No abstract

Colicin E2 purified by conventional methods contains a tightly bound low-molecular-weight protein, as has been found with purified colicin E3 [Jakes, N. & Zinder, N. D. (1974) Proc. Nat. Acad. Sci. USA 71, 3380-33841. Such E2 preparations do not cause DNA cleavage in vitro. After separation from the low-molecular-weight protein, colicin E2 retained the original in vivo killing activity, and in addition showed a high activity in vitro in cleaving various DNA molecules, such as a ColE1 hybrid plasmid and DNAs from Escherichia coli, X phage, 4X174 phage, and simian virus 40. The low-molecular-weight protein ("E2-immunity protein") specifically prevented this in vitro DNA cleavage reaction, i.e., had an "immunity function." The results demonstrate that colicin E2 itself is a DNA endonuclease and explain the in vivo effects caused by E2 in sensitive cells as well as the mechanism of immunity in E2-colicinogenic cells. Colicin E2 (E2) is a protein antibiotic synthesized by certain strains of coliform bacteria that carry the ColE2 plasmid, and is classified in the E group of colicins along with colicins El and E3 (1, 2). Although the physical properties of E2 and E3 are similar (3) and both colicins share the same receptor (1, 2), their apparent mode of action is different. It was first discovered that E2 causes degradation of DNA, while colicin E3 (E3) causes specific inhibition of protein synthesis in sensitive Escherichia coli cells treated with these colicins (4). Subsequent work on the mode of action of E3 has demonstrated that ES inactivates ribosomes in treated cells (5) by causing the cleavage of a fragment from the 3' end of the 16S RNA (6, 7). The same cleavage reaction has also been demonstrated in vitro using "purified" E3 protein (see below) and purified ribosomes (8,9). This in vitro demonstration of E3 action has strongly indicated that E3 acts directly on ribosomes rather than indirectly and that E3 itself is an RNase with a very stringent substrate specificity.In contrast to ES, the mechanism of action of E2 has not been clear. Since the initial discovery of DNA degradation in E2-treated sensitive cells (4), several authors have confirmed and extended the original observations on DNA cleavage reactions (10-13). However, others have suggested that the primary action of E2 is not on the cellular DNA, but on some other targets such as membranes (14) or tRNA (15).Since E2 has properties similar to E3 (3), and E3 causes ribosome inactivation in vitro, one might expect some kind of effects of E2 on cellular DNA in vitro, if DNA is in fact the primary target of E2. Several attempts have been made to demonstrate such effects in vitro using purified E2 (e.g., refs. 16-19). However, published results were either negative or too uncertain to allow a definitive conclusion regarding such direct effects.Recently, Jakes and Zinder observed that the "purified" ES Colicin E2* was separated from the immunity protein by dissociation of the complexed E2 with guanidine-HCl at about 4°. Lyophilized E2 was di...

Colicin M was isolated from Escherichia coli K-12 32T 19F/T1. The purified, biologically active protein had a molecular weight of 27,000. It contained phosphatidyl ethanolamine. The molecular weight found for the polypeptide chain by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 18,000. Colicin M was found to be firmly integrated in the membrane of the producing strain. The action of the colicin seems to be on the membrane, since cells of the susceptible strain E. coli K-12 ROW/V/22.1 lyse rapidly. Using the phase contrast microscope, lysis was followed by decrease in turbidity of the cell culture and release of protein into the medium. Lysis started at about 15 min after addition of colicin M and was completed after 40 to 60 min. At this time, one-third of the protein had been released from the cells. The number of viable cells dropped within 10 min to 0.01%. Colicin M induced formation of spheroplasts in the presence of 16% sucrose. The electron microscope examination revealed that at first bulges in the cell envelope appear, most frequently occurring equatorially but also occurring at sites all over the cell. In the process of spheroplast formation, the cytoplasmic membrane often retreats from one-half of the outer membrane so that the cytoplasm is confined to one hemisphere. Sucrose did not prevent cells from dying unless cells were pregrown in a sucrose containing medium for several generations before colicin M was added. With cells pregrown in the presence of sucrose, the number of survivors was 100 times higher than in the absence of sucrose.Receptors on the cell surface are of interest with regard to conjugation of bacteria, phage infection, and killing of cells by colicins. We isolated the receptor for phage T5 from Escherichia coli B and showed that colicin M binds to the same receptor (4). The receptor activity resides in a protein with a molecular weight of 85,000, which is apparently a single polypeptide chain localized in the outer membrane (5). Colicin M was originally defined by P. Fredericq on the basis that M-producing strains are consistently resistant to Ti and T5 (7). In the course of our studies on the interaction among the receptor, phage T5, and colicin M, we noticed that susceptible cells lyse rapidly in liquid culture when incubated with colicin M. Induction of rapid lysis seemed to be unique for this colicin. Cell lysis caused by other colicins studied so far is the ultimate consequence of primary damages occurring much earlier (15). In the case of colicin M, lysis is an early event. We therefore started to investigate the mode of '

Nikkomycins X and Z (NZ), competitive inhibitors of fungal chitin synthetase, were combined with azoles in a series of in vitro checkerboard assays to test for synergism against Candida spp. All combinations of nikkomycins and azoles tested resulted in marked synergistic activity against an isolate of Candida albicans, with fractional inhibitory concentration indices ranging from 0.016 to 0.28. No synergistic effect was demonstrable with isolates of C. tropicalis, C. parapsilosis, or C. krusei, though results for the latter two were suggestive of an additive effect. In survival models of mice infected intravenously with C. albicans, NZ administered singly in doses ranging from 5 to 50 mg/kg of body weight twice a day was able to delay the onset of mortality but showed no dose-response effect. The combination of NZ and the azole R 3783 administered orally in a ratio of 8:1 to 40:1 or greater (wt/wt) enhanced survival better than did the drugs given individually, but this effect was less evident for combinations involving fluconazole. In short-term organ load assays with outbred mice infected intravenously with C. albicans, high ratios of NZ to R 3783 reduced the CFU per gram in kidneys more significantly than did the drugs individually. Statistically significant reductions were not seen for short-term fungal burden assays using combinations of NZ and fluconazole in outbred mice or in inbred mice more susceptible to candidiasis. In a model of rat vaginal candidiasis, the combination of NZ and R 3783 administered either orally or vaginally was more effective than the drugs used singly. Thus, under certain conditions, combination therapy with nikkomycin and select azoles may offer promise for an increased therapeutic effect in candidiasis.With the increase in the number of immunocompromised patients in the last decade, there has been a concomitant increase in the numbers of life-threatening mycoses (17,20).Unfortunately, antifungal drugs released for clinical use over the last decade have not offered substantive improvements in efficacy over amphotericin B. Recently, several new compounds active against the cell walls of fungi have been evaluated. These compounds include derivatives of echinocandin B, which inhibit beta-glucan synthesis (6,22), and the nikkomycins, which inhibit chitin synthesis (4). While these drugs show promise, their spectrum of activity appears to be limited in comparison to those of the azoles and amphotericin B (6, 12). Previous data obtained with nikkomycin used as therapy in a mouse model of candidiasis showed only modest efficacy (1).One approach to increasing the therapeutic index of anti-infective drugs is to test them in combinations for synergistic interactions. While there have been a number of reports concerning positive interactions of antifungal drugs or antifungal and antibacterial drugs, few combinations have shown substantial improvements over the effects of the single drugs; many have shown antagonism (2,3,5,13,16,19,23).Because of previous reports that azole compounds interfer...

Colicin M of Escherichia coli C1139 was isolated in pure form. It consisted of a single polypeptide with a molecular weight of 27,000 ± 2,000. Colicin M lysed sensitive cells of E. coli but had to act continuously up to the point when lysis commenced (after 20 min). Colicin M was largely resistant to hydrolysis by trypsin except when adsorbed to cells. Within 4 to 5 min after addition of colicin M, cells could be rescued by trypsin or sodium dodecyl sulfate. Later, colicin M was apparently inaccessible to these inactivating agents. Killing of cells by colicin M required Ca2+ ions. Cells could be rescued with ethylene glycol-bis(f-aminoethyl ether)-N,N'-tetraacetate (EGTA) immediately before the onset of lysis. Under these conditions, colicin M remained bound to the cells, and it became again sensitive to trypsin. We conclude that under the influence of EGTA colicin M is removed from its site of action and becomes again accessible to trypsin at the cell surface. Colicin M was originally defined on the basis that resistant cells were cross-resistant to phages Ti and T5 (6). Later it was shown that colicin M causes lysis of sensitive cells (2). Both observations were of interest since the uptake of the toxic protein required the function coded by the