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

Activation of Secondary Metabolite Gene Clusters in Streptomyces clavuligerus by the PimM Regulator of Streptomyces natalensis

Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex

Regulación de la producción de macrólidos antifúngicos = Regulation of antifungal macrolides production

407 p.Streptomyces son un grupo de bacterias del suelo conocido por su capacidad de producir una gran variedad de metabolitos secundarios de interés industrial. Entre ellos, destacan los macrólidos poliénicos, potentes antifúngicos que son comúnmente empleados en la industria alimentaria para prevenir la contaminación de carnes y quesos, y en la práctica clínica para el tratamiento de micosis superficiales y sistémicas. A pesar del indudable interés de los macrólidos polienos como agentes antifúngicos, no existe un esquema establecido que permita entender la red de elementos reguladores que gobiernan la producción de este tipo de moléculas. El objetivo central de este trabajo fue desvelar el papel de distintos elementos reguladores en el control de la biosíntesis de polienos. De manera general, existen dos niveles en el control transcripcional del metabolismo secundario: el ejercido por los reguladores que se encuentran dentro de la agrupación génica del metabolito al que regulan, y el ejercido por aquellos reguladores globales o pleiotrópicos, que afectan a multitud de procesos celulares. La pimaricina es el paradigma de los polienos glicosilados y es producida por S. natalensis, el cual presenta una agrupación génica de gran tamaño dedicada a su biosíntesis. Esta agrupación contiene dos reguladores específicos de ruta, PimM y PimR, que actúan como activadores transcripcionales de los genes biosintéticos y constituyen el prototipo de los reguladores de tipo PAS-LuxR y SARP-LAL respectivamente. En este trabajo se ha profundizado en la caracterización de los operadores de dichos reguladores. La validación experimental de la secuencia de unión de PimM ha mostrado que la mayor afinidad del regulador se produce con el consenso inalterado y cualquier cambio ya supone una disminución considerable en la afinidad, lo que indica que las secuencias diana del regulador han evolucionado de manera que se han optimizado para la unión del mismo. Por su parte, el operador reconocido por PimR contiene un doblete y un triplete de heptanucleótidos, cuya caracterización ha mostrado que el triplete es esencial para la unión del regulador, mientras que el doblete es prescindible. El hecho de que reguladores ortólogos de PimR presenten operadores conservados sugiere que los resultados observados podrían ser extrapolables a la producción de compuestos por otras especies. La filipina, por su parte, es el modelo de los macrólidos poliénicos no glicosilados y por ello resultó interesante estudiar los mecanismos reguladores de su producción. La biosíntesis de este pentaeno por S. filipinensis es el resultado de una compleja ruta biosintética en la que hay dos reguladores ortólogos a pimM y pimR, filF y filR respectivamente, cuya funcionalidad se encuentra conservada. Sin embargo, poco se sabe del efecto de reguladores globales sobre su producción. Por ello, en este trabajo hemos estudiado el papel del regulador PhoP y de las γ-butirolactonas sobre la biosíntesis del antifúngico. La producción de filipina resultó ser altamente sensible a la presencia de fosfato inorgánico en el medio de crecimiento y este efecto está mediado, al menos parcialmente, por el sistema de dos componentes PhoRP. La caracterización de este sistema en S. filipinensis ha mostrado que se encuentra conservado y que PhoP actúa como un represor del metabolismo secundario y un activador del metabolismo primario. Por otro lado, la caracterización de una región cromosómica de S. filipinensis nos llevó a identificar un posible receptor y un posible pseudor-receptor de γ-butirolactonas así como una posible enzima biosintética (sfbR, sfbR2 y sfbA respectivamente). La inactivación de dichos genes desveló que presentan papeles diferenciales sobre la biosíntesis de filipina y la diferenciación morfológica. El estudio de la expresión de estos genes mostró que sfbR y sfbR2 presentan una regulación cruzada y sugiere que mantienen un equilibrio responsable del fino control del metabolismo secundario y la diferenciación morfológica en S. filipinensis. Por último, cabe destacar que durante la biosíntesis de la filipina por S. filipinensis se forman tres intermediarios de la misma: filipina I, 1’-hidroxifilipina I y filipina II. El estudio de la actividad biológica de dichos intermediarios (actividades antifúngica y hemolítica) mostró que la filipina II presenta propiedades similares a la filipina III, mientras que la filipina I resultó ser la menos activa. Sin embargo, el derivado 1’-hidroxifilipina I presentó propiedades mejoradas. Estos resultados la sitúan como un polieno con posible aplicación médica aunque otros parámetros han de ser evaluados para confirmarlo

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis

International audienceThe biosynthesis of small-size polyene macrolides is ultimately con-trolled by acouple of transcriptional regulators that act in a hierarchical way. AStreptomycesantibiotic regulatory protein–large ATP-binding regulator of the LuxRfamily (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encodinggene and activates its transcription, and in turn, the gene product of the latter acti-vates transcription from various promoters of the polyene gene cluster directly. Theprimary operator of PimR, the archetype of SARP-LAL regulators, contains three hep-tameric directrepeats separated by four-nucleotide spacers, but the regulator canalso bind a secondary operator with only two direct repeats separated by a3-nucleotide spacer, both located in the promoter region of its unique target gene,pimM. A similar arrangement of operators has been identified for PimR counterpartsencoded by gene clusters for different antifungal secondary metabolites, includingnot only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cyclohex-imide. Here, we used promoter engineering and quantitative transcriptional analysesto determine the contributions of the different heptameric repeats to transcriptionalactivation and final polyene production. Optimized promoters have thus been devel-oped. Deletion studies and electrophoretic mobility assays were used for the defini-tion of DNA-binding boxes formed by 22-nucleotide sequences comprising twoconserved heptameric direct repeats separated by four-nucleotide less conservedspacers. The cooperative binding of PimRSARPappears to be the mechanism involvedin the binding of regulator monomers to operators, and at least two protein mono-mers are required for efficient binding

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

Pathway-specific regulation revisited: cross-regulation of multiple disparate gene clusters by PAS-LuxR transcriptional regulators

International audiencePAS-LuxR regulators are highly conserved proteins devoted to the control of antifungal production by binding to operators located in given promoters of polyene biosynthetic genes. The canonical operator of PimM, archetype of this class of regulators, has been used here to search for putative targets of orthologous protein PteF in the genome of Streptomyces avermitilis, finding 97 putative operators outside the pentaene filipin gene cluster (pte). The processes putativelyaffected included genetic information processing; energy, carbohydrate, and lipid metabolism; DNA replication and repair; morphological differentiation; secondary metabolite biosynthesis; and transcriptional regulation, among others.Seventeen of these operators were selected, and their binding to PimM DNA-binding domain was assessed by electrophoretic mobility shift assays. Strikingly, the protein bound all predicted operators suggesting a direct control over targeted processes. As a proof of concept, we studied the biosynthesis of the ATP-synthase inhibitor oligomycin whose gene cluster included two operators. Regulator mutants showed a severe loss of oligomycin production, whereas gene complementation of the mutant restored phenotype, and gene duplication in the wild-type strain boosted oligomycin production. Comparative gene expression analyses in parental and mutant strains by reverse transcription-quantitative polymerase chain reaction of selected olm genes corroborated production results. These results demonstrate that PteF is able to crossregulate the biosynthesis of two related secondary metabolites, filipin and oligomycin, but might be extended to all the processes indicated above. This study highlights the complexity of the network of interactions in which PAS-LuxR regulators are involved and opens new possibilities for the manipulation of metabolite production in Streptomycetes

Biotechnological production and application of the antibiotic pimaricin: biosynthesis and its regulation

International audiencePimaricin (natamycin) is a small polyene macrolideantibiotic used worldwide. This efficient antimycotic andantiprotozoal agent, produced by several soil bacterial speciesof the genusStreptomyces, has found application in humantherapy, in the food and beverage industries and as pesticide.It displays a broad spectrum of activity, targeting ergosterolbut bearing a particular mode of action different to other poly-ene macrolides. The biosynthesis of this only antifungal agentwith a GRAS status has been thoroughly studied, which haspermitted the manipulation of producers to engineer the bio-synthetic gene clusters in order to generate several analogues.Regulation of its production has been largely unveiled, con-stituting a model for other polyenes and setting the leads foroptimizing the production of these valuable compounds. Thisreview describes and discusses the molecular genetics, uses,mode of action, analogue generation, regulation and strategiesfor increasing pimaricin production yields

PAS-LuxR transcriptional control of filipin biosynthesis in S. avermitilis

International audienceThe 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

Modulation of Multiple Gene Clusters’ Expression by the PAS-LuxR Transcriptional Regulator PteF

International audiencePAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from Streptomyces avermitilis. Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator. Transcriptomic analyses were performed in S. avermitilis ΔpteF and its parental strain. Both strains were grown in a YEME medium without sucrose, and the samples were taken at exponential and stationary growth phases. A total of 257 genes showed an altered expression in the mutant, most of them at the exponential growth phase. Surprisingly, despite PteF being considered an activator, most of the genes affected showed overexpression, thereby suggesting a negative modulation. The affected genes were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation, among others, but were particularly related to secondary metabolite biosynthesis. Notably, 10 secondary metabolite gene clusters out of the 38 encoded by the genome showed altered expression profiles in the mutant, suggesting a regulatory role for PteF that is wider than expected. The transcriptomic results were validated by quantitative reverse-transcription polymerase chain reaction. These findings provide important clues to understanding the intertwined regulatory machinery that modulates antibiotic biosynthesis in Streptomyces