but not lysine4. The PMBN-dependent bactericidal activity of GPS was abolished by heating or zymosan treatment that inactivate its C but not by lack of the action of the classical pathway of the C in C4-deficient GPS. PMBN formed a bactericidal system also with normal rabbit, rat, and human serum but not with mouse serum. The bactericidal system against E. coli 018:K1 and its derivative EH 817 (018:K1-) was found to require a factor that can be removed from normal sera by absorption with a rough E. coli strain. This factor could be replaced by specific anti-018 antibodies. The bactericidal activity of fetal calf serum plus PMBN against E. coli 018:K1 was enhanced by normal rabbit or anti-E. coli 018 hyperimmune serum. We suggest that PMBN unshields the deep structures and the hydrophobic membrane milieu of the outer membrane and facilitates the insertion of the membrane attack complex of the C into this milieu.