Structural and bioactivity characterization of filipin derivatives from engineered streptomyces filipinensis strains reveals clues for reduced haemolytic action

The rise in the number of immunocompromised patients has led to an increased incidence of fungal infections, with high rates of morbidity and mortality. Furthermore, misuse of antifungals has boosted the number of resistant strains to these agents; thus, there is urgent need for new drugs against these infections. Here, the in vitro antifungal activity of filipin III metabolic intermediates has been characterized against a battery of opportunistic pathogenic fungi—Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Trichosporon cutaneum, Trichosporon asahii, Aspergillus nidulans, Aspergillus niger, and Aspergillus fumigatus—using the Clinical and Laboratory Standards Institute broth microdilution method. Structural characterization of these compounds was undertaken by mass spectrometry (MS) and nuclear magnetic resonance (NMR) following HPLC purification. Complete NMR assignments were obtained for the first time for filipins I and II. In vitro haemolytic assays revealed that the haemolytic action of these compounds relies largely on the presence of a hydroxyl function at C26, since derivatives lacking such moiety show remarkably reduced activity. Two of these derivatives, 1′-hydroxyfilipin I and filipin I, show decreased toxicity towards cholesterol-containing membranes while retaining potent antifungal activity, and could constitute excellent leads for the development of efficient pharmaceuticals, particularly against CryptococcosisThis research was funded by the Spanish Ministerio de Economía, Industria y Competitividad (grants BIO2013-42983-P and PCIN-2016-190 to J.F.A.), an FPU contract of the Ministerio de Educación, Cultura y Deporte (FPU13/01537 to A.P.), and contracts from the Junta de Castilla y León (to E.G.B.) and from the youth employment initiative (to E.J.), both co-financed by the European Social Fun

PimT, an amino acid exporter controls polyene production via secretion of the quorum sensing pimaricin-inducer PI-factor in Streptomyces natalensis

<p>Abstract</p> <p>Background</p> <p>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 <it>Streptomyces natalensis</it>, which elicits polyene production in a manner characteristic of quorum sensing. Here, we describe the involvement of an amino-acid exporter from <it>S. natalensis </it>in modulating the expression of pimaricin biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>This report describes the involvement of an amino acid exporter (encoded by <it>pimT </it>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.</p

LAL Regulators SCO0877 and SCO7173 as Pleiotropic Modulators of Phosphate Starvation Response and Actinorhodin Biosynthesis in Streptomyces coelicolor

LAL regulators (Large ATP-binding regulators of the LuxR family) constitute a poorly studied family of transcriptional regulators. Several regulators of this class have been identified in antibiotic and other secondary metabolite gene clusters from actinomycetes, thus they have been considered pathway-specific regulators. In this study we have obtained two disruption mutants of LAL genes from S. coelicolor (Δ0877 and Δ7173). Both mutants were deficient in the production of the polyketide antibiotic actinorhodin, and antibiotic production was restored upon gene complementation of the mutants. The use of whole-genome DNA microarrays and quantitative PCRs enabled the analysis of the transcriptome of both mutants in comparison with the wild type. Our results indicate that the LAL regulators under study act globally affecting various cellular processes, and amongst them the phosphate starvation response and the biosynthesis of the blue-pigmented antibiotic actinorhodin. Both regulators act as negative modulators of the expression of the two-component phoRP system and as positive regulators of actinorhodin biosynthesis. To our knowledge this is the first characterization of LAL regulators with wide implications in Streptomyces metabolism

Phosphate effect on filipin production and morphological differentiation in Streptomyces filipinensis and the role of the PhoP transcription factor.

The biosynthesis of the antifungal filipin in Streptomyces filipinensis is very sensitive to phosphate regulation. Concentrations as low as 2.5 mM block filipin production. This effect is, at least in part, produced by repression of the transcription of most filipin biosynthetic genes. The role of the two-component PhoRP system in this process was investigated. The phoRP system of S. filipinensis was cloned and transcriptionally characterised. PhoP binds to two PHO boxes present in one of its two promoters. Filipin production was greatly increased in ΔphoP and ΔphoRP mutants, in agreement with a higher transcription of the fil genes, and the effect of phosphate repression on the antibiotic production of these strains was significantly reduced. No PhoP binding was observed by electrophoretic mobility gel shift assays (EMSAs) with the promoter regions of the fil gene cluster thus suggesting an indirect effect of mutations. Binding assays with cell-free extracts from the wild-type and mutant strains on fil genes promoters revealed retardation bands in the parental strain that were absent in the mutants, thus suggesting that binding of the putative transcriptional regulator or regulators controlled by PhoP was PhoP dependent. Noteworthy, PhoP or PhoRP deletion also produced a dramatic decrease in sporulation ability, thus indicating a clear relationship between the phosphate starvation response mediated by PhoP and the sporulation process in S. filipinensis. This effect was overcome upon gene complementation, but also by phosphate addition, thus suggesting that alternative pathways take control in the absence of PhoRP

Hierarchical Control on Polyene Macrolide Biosynthesis: PimR Modulates Pimaricin Production via the PAS-LuxR Transcriptional Activator PimM

<div><p>Control of polyene macrolide production in <em>Streptomyces natalensis</em> is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the <em>Streptomyces</em> antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. The PimR SARP domain (PimR^SARP) was expressed in <em>Escherichia coli</em> as a glutathione S-transferase (GST)–fused protein. Electrophoretic mobility shift assays showed that GST-PimR^SARP binds a single target, the intergenic region between the regulatory genes <em>pimR</em> and <em>pimM</em>s in the pimaricin cluster. The PimR^SARP-binding site was investigated by DNaseI protection studies, revealing that it contains three heptameric direct repeats adjusting to the consensus 5′-CGGCAAG-3′. Transcription start points of <em>pimM</em> and <em>pimR</em> promoters were identified by 5′-RACE, revealing that unlike other SARPs, PimR^SARP does not interact with the -35 region of its target promoter. Quantitative transcriptional analysis of these regulatory genes on mutants on each of them has allowed the identification of the <em>pimM</em> promoter as the transcriptional target for PimR. Furthermore, the constitutive expression of <em>pimM</em> restored pimaricin production in a pimaricin-deficient strain carrying a deletion mutant of <em>pimR</em>. These results reveal that PimR exerts its positive effect on pimaricin production by controlling <em>pimM</em> expression level, a regulator whose gene product activates transcription from eight different promoters of pimaricin structural genes directly.</p> </div

Organization of the pimaricin gene cluster and GST-PimR^SARP DNA binding assay results.

<p>A) Pointed boxes indicate the direction of transcription. Transcriptional regulatory genes (<i>pimR</i> and <i>pimM</i>) are indicated in red, other regulators are indicated in purple, and the polyketide synthase genes in green. The remaining genes (in grey) are involved in polyene tailoring or export. B) Electrophoretic mobility analysis (EMSA) of GST-PimR^SARP binding to different putative promoter regions. The arrow indicates the DNA–protein complex. Promoter names are indicated above the picture. All experiments were carried out with 2 ng labeled DNA probe. Left lane, control without protein; right lane, 10 µM of GST-PimR^SARP protein.</p

Gene expression analysis of the genes <i>SCO0877</i> and <i>SCO7173</i> by RT-PCR.

<p>Analysis was carried out on <i>S.coelicolor</i> M145 strain as indicated in the Methods section after 28 (<i>SCO0877</i>) or 32 (<i>SCO7173</i>) PCR amplification cycles. RNA was extracted from cultures after growth for 48 h and 60 h in defined (M) and complex (C) MG medium. The identity of each amplified product was corroborated by direct sequencing. The absence of contaminating DNA in the RNA samples was assessed by PCR. Transcription of the <i>hrdB</i> gene (<i>SCO5820</i>) was assessed as an internal control (bottom).</p

Binding of GST-PimR^SARP to its target is specific.

<p>Analysis by EMSA of the binding of GST-PimR^SARP to the <i>pimR-M</i> promoter region. Arrows indicate the DNA–protein complexes. All experiments were carried out with 2 ng labeled DNA probe. Lane P, control without protein. A) Decreasing gradient of protein. Lane 1, 5 µM of protein; lane 2, 2.5 µM; lane 3, 1.25 µM; lane 4, 625 nM; lane 5, 312 nM; lane 6, 160 nM; lane 7, 80 nM; lane 8, 40 nM of protein. B) Control reaction with 10 µM of pure GST protein. C) Competition experiment between labeled <i>pimM-R</i> promoter and unlabeled <i>pimM-R</i> promoter. The experiment was performed with 1.25 µM of GST-PimR^SARP. D) Competition experiment between <i>pimM-R</i>p and <i>pimJ</i>p. Note that 200-fold-higher concentrations of unlabeled <i>pimJ</i>p competitor DNA failed to decrease the intensities of the <i>pimM-R</i>p retardation bands. The experiment was performed with 80 nM of GST-PimR^SARP.</p