Dataset Information

Myricanol Inhibits the Type III Secretion System of Salmonella enterica Serovar Typhimurium by Interfering With the DNA-Binding Activity of HilD.

ABSTRACT: The type III secretion system (T3SS) consists of a syringe-like export machine injecting effectors from the bacterial cytosol directly into host cells to establish infection. This mechanism is widely distributed in gram-negative bacteria and can be targeted as an innovative strategy for the developing of anti-virulence drugs. In this study, we present an effective T3SS inhibitor, myricanol, inspired by the use of folk medicinal plants traditionally used against infections. Myricanol is a cyclic diarylheptanoid isolated from the medicinal plant Myrica nagi, which is found in South and East Asia. Bioassay-guided fractionation revealed that myricanol inhibited not only the secretion of type III effector proteins of Salmonella enterica serovar Typhimurium UK-1 ?8956 (S. Typhimurium) but also the invasion of S. Typhimurium into mammalian cells, but showed no toxicity to bacterial growth or the host cells. RNA-Seq data analysis showed that the transcription of the pathogenesis-related SPI-1 gene was significantly inhibited by myricanol. Further study demonstrated that myricanol binds physically to HilD and interferes with its DNA-binding activity to the promoters of the hilA and invF genes. In conclusion, we propose that myricanol is responsible for the anti-infectious properties of M. nagi and is a novel T3SS inhibitor of S. Typhimurium through a previously unappreciated mechanism of action.

SUBMITTER: Wu Y

PROVIDER: S-EPMC7546796 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Myricanol Inhibits the Type III Secretion System of <i>Salmonella enterica</i> Serovar Typhimurium by Interfering With the DNA-Binding Activity of HilD.

Wu Yang Y Yang Xuefei X Zhang Dongdong D Lu Chunhua C

Frontiers in microbiology 20200925

The type III secretion system (T3SS) consists of a syringe-like export machine injecting effectors from the bacterial cytosol directly into host cells to establish infection. This mechanism is widely distributed in gram-negative bacteria and can be targeted as an innovative strategy for the developing of anti-virulence drugs. In this study, we present an effective T3SS inhibitor, myricanol, inspired by the use of folk medicinal plants traditionally used against infections. Myricanol is a cyclic ...[more]

PMID: 33101243

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Project description:The Salmonella type three secretion system (T3SS), encoded in the Salmonella pathogenicity island 1 (SPI1) locus, mediates the invasion of the host intestinal epithelium. SPI1 expression is dependent upon three AraC-like regulators: HilD, HilC, and RtsA. These regulators act in a complex feed-forward loop to activate each other and hilA, which encodes the activator of the T3SS structural genes. HilD has been shown to be the major integration point of most signals known to activate the expression of the SPI1 T3SS, acting as a switch to control induction of the system. HilE is a negative regulator that acts upon HilD. Here we provide genetic and biochemical data showing that HilE specifically binds to HilD but not to HilC or RtsA. This protein-protein interaction blocks the ability of HilD to bind DNA as shown by both an in vivo reporter system and an in vitro gel shift assay. HilE does not affect HilD dimerization, nor does it control the stability of the HilD protein. We also investigated the role of HilE during the infection of mice using competition assays. Although deletion of hilE does not confer a phenotype, the hilE mutation does suppress the invasion defect conferred by loss of FliZ, which acts as a positive signal controlling HilD protein activity. Together, these data suggest that HilE functions to restrict low-level HilD activity, preventing premature activation of SPI1 until positive inputs reach a threshold required to fully induce the system.IMPORTANCESalmonella is a leading cause of gastrointestinal and systemic disease throughout the world. The SPI1 T3SS is required for Salmonella to induce inflammatory diarrhea and to gain access to underlying tissue. A complex regulatory network controls expression of SPI1 in response to numerous physiological inputs. Most of these signals impinge primarily on HilD translation or activity. The system is triggered when HilD activity crosses a threshold that allows efficient activation of its own promoter. This threshold is set by HilE, which binds to HilD to prevent the inevitable minor fluctuations in HilD activity from inappropriately activating the system. The circuit also serves as a paradigm for systems that must integrate numerous environmental parameters to control regulatory output.

Project description:UNLABELLED:Type III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the "switch protein," a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of the Salmonella enterica serovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage event per se does not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function. IMPORTANCE:Bacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally diverse bacterial proteins into the host cell. Critical to the function of these systems is their ability to secrete proteins in a strict hierarchical order, but it is unclear how the mechanism of switching works. Central to the switching mechanism is a highly conserved inner membrane protease that undergoes autocatalytic cleavage. Although it has been suggested previously that the autocleavage event is the trigger for substrate switching, we show here that this is not the case. Rather, our results show that cleavage allows the proper conformation of the protein to render it competent for its switching function. These findings may help develop inhibitors of type III secretion machines that offer novel therapeutic avenues to treat various infectious diseases.

Dataset Information

Myricanol Inhibits the Type III Secretion System of Salmonella enterica Serovar Typhimurium by Interfering With the DNA-Binding Activity of HilD.

Publications

Myricanol Inhibits the Type III Secretion System of <i>Salmonella enterica</i> Serovar Typhimurium by Interfering With the DNA-Binding Activity of HilD.

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