The gene cluster responsible for pimaricin biosynthesis in Streptomyces natalensis contains a cholesterol oxidase-encoding gene (pimE) surrounded by genes involved in pimaricin production. Gene-inactivation and -complementation experiments revealed that pimE encodes a functional cholesterol oxidase and, surprisingly, that it is also involved in pimaricin biosynthesis. This extracellular enzyme was purified from S. natalensis culture broths to homogeneity, and it was shown to restore pimaricin production when added to the mutant culture broths. Other cholesterol oxidases also triggered pimaricin production, suggesting that these enzymes could act as signaling proteins for polyene biosynthesis. This finding constitutes the description of a cholesterol oxidase gene with an involvement in antibiotic biosynthesis, and it broadens the scope of the biological functions for this type of oxidase.

We present the X-ray structure of PimD, both substrate-free and in complex with 4,5-desepoxypimaricin. PimD is a cytochrome P450 monooxygenase with native epoxidase activity that is critical in the biosynthesis of the polyene macrolide antibiotic pimaricin. Intervention in this secondary metabolic pathway could advance the development of drugs with improved pharmacologic properties. Epoxidation by P450 typically includes formation of a charge-transfer complex between an oxoferryl pi-cation radical species (Compound I) and the olefin pi-bond as the initial intermediate. Catalytic and structural evidence presented here suggest that epoxidation of 4,5-desepoxypimaricin proceeds via a hydroperoxoferric intermediate (Compound 0). The oxygen atom of Compound 0 distal to the heme iron may insert into the double bond of the substrate to make an epoxide ring. Stereoelectronic features of the putative transition state suggest substrate-assisted proton delivery.

Wide differences exist among the polyene antibiotics, nystatin, rimocidin, filipin, pimaricin, and amphotericin B, with reference to steroid interference with their antifungal activities against Candida albicans. Of the numerous steroids tested, ergosterol was the only one which effectively antagonized the antifungal activity of all five polyene antibiotics. The antifungal activities of nystatin and amphotericin B were the least subject to vitiation by the addition of steroids other than ergosterol, and those of filipin, rimocidin, and pimaricin were the most sensitive to interference. Attempts to delineate the structural requirements of steroids possessing polyene-neutralizing activity in growing cultures of C. albicans are discussed. The ultraviolet absorbance of certain antibiotic steroid combinations was also studied.

Fourteen clinical isolates of Zygomycetes were tested for their in vitro susceptibility to nine antifungal agents. Susceptibility assessment was performed using a microtiter broth dilution method. Synthetic broth with YNB and glucose was used for 5-fluorocytosine and BHI broth for all the other antimycotics. Amphotericin B exhibited the strongest activity against all isolates tested. MIC values of other two polyenes--nystatin and pimaricin--ranged within the susceptibility limits, with a little pronounced higher activity of pimaricin. The isolates of the genus Absidia and Syncephalastrum were well sensitive to all antimycotics with the exception of 5-fluorocytosine and naftifine. A very weak or zero growth inhibitory effect against all members of the genera Mucor and Rhizopus was found in azoles, 5-fluorocytosine and naftifine.

About 50 polyenic antifungal antibiotics produced by actinomycetes have been isolated and described. Among these are the most effective antimonilial agents so far known. These polyenes can be classified into four broad groups depending on the number of conjugated carbon-to-carbon double bonds that are present in their chromophores. Only some of the tetraenes (four conjugated double bonds), such as nystatin and pimaricin, and a few heptaenes (seven conjugated double bonds), such as amphotericin B, Trichomycin candicidin and hamycin, have found practical application in the treatment of infectious diseases caused by fungi. Nystatin and pimaricin show the least in vitro activity against fungi, while trichomycin and candicidin are the most active. Recent clinical investigations carried out in the USA have shown that candicidin is very effective in the treatment of vaginal monilial infections.

1. The effect of pimaricin, etruscomycin and amphotericin B on the K+ release from liposomes is strongly dependent on their sterol concentration. Pimaricin and etruscomycin induce K+ release from egg lecithin liposomes with cholesterol contents of more than 25 and 10 mol%, respectively, at polyene concentrations of 100 and 10 microgram/ml, respectively. Amphotericin B shows a maximal effect at a cholesterol content of 20 mol% at a concentration of 0.4 microgram/ml. 2. For liposomes containing ergosterol the sensitivity is shifted to a lower sterol content. All three polyenes show activity at 10 mol% ergosterol. The sensitivity for amphothericin B is increased approx. 15 times by the incorporation of ergosterol compared to cholesterol. The increase in sensitivity is much less for pimaricin and etruscomycin. The K+ release is maximal at an ergosterol concentration of 30 mol%. 3. Pimaricin, etruscomycin and amphotericin B can induce K+ release from erythrocytes without the release of haemoglobin at concentrations of 20, 2 and 1 microgram/ml, respectively. For these polyenes a selective permeability change is also demonstrated for liposomes since K+ is released but no [14C]dextran. Filipin shows a nonselective release of solutes from erythrocytes and liposomes. 4. At cholesterol concentrations higher than 20 mol% and ergosterol concentrations higher than 10 mol%, etruscomycin, pimaricin and amphotericin B show little dependence of the bilayer thickness and are able to release K+ from didocosenoyl phosphatidylcholine liposomes after addition of the polyene to one side of the membrane. A possible mechanism is discussed.

The distribution of Legionella longbeachae and Legionella spp. in Japanese potting soils was examined. Thirty samples were collected: 13 were composted wood products, 11 were potting mixes (containing composted wood products, sand, mineral fertilizer, and manure), 2 were peatmoss, 3 were peatmoss-sand mixes (containing peatmoss and sand), and 1 was a sample of hydroponic clay balls. A suspension of each sample was made in sterile distilled water and acidified, and 100 microl was plated on buffered charcoal yeast extract alpha (BCYEalpha) agar containing modified wadowsky yee (MWY) supplement and pimaricin (direct method). In parallel, each suspension was incubated at 33 degrees C for several months to allow for amebic enrichment, if present; the suspensions were then plated onto culture media as described above (enrichment method). A total of 46 strains of legionellae were isolated from 22 of the 24 samples (13 composted wood products, 11 potting mixes). L. longbeachae was isolated from 9 samples. The most predominant species of legionellae in potting soils was Legionella bozemanii, which was isolated from 13 samples. Legionella spp. and L. micdadei were isolated from 8 and 7 samples each. Compared with findings in potting soils in Australia (26/45; 58%), Japanese potting soils had an only 8.3% (2/24) isolation rate for L. longbeachae by the direct method. The components of composted wood products were broadleaves such as oak and Japanese oak, in contrast to the pine and eucalypt used in Australia, which may account for the different isolation rates. However, the amebic enrichment method was useful in increasing the recovery of legionellae in potting soils. Legionellae were not isolated from the peatmoss samples, a result identical to findings in surveys of similar material in Europe.

World literature on the subject reports 112 cases of keratomycosis by Fusarium, including the two cases described in this paper and some personal communications. In most of these cases (60--70%) there is a history of trauma inflicted by vegetable matter or by foreign bodies introduced into the eye (metal, sand, stone, etc.). The different species of Fusarium isolated are F. episphaeria, F. dimerum, F. moniliforme, F. nivale, F. oxysporum, and F. solani. The treatment recommended in a 5-percent suspension or unguent of pimaricin (natamycin).

The DNA region encoding the filipin gene cluster in Streptomyces avermitilis (pte) contains a PAS-LuxR regulatory gene, pteF, orthologue to pimM, the final pathway-specific positive regulatory protein of pimaricin biosynthesis in Streptomyces natalensis. Gene replacement of the gene from S. avermitilis chromosome resulted in a severe loss of filipin production and delayed spore formation in comparison to that of the wild-type strain, suggesting that it acts as a positive regulator of filipin biosynthesis and that it may also have a role in sporulation. Complementation of the mutant with a single copy of the gene integrated into the chromosome restored wild-type phenotypes. Heterologous complementation with the regulatory counterpart from S. natalensis also restored parental phenotypes. Gene expression analyses in S. avermitilis wild-type and the mutant by reverse transcription-quantitative polymerase chain reaction of the filipin gene cluster suggested the targets for the regulatory protein. Transcription start points of all the genes of the cluster were studied by 5'-rapid amplification of complementary DNA ends. Transcription start point analysis of the pteF gene revealed that the annotated sequence in the databases is incorrect. Confirmation of target promoters was performed by in silico search of binding sites among identified promoters and the binding of the orthologous regulator for pimaricin biosynthesis PimM to gene promoters by electrophoretic mobility shift assays. Precise binding regions were investigated by DNAse I protection studies. Our results indicate that PteF activates the transcription from two promoters of polyketide synthase genes directly, and indirectly of other genes of the cluster.

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM. This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. PimM, truncated forms of the protein without the PAS domain (PimM(ΔPAS)), and forms containing just the DNA-binding domain (DBD) (PimM(DBD)) were overexpressed in Escherichia coli as GST-fused proteins. GST-PimM binds directly to eight promoters of the pimaricin cluster, as demonstrated by electrophoretic mobility shift assays. Assays with truncated forms of the protein revealed that the PAS domain does not mediate specificity or the distinct recognition of target genes, which rely on the DBD domain, but significantly reduces binding affinity up to 500-fold. Transcription start points were identified by 5'-rapid amplification of cDNA ends, and the binding regions of PimM(DBD) were investigated by DNase I protection studies. In all cases, binding took place covering the -35 hexamer box of each promoter, suggesting an interaction of PimM and RNA polymerase to cause transcription activation. Information content analysis of the 16 sequences protected in target promoters was used to deduce the structure of the PimM-binding site. This site displays dyad symmetry, spans 14 nucleotides, and adjusts to the consensus TVGGGAWWTCCCBA. Experimental validation of this binding site was performed by using synthetic DNA duplexes. Binding of PimM to the promoter region of one of the polyketide synthase genes from the Streptomyces nodosus amphotericin cluster containing the consensus binding site was also observed, thus proving the applicability of the findings reported here to other antifungal polyketides.

Streptomyces natalensis produces the antifungal polyene macrolide pimaricin. Genetic manipulation of its biosynthetic genes has been hampered by the lack of efficient gene transfer systems. We have developed a gene transfer system based on intergeneric conjugation from Escherichia coli. Using this approach, we managed to attain transformation efficiencies of 1 x 10(-4) exconjugants per recipient when using self-replicating vectors such as pHZ1358. The use of integrative vectors such as pSET152 or pSOK804 resulted in significantly lower efficiencies. Site-specific integration or the use of self-replicating plasmids did not affect pimaricin production or the essential functions of S. natalensis. Use of DNA methylation proficient E. coli donor strains resulted in no transformants, indicating the presence of methyl-specific restriction systems in S. natalensis. This methodology will enable easier manipulation of the genes responsible for pimaricin biosynthesis, and could prove valuable for the generation of new designer polyene macrolides with better antifungal activity and pharmacological properties. As an example of the validity of the method, we describe the introduction of Supercos-1-derived cosmid vectors into S. natalensis in order to promote gene replacements by double crossover recombination.

The importance of opportunistic fungal pathogens in causing ocular infections is emphasized. A study was conducted over a period of 4 years (1974--1977) to investigate the role of opportunistic fungi in causing mycotic keratitis and to elucidate certain aspects of epidemiology of this disease in Nigeria. Fifty-nine cases of corneal ulcers of suspected mycotic etiology were investigated. Fungal etiology was confirmed in 42 of these cases. The predominant etiological agent was Fusarium solani in 14 cases (33.33%) followed by Penicillium citrinum in 8 cases (19.04%) and Aspergillus fumigatus in 5 cases (11.90%). The yeasts were responsible for only 3 cases (7.14%) i.e. one each caused by Candida albicans, C. parapsilosis and C. guilliermondii. Among the remaining 12 cases, one was caused by F. moniliforme, 3 by A. flavus, 2 each by A. niger, Penicillium expansum and Penicillium sp., and one each by Cladosporium cladosporioides and Cladosporium sp. The clinical features of the cases are briefly described. The incidence of mycotic keratitis in relation to sex, age, occupation, trauma and other factors has been analysed. Corneal trauma appeared to be an important predisposing factor as 27 (67.28%) of the patients gave a history of injuries to the eye. Notably, a large number of patients were farmers and trauma was most often from palm tree leaf, thorn, kernel or other plant objects. Topical application of corticosteroids or broad spectrum antibiotics did not seem to play an important role in the etiology of keratomycosis. Cases were recorded throughout the year although the number of cases was higher in the months of March--May, and November--December than that during the rest of the year. The isolates of the causative agents were studied in detail for their morphological and cultural characters. The isolates of F. solani grew well at 37 degrees C and survived at 40 degrees C for more than 3 weeks. In vitro drug sensitivity tests indicated good antifungal activity of pimaricin and econazole for F. solani, clotrimazole and econazole for Aspergillus fumigatus. A. flavus and Penicillium citrinum, and 5-fluorocytosine for Candida spp. Investigations on the incidence of fungi in normal healthy eyes of 450 persons comprising 204 adults and 246 children yielded 204 isolates belonging to 21 genera of fungi. Cladosporium was most frequent (12.88%) followed by Penicillium (10.22%) and Aspergillus (6.66%). Another important fungus was Fusarium represented by 10 isolates, viz. 4 of F. solani, 2 of F. moniliforme, 1 of F. exysporum, and 3 of Fusarium sp. The yeasts were represented by two isolates each of Candida tropicalis, C. pseudotropicalis, C. krusei, Trichosporon sp and Cryptococcus albidus, and one of Candida guilliermondii. Successive culturing of fungi from normal eyes in a small group indicated that fungi occur in the outer eye generally as transients. The epidemiology of mycotic keratitis has been discussed in relation to the present findings and in comparison with observations of other investigators.

Fungal inhibition in four commonly used agar media was improved by substituting natamycin (pimaricin) for cycloheximide. The recovery of bacteria was not affected by natamycin, whereas fungal contamination from a variety of samples was significantly suppressed. Furthermore, natamycin lacks the occupational health hazards of cycloheximide. Medium-dependent natamycin degradation occurred during the preparation and refrigerated storage of agar plates, but the addition of natamycin at 21.6 mug/ml resulted in effective residual activity.

BACKGROUND

Polyenes represent a major class of antifungal agents characterised by the presence of a series of conjugated double bonds in their planar hydroxylated macrolide ring structure. Despite their general interest, very little is known about the factors that modulate their biosynthesis. Among these factors, we have recently discovered a new inducing compound (PI-factor) in the pimaricin producer Streptomyces natalensis, which elicits polyene production in a manner characteristic of quorum sensing. Here, we describe the involvement of an amino-acid exporter from S. natalensis in modulating the expression of pimaricin biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.

RESULTS

Adjacent to the pimaricin gene cluster lies a member of the RhtB family of amino-acid exporters. Gene deletion and complementation experiments provided evidence for a role for PimT in the export of L-homoserine, L-serine, and L-homoserine lactone. Expression of the gene was shown to be induced by homoserine and by the quorum-sensing pimaricin-inducer PI-factor. Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.

CONCLUSIONS

This report describes the involvement of an amino acid exporter (encoded by pimT in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor. The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation. Additionally, this finding constitutes the first detailed characterization of an amino-acid exporter in an Actinomycete, and to our knowledge, the first evidence for the implication of this type of exporters in quorum sensing.

Streptomyces secondary metabolism is strongly affected by oxygen availability. The increased culture aeration enhances pimaricin production in S. natalensis, however the excess of O(2) consumption can lead to an intracellular ROS imbalance that is harmful to the cell. The adaptive physiological response of S. natalensis upon the addition of exogenous H(2)O(2) suggested that the modulation of the intracellular ROS levels, through the activation of the H(2)O(2) inducible catalase during the late exponential growth phase, can alter the production of pimaricin. With the construction of defective mutants on the H(2)O(2) related enzymes SodF, AhpCD and KatA1, an effective and enduring modulation of intracellular ROS was achieved. Characterization of the knock-out strains revealed different behaviours regarding pimaricin production: whilst the superoxide dismutase defective mutant presented low levels of pimaricin production compared to the wild-type, the mutants defective on the H(2)O(2)-detoxifying enzymes displayed a pimaricin overproducer phenotype. Using physiological and molecular approaches we report a crosstalk between oxidative stress and secondary metabolism regulatory networks. Our results reveal that the redox-based regulation network triggered by an imbalance of the intracellular ROS homeostasis is also able to modulate the biosynthesis of pimaricin in S. natalensis.

Mutants of Aspergillus fennelliae (Neosartorya fennelliae) resistant to relatively high levels of amphotericin B and low levels of nystatin were obtained by successive transfers of wild type in the presence of increasing concentrations of the polyenes. The resistance of the mutants to the polyenes was accompanied by both qualitative and quantitative changes in the sterol composition of the cells. Those resistant to amphotericin B (AF5-AB1 and p-AB1) lacked ergosterol, the major sterol of the wild type, but contained a new sterol clearly distinguished by the pattern of ultraviolet spectrophotometry and thin-layer chromatography. The mutants resistant to nystatin, however, contained both ergosterol and a new sterol, but the former was produced in a much reduced amount, as compared with the wild type. Genetic analysis indicated that the lack of ergosterol is closely associated with a reduced growth rate, poor asexual reproduction, and the loss of sexual reproduction. Growth studies revealed that the addition of ergosterol to the media did not affect the growth pattern of the mutants. Mutants resistant to amphotericin B showed an increased minimal inhibitory concentration for nystatin, pimaricin, and filipin. Mutants resistant to nystatin, however, conferred increased minimal inhibitory concentration for pimaricin and filipin but not for amphotericin B.

The recombinant polyketide synthase thioesterase domains from the pimaricin and 6-deoxyerythronolide B biosynthetic pathways catalyze hydrolysis of a number of simple N-acetylcysteamine thioester derivatives. This study demonstrates that thioesterases are not highly substrate selective in formation of the acyl-enzyme intermediate, in contrast to non-ribosomal peptide synthase thioesterase domains that show very high specificity for substrate loading. This observation has important implications for the engineering of biosynthetic pathways to produce polyketide products.

Glycosylated polyene macrolide antibiotics, as nystatins and amphotericins, are amphiphilic structures known to exert antifungal activity by disrupting the fungal cell membrane, leading to leakage of cellular materials, and cell death. This membrane disruption is strongly influenced by the presence and the exact nature of the membrane sterols. The solution structures of five representative glycosylated members, three tetraenes (pimaricin, nystatin A1 and rimocidin) and two heptaenes (candidin and vacidin A) have been calculated using geometric restraints derived from 1H-NMR data and random searches of their conformational space. Despite a different apparent structural order, the NMR solutions structure indicate that the hydroxyl groups all clustered on one side of the rod-shaped structures, and the glycosyl moieties are structurally conserved both in their conformation and their apparent order. The molecular structures afford an understanding of their selective interaction with the membrane sterols and the design of new polyene macrolides with improved activities.

The polyene antibiotics, including nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very valuable antifungal polyketide compounds, and they are typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain genes potentially encoding polyene biosynthesis. Here, sequence information of an approximately 125.7-kb contiguous DNA region in five overlapping cosmids isolated from the P. autotrophica KCTC9441 genomic library revealed a total of 23 open reading frames, which are presumably involved in the biosynthesis of a nystatin-like compound tentatively named NPP. The deduced roles for six multi-modular polyketide synthase (PKS) catalytic domains were found to be highly homologous to those of previously identified nystatin biosynthetic genes. Low NPP productivity suggests that the functionally clustered NPP biosynthetic pathway genes are tightly regulated in P. autotrophica. Disruption of a NPP PKS gene completely abolished both NPP biosynthesis and antifungal activity against Candida albicans, suggesting that polyene-specific genome screening may constitute an efficient method for isolation of potentially valuable previously identified polyene genes and compounds from various rare actinomycetes widespread in nature.

Candida albicans was the most common fungus responsible for mycotic keratitis in our series from a northern climate, as opposed to southern climates where other fungi were more common. Pimaricin was effective in our patients with Candida infections and in one patient with Aspergillus infection that had been unresponsive to previous amphotericin B.

Phialophora verrucosa and Cladosporium cladosporioides were isolated from a corneal ulcer. The lesion had been previously treated with antivirals and steroids and did not respond to Pimaricin therapy or conjunctival flap. A penetrating keratoplasty was performed with good functional and optical results. Chromomycosis of the cornea is an unusual infection. This is the second case of Phialophora in Florida and the first case of Cladosporium infection.