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Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor.

ABSTRACT: Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC(50) of 14 nM, guadinomine B is the most potent known inhibitor of the type III secretion system (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli , Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase, a nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-acyl carrier protein extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.

SUBMITTER: Holmes TC

PROVIDER: S-EPMC3483642 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor.

Holmes Tracy C TC May Aaron E AE Zaleta-Rivera Kathia K Ruby J Graham JG Skewes-Cox Peter P Fischbach Michael A MA DeRisi Joseph L JL Iwatsuki Masato M Ōmura Satoshi S Khosla Chaitan C

Journal of the American Chemical Society 20121010 42

Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC(50) of 14 nM, guadinomine B is the most potent known inhibitor of the type III secretion system (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli , Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guad ...[more]

PMID: 23030602

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Project description:The global increase in antimicrobial-resistant infections means that there is a need to develop new antimicrobial molecules and strategies to combat the issue. Aurodox is a linear polyketide natural product that is produced by Streptomyces goldiniensis, yet little is known about aurodox biosynthesis or the nature of the biosynthetic gene cluster (BGC) that encodes its production. To gain a deeper understanding of aurodox biosynthesis by S. goldiniensis, the whole genome of the organism was sequenced, revealing the presence of an 87 kb hybrid polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) BGC. The aurodox BGC shares significant homology with the kirromycin BGC from S. collinus Tϋ 365. However, the genetic organization of the BGC differs significantly. The candidate aurodox gene cluster was cloned and expressed in a heterologous host to demonstrate that it was responsible for aurodox biosynthesis and disruption of the primary PKS gene (aurAI) abolished aurodox production. These data supported a model whereby the initial core biosynthetic reactions involved in aurodox biosynthesis followed that of kirromycin. Cloning aurM* from S. goldiniensis and expressing this in the kirromycin producer S. collinus Tϋ 365 enabled methylation of the pyridone group, suggesting this is the last step in biosynthesis. This methylation step is also sufficient to confer the unique type III secretion system inhibitory properties to aurodox. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) is a significant global pathogen for which traditional antibiotic treatment is not recommended. Aurodox inhibits the ability of EHEC to establish infection in the host gut through the specific targeting of the type III secretion system while circumventing the induction of toxin production associated with traditional antibiotics. These properties suggest aurodox could be a promising anti-virulence compound for EHEC, which merits further investigation. Here, we characterized the aurodox biosynthetic gene cluster from Streptomyces goldiniensis and established the key enzymatic steps of aurodox biosynthesis that give rise to the unique anti-virulence activity. These data provide the basis for future chemical and genetic approaches to produce aurodox derivatives with increased efficacy and the potential to engineer novel elfamycins.

Project description:BackgroundWe previously identified a plant-derived phenolic compound ortho-coumaric acid (OCA) as an inhibitor of type III secretion system (T3SS) of Xanthomonas oryzae pv. oryzae (Xoo), the pathogen causing bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. However, the molecular mechanisms by which OCA suppresses T3SS and the transcriptional responses to the OCA treatments in Xoo remains unclear.ResultsThe present study conducted the RNA-seq-based transcriptomic analysis to reveal changes in gene expression in Xoo in response to 30 min, 1 h, 3 h, and 6 h of OCA treatment. Results showed that OCA significantly inhibited the expression of T3SS genes after 30 min, and the inhibition also existed after 1 h, 3 h, and 6 h. After treatment for 30 min, membrane proteins in the functional category of cellular process was the predominant group affected, indicating that Xoo was in the early stress stage. Over time, more differentially-expressed genes (DEGs) gathered in the functional category of biological process. Analysis of common DEGs at all four of time points revealed the core elements of Xoo during the response to OCA treatment. Notable, a multidrug transporter cluster that consisted of a MarR-family protein (PXO_RS13760), a multidrug RND transporter (PXO_RS13755), a multidrug transporter (PXO_RS13750), and an MFS transporter (PXO_RS13745) were significantly up-regulated at all four of the time points. Although these three transporter genes were not upregulated by OCA in the PXO_RS13760 deletion mutant, the deficiency of PXO_RS13760 in Xoo did not affect T3SS transcript, and OCA still had the ability to inhibit the expression of T3SS in the mutant, suggesting that the MarR-family protein was involved in bacterial responses to OCA, but not direct OCA inhibition of T3SS in Xoo.ConclusionsWe analyzed the transcriptome of Xoo during OCA treatment at both early and late stages, which revealed the landscape of Xoo responses to OCA at the whole-genome transcription level. A multidrug transporter cluster was identified to be involved in the response process, but had no direct relation to T3SS in Xoo.

Dataset Information

Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor.

Publications

Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor.

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