Mutual Relationship between Antibiotics and Resting Spores of Bacillus subtilis: Binding of Cyclic Polypeptide and Aminoglycoside Antibiotics to Spores and Their Inhibitory Effect on Outgrowth and Vegetative Growth

Not only cyclic polypeptide antibiotics such as polymyxin B, colistin and gramicidin S but also aminoglycoside antibiotics such as streptomycin, kanamycin, gentamicin and kanamycin derivatives combined with the resting spores of Bacillus subtilis and inhibited outgrowth or vegetative growth after germination. All the antibiotics other than gramicidin S were released from the resting spores and their inhibitory action was reversed by the addition of Ca²⁺ and Fe³⁺. As the above antibiotics have free amino (or guanidine) groups in common, it was assumed that such groups play an important role in binding of the antibiotics to the resting spores. Moreover, it was shown that protamine and poly-l-lysine were also bound to the resting spores and were released from them by Ca²⁺. On the other hand, free carboxyl groups had been demonstrated in the outermost surface of the resting spores in a previous study. Thus, we assume that the mode of binding of the antibiotics to the resting spores may be due to the formation of reinforced ionic bonds between amino (or guanidine) groups in the antibiotics and carboxyl groups on the spore surface.     

Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms

During Pseudomonas aeruginosa flow cell biofilm development, the cell population differentiates into a nonmotile subpopulation which forms microcolonies and a migrating subpopulation which eventually colonizes the top of the microcolonies, resulting in the development of mushroom-shaped multicellular structures. The cap-forming subpopulation was found to develop tolerance to membrane-targeting antimicrobial agents, such as the cyclic cationic peptide colistin and the detergent sodium dodecyl sulfate. The stalk-forming subpopulation, on the other hand, was sensitive to the membrane-targeting antibacterial agents. All biofilm-associated cells were sensitive to the antibacterial agents when tested in standard plate assays. A mutation eliminating the production of type IV pili, and hence surface-associated motility, prevented the formation of regular mushroom-shaped structures in the flow cell biofilms, and the development of tolerance to the antimicrobial agents was found to be affected as well. Mutations in genes interfering with lipopolysaccharide modification (pmr) eliminated the biofilm-associated colistin tolerance phenotype. Experiments with a PAO1 strain harboring a pmr-gfp fusion showed that only the cap-forming subpopulation in biofilms treated with colistin expresses the pmr operon. These results suggest that increased antibiotic tolerance in biofilms may be a consequence of differentiation into distinct subpopulations with different phenotypic properties.     

Synthesis and characterization of the colistin peptide polymyxin E1 and related antimicrobial peptides.

Two strategies were developed to synthesize the acylated cyclic peptides know as polymyxins. Synthesis of polymyxin E1 and several analogs enabled us to evaluate the minimum inhibitory concentration of individual compounds against Gram-negative bacteria. In this study we also report the first identification of two component peptides in the complex polymyxin fermentation product colistin, a Thr2Ser isoform and an acyl group isomer. Both of these peptides, as well as a known component peptide, Leu7Ile, were similar to polymyxin E1 in potency, suggesting that conservative mutations in the colistin family are functionally inconsequential. In contrast, the acyclic analogs of all of these peptides were inactive, indicating that the characteristic lariat structure of the polymyxins is necessary for antimicrobial activity.     

Polymyxins as Novel and Safe Mucosal Adjuvants to Induce Humoral Immune Responses in Mice

There is currently an urgent need to develop safe and effective adjuvants for enhancing vaccine-induced antigen-specific immune responses. We demonstrate here that intranasal immunization with clinically used polypeptide antibiotics, polymyxin B (PMB) and colistin (CL), along with ovalbumin (OVA), increases OVA-specific humoral immune responses in a dose-dependently manner at both mucosal and systemic compartments. Enhanced immunity by boosting was found to persist during 8 months of observation. Moreover, mice intranasally immunized with OVA plus various doses of PMB or CL showed neither inflammatory responses in the nasal cavity and olfactory bulbs nor renal damages, compared to those given OVA alone. These data suggest that polymyxins may serve as novel and safe mucosal adjuvants to induce humoral immune responses. The polymyxin adjuvanticity was found to be independent of endotoxins liberated by its bactericidal activity, as indicated by similar enhancing effects of PMB in lipopolysaccharide (LPS)-hyporesponsive and LPS-susceptible mice. However, despite the presence of preexisting anti-PMB antibodies, we observed no reduction in the adjuvant function of polymyxins when they were given intranasally. Furthermore, the titers of OVA-specific Abs in mice intranasally immunized with OVA plus PMB or CL were significantly higher than those in mice administered with polymyxin analogues, such as polymyxin B nonapeptide and colistin methanesulfonate. The levels of released β-hexosaminidase and histamine in mast cell culture supernatants stimulated by PMB or CL were also significantly higher than those stimulated by their analogues. These results suggest that both the hydrophobic carbon chain and hydrophilic cationic cyclic peptide contribute to the mucosal adjuvanticity of PMB and CL.     

Gene transfer into intact vertebrate embryos.

Intact chick embryos at 40 h incubation were transfected in vivo with chimeric vectors expressing chloramphenicol acetyl transferase (CAT) under different promoter sequences. The cationic lipid, dioctadecylamidoglycyl spermine (DOGS) used as the transfecting agent had no noticeable toxic effects on embryonic development. CAT activity was monitored 48 h post-transfection on homogenates of embryos dissected free of all annexes. Of the various constructs tested, those containing the AP-1 response element linked to CAT (TRE-tk-CAT) gave high expression and consistent enzyme responses within groups. Co-transfection experiments in which embryos were exposed simultaneously to a CAT vector containing the cAMP response element and to a vector expressing the catalytic subunit of protein kinase A showed that the promoters of the introduced genes can be regulated by their respective transacting factors. This method may therefore represent a general tool for introducing genes into intact vertebrate embryos at precise developmental times.     

Chemical Synthesis and Characterization of α-N-Octanoyl and Other α-N-Acyl Colistin Nonapeptide Derivatives

Eight α-N-acyl colistin nonapeptide derivatives including three aliphatic, four aromatic and one alicyclic derivatives were synthesized by the reaction of colistin nonapeptide with corresponding acid chlorides. This acylation reaction was carried out under the condition kept restrictedly at pH 5,0 in order to introduce an acyl group only to α-amino group but not to γ-amino group existing in a colistin nonapeptide molecule. Synthetic method and several physico-chemical natures of these acyl colistin nonapeptide derivatives are given in this paper.

All of the acylated derivatives thus synthesized exhibited characteristic antimicrobial activities. Antimicrobial spectra were substantially based upon a gram-negative type and not so much altered by chemical structures of acyl groups which were considerably differentiated from each other such as cyclic or chain form. Thus, more possible response of carbon size than its structure to the antimicrobial effectiveness was inferred. In spite of almost no toxicity and feeble antimicrobial activity of colistin nonapeptide itself, these acylated colistin nonapeptide derivatives showed a toxicity against mice at a dose of 16.9~70 mg/kg in LD₅₀, which, however, was inferior to the toxicity of colistin sulfate, possibly correspondent to their much weaker antimicrobial activities, as a whole. Hence, it seems likely that acyl part of these acylated colistin nonapeptide derivatives including that of colistin is seriously responsible for the biological activities.     

Factors influencing the assessment of anticoccidial activity in cell culture

A comparative study has been made of the factors influencing the assessment of anticoccidial potency in vitro against Eimeria tenella using established anticoccidials and arprinocid and some of its analogues. Drugs whose potency depended upon medium composition were amprolium, lasalocid and halofuginone. There was a difference in strain sensitivity with robenidine. Host cell type had an important effect on potency of monensin, decoquinate, arprinocid and its analogues. Arprinocid was active in chick liver cell systems but totally inactive in chick kidney cell systems, although its N-oxide was active in both cell types. Arprinocid-containing medium, conditioned by supporting the growth of chick embryo liver cell cultures, had an anticoccidial effect on E. tenella growing in chick kidney cells. It is deduced that the anticoccidial activity of arprinocid in the chick is due to a metabolite.     

Lethality for mice and chick embryos, pyrogenicity in rabbits and ability to gelate lysate from amoebocytes of Limulus polyphemus by lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella

Phenol-water extracted lipopolysaccharides (LPS) from Veillonella, Fusobacterium nucleatum, Bacteroides fragilis and Bacteroides melaninogenicus were lethal for mice and 11-days-old chick embryos, pyrogenic in rabbits, and gelated Limulus amoebocyte lysate. Mouse lethality was considerably enhanced by actinomycin-D. In all test systems the endotoxin activity of Veillonella and Fusocbacterium LPS was comparable to that of LPS from Salmonella enteritidis, which was included as a reference endotoxin. The endotoxicity of the Bacteroides LPS was very low. While nanograms of the Veillonella and Fusobacterium LPS killed the chick embryos and gelated the Limulus lysates, microgram amounts of the Bacteroides LPS were needed to give positive reaction in the same test systems. As much as 74 microgram of the most active B. fragilis LPS were required to give a typical biphasic fever response in rabbits. A significant correlation was found between all test results (r = 0.90-0.98, p less than 0.001).     

Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity

Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.     

Expression of A1 Adenosine Receptors Modulating Dopamine-Dependent Cyclic AMP Accumulation in the Chick Embryo Retina

Dopamine and 2-chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16-day-old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine-dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12-day-old chick embryo retinas, with an IC50 of approximately 1 microM. This effect was not blocked by dipyridamole. N6-(l-Phenylisopropyl)adenosine, (l-PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 microM which was two orders of magnitude lower than its stereoisomer d-PIA (10 microM). The maximal inhibition of the dopamine-elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3-isobutyl-1-methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective; whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11- to 12-day-old embryos and 2-chloroadenosine did not interfere with the apomorphine-mediated shift in cyclic AMP levels. In the retinas from 5-day-old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation

Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified from tendon cells of 17-day-old chick embryos, one of which (alpha) appears to be identical in molecular weight and carbohydrate content with the single alpha of enzyme from 13-day-old chick embryos, as well as another form (alpha') that contains two oligosaccharides, each containing eight mannosyl units [see Kedersha, N. L., Tkacz, J. S., & Berg, R. A. (1985) Biochemistry (preceding paper in this issue)]. Biosynthetic labeling studies were performed with chick tendon cells using [2-3H]mannose, [6-3H]glucosamine, [14C(U)]mannose, and [14C(U)]glucose. Analysis of the labeled products using polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that only the oligosaccharides on alpha' incorporated measurable mannose or glucosamine isotopes; however, both alpha subunits incorporated 14C amino acid mix and [14C(U)]glucose [metabolically converted to [14C(U)]mannose] under similar conditions. Pulse-chase labeling studies using 14C amino acid mix demonstrated that both glycosylated polypeptide chains alpha and alpha' were synthesized simultaneously and that no precursor product relationship between alpha and alpha' was apparent. In the presence of tunicamycin, neither alpha nor alpha' was detected; a single polypeptide of greater mobility appeared instead. Incubation of the cells with inhibitory concentrations of glucosamine partially depressed the glycosylation of alpha' but allowed the glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2 Gy of gamma rays delivered at a dose rate of 0.316 Gy/min using a ⁶⁰Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney.Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.     

The functional association of polymyxin B with bacterial lipopolysaccharide is stereospecific: studies on polymyxin B nonapeptide

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a major inducer of sepsis. The natural cyclic peptide polymyxin B (PMB) is a potent antimicrobial agent, albeit highly toxic, by virtue of its capacity to neutralize the devastating effects of LPS. However, the exact mode of association between PMB and LPS is not clear. In this study, we have synthesized polymyxin B nonapeptide, the LPS-binding cyclic domain of PMB, and its enantiomeric analogue and studied several parameters related to their interaction with LPS and their capacity to sensitize Gram-negative bacteria toward hydrophobic antibiotics. The results suggest that whereas the binding of the two enantiomeric peptides to E. coli and to E. coli LPS is rather similar, functional association with the bacterial cell is stereospecific. Thus, the L-enantiomer is capable of synergism with the hydrophobic antimicrobial drugs novobiocin and erythromycin, whereas the D-enantiomer is devoid of such activity. The potential of understanding and consequently utilizing the PMB-LPS association for novel, nontoxic PMB-derived drugs is discussed.     

A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides.

The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and Candida albicans cells in phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC50/IC50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC50 = 0.6 microM) and had the lowest therapeutic index (HC50/IC50 = 0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.     

Calcium transport and related functions in the chorioallantoic membrane of cultured shell-less chick embryos

In order to investigate the influence of the egg shell on the process of shell calcium mobilization by the chorioallantoic membrane (CAM), chick embryos were maintained in long-term cultures in vitro without the shells. The shell-less embryos were severely calcium deficient and showed signs of retarded development and anomalous skeletal calcification. Throughout development, calcium transport and calcium-binding protein (CaBP) activities were diminished in the CAM of shell-less embryos as compared to those of control embryos which developed in ovo. The levels of developmentally expressed carbonic anhydrase activity remained, however, similar. By means of a single radial immunodiffusion assay of CaBP using a specific anti-CaBP antiserum, the level of immunoreactive CaBP was found to be significantly increased in the CAM of the shell-less embryos. These studies indicate that the CAM of chick embryos cultured under shell-less conditions is defective in calcium transport, probably as a result of the expression of an inactive form of the CaBP.     

Effects of prolonged antitubulin culture on the ultrastructure of anterior limb bud cells of the chick embryo

The anterior limb bud mesenchyme cells of stage 24 chick embryos were dissociated by trypsinization followed by gentle pipetting, and placed in a tissue culture medium of F12 containing 10% fetal calf serum and antibiotics. As the cells became nearly confluent, some of them were exposed to colchicine or vinblastine sulfate for durations as long as 48 hr. The control and antitubulin-treated cells were processed for transmission electron microscopy and the ultrastructure of the cells was compared. Annulate lamellae (AL) were observed in small amounts in both control and antitubulin-treated cells. The amount of AL did not markedly differ in the control versus antitubulin-treated cells. Furthermore, few multinucleated cells were observed in antitubulin-treated cultures. These results indicate that prolonged culture of cells in antitubulins need not, in itself, lead to a condition of enhanced AL development as reported in several other studies using various cell types.     

Colistin interactions with the mammalian urothelium

Here we describe the effect of colistin on the barrier function of the mammalian urinary bladder epithelium. Addition of colistin to the mucosal solution of the rabbit urinary bladder epithelium (urothelium) resulted in an increase in the transepithelial conductance. The magnitude of the increase in transepithelial conductance was dependent on the membrane voltage, concentration of colistin, and presence of divalent cations in the bath solution. The initial site of action of colistin was at the apical membrane. Colistin increased the membrane conductance only when the apical membrane potential was cell interior negative. The more negative the membrane potential, the larger the conductance increase. The concentration dependence of the conductance increase saturated, suggesting a membrane binding site. Divalent cations decreased the magnitude of the conductance increase. This divalent cation action occurred at two sites: one in competition with colistin for a membrane binding site, and the other by rapidly blocking the induced conductance. At short exposure times, the increase in conductance was reversed by either removing colistin from the bath or changing the voltage so that the apical membrane was cell interior positive. At long exposure times, the increase was only partially reversible by voltage or removal from the bath. This finding suggests that at long exposure times, there is a toxic effect of colistin on the urothelium. bladder epithelium; epithelial transport; tight junctions; antibiotics; cationic proteins     

Alprenolol binding and cyclic AMP production in embryonic chick red cells during erythropoiesis

We have measured alprenolol binding and cyclic AMP production in erythroid cells taken from chick embryos incubated from 8 days to hatching and in cells from the adult. Beta-adrenergic receptor number and affinity measured by alprenolol binding are essentially unchanged in red cell membranes prepared from 8- through 17-day embryos. Receptor number was found to be half as much in the adult. Erythroid cells from embryos of all ages studied show stimulation of cyclic AMP production when incubated with epinephrine, and most of the cyclic AMP produced remains intracellular. Inasmuch as the cells from younger embryos can in fact produce cyclic AMP, the previously-reported lack of epinephrine sensitivity of cation transport in the red cells of younger embryos (Wacholtz et al., 1978) cannot be attributed to the lack of functional receptors or to an impairment of cyclic AMP production.     

Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure-activity relationships in the bacterial membrane disruptor betapep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of betapep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel beta-sheet structure is the bioactive conformation of betapep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, betapep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.     

Monoclonal antibodies to the nuclear matrix of chick embryonal erythrocytes

Monoclonal antibodies were prepared from mice that had been immunized with the nuclear matrix from chick embryonal erythrocytes. Seven stable clones were obtained by an ELISA that used nuclear lysate as the solid phase. Six clones of them reacted with the nuclear matrix, and one reacted with nuclear components other than the matrix. Immunoblotting showed that one clone recognized the 72K polypeptide, two clones recognized the 69K polypeptide and three clones recognized both the 69K and 44K polypeptides. Indirect immunofluorescence that used antibodies to the nuclear matrix showed homogeneous nuclear fluorescence in cultured chick embryonal fibroblasts, and intense fluorescence was present in the peripheral part of the nucleus in thin-sectioned chick embryos. Only weak nuclear fluorescence was seen in fibroblasts from humans and rats when two of the antibodies which recognized only 69K polypeptide were used. The rest of the antibodies to the nuclear matrix produced no nuclear fluorescence in human and rat fibroblasts. The metaphase-rich population of chick embryonal fibroblasts were stained diffusely over the entire cytoplasm, but not the chromosomes, when antibodies to the nuclear matrix were used. These results indicate that monoclonal antibodies we prepared are directed to the major proteins of the nuclear matrix that correspond to the lamin A and B defined in rat liver.