Amphotericin B potentiates the antifungal effects of 5-fluorocytosine and rifampicin, probably by increasing the penetration of these agents through the fungal cytoplasmic membrane.

Prior work has indicated that the polycistronic lacZYA mRNA of Escherichia coli is cleaved during decay at approximately intergenic sites (L. W. Lim and D. Kennell, J. Mol. Biol. 135: 369-390, 1979). In this work, we characterized the products by using probes specific for the different cistrons. This analysis indicated that six lac mRNA species are present in the following order of decreasing abundance: lacZ, -A, -ZYA, -ZY, -YA, and -Y. Very little lacYA and lacY mRNAs were present, whereas in cells induced to steady state, there was 10 times more lacZ than lacZYA mRNA. The lacZ mRNA appeared as a discrete species extending to a site in the lacZ-Y intergenic space (ca. residue 3150). This site is just distal to a potential rho-independent termination sequence. We examined the function of this sequence to determine whether it contributes to the distribution of the mRNAs. Although the termination sequence was shown to function in vitro, when it was recloned into an expression vector, no termination was seen in vivo. Moreover, direct examination of the kinetics of lac messenger synthesis revealed that after initiation, most transcription continued to the end of the operon. We conclude that during normal growth, the operon is transcribed in its entirety and that the individual lac mRNAs are formed by cleavage. These results confirm earlier work implying that the lac operon is transcribed in its entirety but are in conflict with several recent reports suggesting that internal termination occurs. Our findings indicate that the natural polarity of the operon (lacZ is expressed sixfold more strongly than lacA) is based on posttranslational effects and not on polarity of transcription.There have been extensive studies of the transcription and decay of the lac operon mRNA (reviewed in reference 18). According to current understanding, the lacZYA mRNA is transcribed polycistronically and then is cleaved at approximately intergenic sites. These cleavages, near the 5' ends of the lacY and lacA transcripts and at an additional site near the 5' end of the lacZ transcript, inactivate the distal mRNAs. The further chemical decay of these mRNAs then occurs as a net 5'-to-3' process. In this process, endonucleolytic cleavages occur on the mRNA that is exposed as ribosomes run off inactivated messages. The resulting fragments are then removed by the 3'-to-5' exonucleases, RNase II, polynucleotide phosphorylase and possibly other enzymes (1,14,18). The endonucleolytic cleavage sites on the lacZ and lacY species have been characterized but the enzyme(s) responsible for the endonucleolytic cleavages has not been identified (7,34). It appears, however, that RNase III is not involved in lac mRNA decay, although it does inactivate a few other mRNA species (2, 18, 27a). A newly described endonuclease has some properties inferred for the enzyme that cleaves the lac transcripts (6).The cleavage of the lacZYA mRNA was first deduced from the observation that the lacA mRNA decays about twice as fast as lacZ mRNA (19). Subsequently, the cleavag...

High concentrations of polymyxin B inhibited the growth of Candida albicans and Saccharomyces cerevisiae. When these yeasts were incubated with concentrations of polymyxin B too low to affect growth, and were then exposed to tetracycline, protein synthesis was inhibited and at least 99% of the organisms were killed. Neither inhibition of protein synthesis nor cell death occurred in cultures treated with high concentrations of tetracycline alone. We conclude that polymyxin B at high concentrations affects the cell membrane of yeasts, which results in inhibition of growth. At low concentrations, it increases the permeability of the yeast cell membrane to tetracycline, which then inhibits protein synthesis and leads to cell death.Polymyxin B is a surface-active bactericidal antibiotic which alters the permeability of the bacterial cell envelope by binding to the negatively charged phospholipid component of the membrane and causing cell lysis (4). Because eukaryotic cells also contain phospholipids in their cell membranes (7), it was reasonable to expect that polymyxin B would disrupt permeability barriers in them as well. Two previous studies have shown this to be true in a protozoan (6) and in the yeast Candida tropicalis (5). The latter study, however, failed to demonstrate a polymyxin B effect in a variety of other yeasts.Several agents have been shown to be ineffective against yeasts because of failure to penetrate the cytoplasmic membrane and gain access to their site of action. For example, tetracycline has been shown to inhibit protein synthesis in extracts of yeasts (1) but was ineffective against whole organisms (C. N. Kwan et al., Bacteriol. Proc., p. 179, 1972). In this report, we have investigated the antifungal properties of polymyxin B against certain yeasts, and have exploited the changes in membrane permeability caused by this antibiotic to potentiate the antifungal effect of tetracycline. Synergism studies. The susceptibility of each organism to polymyxin B and tetracycline was determined by the broth-dilution method, as previously described (2). A wide range of antibiotic concentrations was examined for each organism, and cell viability was determined by colony counts after 1, 2, 3, and 7 days of incubation. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of drug required to inhibit completely the growth of the organism over the designated time interval.Synergism was also determined by colony counts of each organism in the presence of the antibiotics used singly and in combination. Exposure to the two drugs was done either by 24-hr pretreatment of the yeasts with polymyxin B or by simultaneous addition of the polymyxin B and tetracycline. Our definition of antifungal drug synergy was a decrease of 100-fold or more in colony counts caused by the drugs in combination as compared with the counts when the drugs were used singly (3). The level of each antibiotic used in the combined drug studies was well below the respective MIC. In the MIC and synergism studies, a s...

Amphotericin B was found to potentiate the antifungal effects of mycophenolic acid glucuronide, tetracycline, and actinomycin D by increasing the penetration of these antibiotics through the fungal cytoplasmic membrane. Because the fungi were over 100 times more susceptible than animal cells to these effects of amphotericin B, these observations might have clinical application. Amphotericin B is a polyene which binds to sterols and changes the permeability properties of yeast and animal cell membranes (5). We have previously used this agent to make yeast cells more susceptible to the action of 5-fluorocytosine and rifampin (6, 7). Here, we report studies of the specificity and nature of the amphotericin B effect.In these experiments, we used mycophenolic acid glucuronide, tetracycline, and actinomycin D, all of which inhibit synthesis of specific macromolecules in yeast extracts, but are relatively ineffective against whole cells because of their inability to pass through the cytoplasmic membrane (1, 3). Amphotericin B made Saccharomyces cerevisiae susceptible to much lower concentrations of these agents by increasing their uptake into the cell, whereas there was no apparent increased uptake of a number of other compounds tested. Also, in the tests performed, the effects of amphotericin B were produced by orders of magnitude less drug with yeast cells than with a human tumor cell line, providing the first evidence that the synergy observed with the drugs might have clinical usefulness. done in Sabaroud broth, and studies of the incorporation of radioactive labels were done in YM5 broth (4) at 30 C. YM5 broth contains 0.01% uracil, so that incorporation of added [3H] uracil in the labeling experiments was linear in all instances. The established line of human tumor cells (KB) was grown and maintained in suspension cultures in Eagle's minimal essential medium with 5% horse serum. The incubations of animal cells with drugs were carried out in 50-ml Erlenmeyerflasks incubated in a New Brunswick gyratory water bath at 37 C.Synergism studies. The susceptibility of the yeast to each agent used alone and in combination with amphotericin B was determined as previously described (6, 7). The minimal inhibitory concentration (MIC) was that concentration of drug which completely inhibited cell multiplication. The criterion for antifungal synergy was also as previously defined: a decrease of 100-fold or more in colony count caused by the drugs in combination, as compared with the count when the drugs were used singly.Measurement of RNA and protein synthesis. Ribonucleic acid (RNA) and protein synthesis was determined by incubating the yeast or KB cells in [3H] uracil or [3H] leucine in the presence of each of the drugs alone, or with amphotericin B in combination with each. Samples of 0.5 ml of cells were taken at times noted, precipitated with 0.5 ml of 10% trichloroacetic acid, filtered, and counted.

Rifampicin, at high concentrations, inhibited growth and RNA synthesis in the yeast phase of Histoplasma capsulatum. These effects were potentiated by low concentrations of amphotericin B. The combination of the two agents was fungicidal, whereas each alone, at much higher concentrations, was only fungistatic.