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Characterization of the Mode of Action of Aurodox, a Type III Secretion System Inhibitor from Streptomyces goldiniensis.

ABSTRACT: Recent work has demonstrated that the polyketide natural product Aurodox from Streptomyces goldiniensis is able to block the pathogenesis of the murine pathogen Citrobacter rodentium In this work, we aimed to gain a better understanding of the mechanism of action of the compound. We show that Aurodox downregulates the expression of the type III secretion systems of enteropathogenic and enterohemorrhagic Escherichia coli Furthermore, we have used transcriptomic analysis to show that Aurodox inhibits the expression at the transcriptional level by repressing the master regulator, ler Our data support a model in which Aurodox acts upstream of ler and not directly on the secretion system itself. Finally, we have shown that Aurodox, unlike some traditional antibiotics, does not induce expression of RecA, which is essential for the production of Shiga toxin. We propose that these properties nominate Aurodox as a promising antivirulence therapy for the treatment of these infections.

SUBMITTER: McHugh RE

PROVIDER: S-EPMC6346137 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Characterization of the Mode of Action of Aurodox, a Type III Secretion System Inhibitor from Streptomyces goldiniensis.

McHugh Rebecca E RE O'Boyle Nicky N Connolly James P R JPR Hoskisson Paul A PA Roe Andrew J AJ

Infection and immunity 20190124 2

Recent work has demonstrated that the polyketide natural product Aurodox from Streptomyces goldiniensis is able to block the pathogenesis of the murine pathogen Citrobacter rodentium In this work, we aimed to gain a better understanding of the mechanism of action of the compound. We show that Aurodox downregulates the expression of the type III secretion systems of enteropathogenic and enterohemorrhagic Escherichia coli Furthermore, we have used transcriptomic analysis to sh ...[more]

PMID: 30455200

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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:Chlamydia trachomatis Scc4 (formerly CT663) engages the transcription machinery and the pathogenic type III secretion system (T3SS). Both machines are required for Chlamydia infection. These requirements and the limited ability for genetic manipulation in Chlamydia have hampered dissection of Scc4's contributions. Here, by developing bacterial systems that permit the controlled expression and stable maintenance of Scc4, we assess Scc4's effects on chlamydial growth phenotype, secretion, and the patterns of T3SS gene expression. Expressing Scc4 in Escherichia coli lacking a T3SS injectisome causes a growth defect. This deficiency is rescued by overexpressing the β-subunit of RNA polymerase (RNAP) or by exploiting sigma 70 (σ70) (homologous to chlamydial σ66) mutants that strengthen the interaction between σ70 region 4 and the β-flap, confirming Scc4's distinction as a module of RNAP holoenzyme capable of modulating transcription. Yersinia pestis expressing Scc4 sustains a functional T3SS, through which CopN secretion is boosted by cooption of Scc4 and Scc1. Finally, conditional expression of Scc4 in C. trachomatis results in fast expansion of the Chlamydia-containing vacuole and accelerated chlamydial development, coupled to selective up- or downregulation of gene expression from different T3SS genes. This work reveals, for the first time, the context-dependent action of Scc4 linking it to diverse protein networks in bacteria. It establishes that Scc4, when overexpressed, exerts incredible effects on chlamydial development by reinforcing control of the T3SS.IMPORTANCE The T3SS is a key virulence factor required for C. trachomatis infection. The control of the T3SS has not been well studied in this obligate intracellular pathogen. Here, we show that Scc4 plays a major role for precise control of the pathogenic T3SS at the levels of gene expression and effector secretion through genetically separable protein networks, allowing a fast adaptive mode of C. trachomatis development during infection in human epithelial cells.

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

Characterization of the Mode of Action of Aurodox, a Type III Secretion System Inhibitor from Streptomyces goldiniensis.

Publications

Characterization of the Mode of Action of Aurodox, a Type III Secretion System Inhibitor from Streptomyces goldiniensis.

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