Dataset Information

Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro) Biosynthetic Pathway in Streptomyces sp. US24 Strain.

ABSTRACT: We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro) diketopiperazine (DKP). To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp) was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS). The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro) biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.

SUBMITTER: Sioud S

PROVIDER: S-EPMC1940057 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro) Biosynthetic Pathway in Streptomyces sp. US24 Strain.

Sioud Samiha S Karray-Rebai Ines I Aouissaoui Hedi H Aigle Bertrand B Bejar Samir S Mellouli Lotfi L

Journal of biomedicine & biotechnology 20070101 6

We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro) diketopiperazine (DKP). To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp) was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases ...[more]

PMID: 17710112

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Project description:The anti-inflammatory, antiviral, and anti-cancer properties, as well as the mechanism of action of cyclo-[Pro-Pro-β3-HoPhe-Phe-] tetrapeptide (denoted as 4B8M), were recently described. The aim of this work was to synthesize and evaluate the immunosuppressive actions of the stereochemical variants of 4B8M by sequential substitution of L-amino acids by D-amino acids (a series of peptides denoted as P01-P07) using parent 4B8M as a reference compound. In addition, diverse available bioinformatics tools using machine learning and artificial intelligence were tested to find the bio-pharmacokinetic and polypharmacological attributes of analyzed stereomers. All peptides were non-toxic to human peripheral blood mononuclear cells (PBMCs) and only cyclo-[D-Pro-Pro-β3-HoPhe-Phe-] peptide (P03) was capable of inhibiting mitogen-induced PBMC proliferation. The peptides inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α) to various degrees, with P04 (cyclo-[Pro-Pro-D-β3-HoPhe-Phe-]) and P03 being the most potent. For further in vivo studies, P03 was selected because it had the combined properties of inhibiting cell proliferation and TNF-α production. P03 demonstrated a comparable ability to 4B8M in the inhibition of auricle edema and lymph node cell number and in the normalization of a distorted blood cell composition in contact sensitivity to the oxazolone mouse model. In the mouse model of carrageenan-induced inflammation of the air pouch, P03 exhibited a similar inhibition of the cell number in the air pouches as 4B8M, but its inhibitory effects on the percentage of neutrophils and eosinophils in the air pouches and blood, as well as on mastocyte degranulation in the air pouches, were stronger in comparison to 4B8M. Lastly, in a mouse model of dextran sulfate-induced colitis, similar effects to 4B8M regarding thymocyte number restoration and normalization of the blood cell pictures by P03 were observed. In summary, depending on either experimental findings or in silico predictions, P03 demonstrated comparable, or even better, anti-inflammatory and bio-pharmacokinetic properties to 4B8M and may be considered as a potential therapeutic. The possibility of P00 and P03 identification by circular dichroism measurements was tested by quantum-chemical calculations.

Project description:UnlabelledVibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue.ImportanceThe ToxR regulon has been a focus of cholera research for more than three decades. During this time, a model has emerged wherein ToxR functions to activate the expression of Vibrio cholerae virulence factors upon host entry. V. cholerae and other enteric bacteria produce cyclo(Phe-Pro), a cyclic dipeptide that we identified as an inhibitor of V. cholerae virulence factor production. This finding suggested that cyclo(Phe-Pro) was a negative effector of virulence factor production and represented a molecule that could potentially be exploited for therapeutic development. In this work, we investigated the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We found that cyclo(Phe-Pro) signaled through ToxR to activate the expression of leuO, a new virulence regulator that functioned to repress virulence factor production. Our results have identified a new arm of the ToxR regulon and suggest that ToxR may play a broader role in pathogenesis than previously known.

Dataset Information

Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro) Biosynthetic Pathway in Streptomyces sp. US24 Strain.

Publications

Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro) Biosynthetic Pathway in Streptomyces sp. US24 Strain.

Similar Datasets

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