Visible Fluorescence Detection of Type III Protein Secretion from Bacterial Pathogens

Yount, Jacob S.; Tsou, Lun K.; Dossa, Paul D.; Kullas, Amy L.; Velden, Adrianus W. M. van der; Hang, Howard C.

doi:10.1021/ja102257v

Cited by 15 publications

(28 citation statements)

References 14 publications

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“…To evaluate the activity of TCMs on type III protein secretion in S. Typhimurium, we screened 120 TCMs with previously suggested anti-infective activity 4 using a high-throughput fluorescence previously developed by our laboratory (Figure 1A) 8 . The type III protein secretion assay employs carboxypeptidase G2 (CPG2) as an enzyme reporter that it is fused to the C-terminus of a known S. Typhimurium T3SS protein effector (SopE2).…”

Section: Resultsmentioning

confidence: 99%

“…The type III protein secretion assay employs carboxypeptidase G2 (CPG2) as an enzyme reporter that it is fused to the C-terminus of a known S. Typhimurium T3SS protein effector (SopE2). SopE2-CPG2-HA is secreted and specifically reports on type III protein secretion through cleavage of fluorogenic substrates (Glu-CyFur), which provides a sensitive method for monitoring the inhibitory activity of small molecules targeted at T3SSs (Figure 1A) 8 . Our screen of anti-infective TCMs at 5 mg/mL revealed 30 medicinal plant extracts with significant S. Typhimurium pathogenicity island-1 (SPI-1) T3SS inhibitory activity (Figure 1B and Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…S. Typhimurium strain expressing SopE2-CPG2-HA fusion protein 8 and the S. Typhimurium strains SB905 46 , SB300 46 , IR715 47 and the T3SS-defective invA mutant 14 have been previously described. All LB media used were made from BD Difco ™ LB (Luria-Bertani) Broth Miller, which contains 10 g/L NaCl.…”

Section: Methodsmentioning

confidence: 99%

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Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates

et al. 2016

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Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections. However, the molecules responsible for these anti-infective properties and their potential mechanisms of action have remained elusive. Using a high-throughput assay for type III protein secretion in Salmonella enterica serovar Typhimurium, we discovered that several TCMs can attenuate this key virulence pathway without affecting bacterial growth. Amongst the active TCMs, we discovered that baicalein, a specific flavonoid from Scutellaria baicalensis, targets S. Typhimurium pathogenicity island-1 (SPI-1) type III secretion system (T3SS) effectors and translocases to inhibit bacterial invasion of epithelial cells. Structurally related flavonoids present in other TCMs, such as quercetin also inactivated the SPI-1 T3SS and attenuated S. Typhimurium invasion. Our results demonstrate that specific plant metabolites from TCMs can directly interfere with key bacterial virulence pathways and reveals a previously unappreciated mechanism of action for anti-infective medicinal plants.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates

et al. 2016

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“…18 This two-component assay takes advantage of the unique enzyme activity of carboxypeptidase G2 (CPG2) that when attached to the C-terminus of a known S. Typhimurium bacterial effector (SopE2) can rapidly and specifically report on type III protein secretion through cleavage of fluorogenic substrates (Glu-CyFur) (Fig 1A). 18 Amongst the plant metabolites we explored, polyphenolic catechins such as catechin gallate (CG) and epicatechin gallate (ECG) completely inhibited SopE2-CPG2-HA reporter activity, whereas catechin and gallic acid had less than 20% inhibitory activity at 25 µM (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Epigallocatechin-3-gallate inhibits bacterial virulence and invasion of host cells

Tsou

Yount

Hang

2017

Bioorganic & Medicinal Chemistry

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Increasing antibiotic resistance and beneficial effects of host microbiota has motivated the search for anti-infective agents that attenuate bacterial virulence rather than growth. For example, we discovered that specific flavonoids such as baicalein and quercetin from traditional medicinal plant extracts could attenuate Salmonella enterica serovar Typhimurium type III protein secretion and invasion of host cells. Here, we show epigallocatechin-3-gallate from green tea extracts also inhibits the activity of S. Typhimurium type III protein effectors and significantly reduces bacterial invasion into host cells. These results reveal additional dietary plant metabolites that can attenuate bacterial virulence and infection of host cells.

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“…These compounds were then widely tested across Gram-negative pathogens, and demonstrated to have activity against E. coli [69], Yersinia [18,20], Chlamydia [17,26], Salmonella [23,24,70], and Shigella [16] species, lending credence to the concept of the T3SS as a potentially broad spectrum Gram-negative target. Despite the value of these pioneering compounds for the study of the T3SS, the chemotype itself appears to have some liabilities as a preclinical candidate for an anti-virulence drug.…”

Section: Acyl Salicylaldehyde Hydrazonesmentioning

confidence: 99%

The Type III Secretion System as a Source of Novel Antibacterial Drug Targets

Kline¹,

Felise²,

Sanowar³

et al. 2012

CDT

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Type III Secretion Systems (T3SSs) are highly organized multi-protein nanomachines which translocate effector proteins from the bacterial cytosol directly into host cells. These systems are required for the pathogenesis of a wide array of Gram-negative bacterial pathogens, and thus have attracted attention as potential antibacterial drug targets. A decade of research has enabled the identification of natural products, conventional small molecule drug-like structures, and proteins that inhibit T3SSs. The mechanism(s) of action and molecular target(s) of the majority of these inhibitors remain to be determined. At the same time, structural biology methods are providing an increasingly detailed picture of the functional arrangement of the T3SS component proteins. The confluence of these two research areas may ultimately identify non-classical drug targets and facilitate the development of novel therapeutics.

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Visible Fluorescence Detection of Type III Protein Secretion from Bacterial Pathogens

Cited by 15 publications

References 14 publications

Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates

Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates

Epigallocatechin-3-gallate inhibits bacterial virulence and invasion of host cells

The Type III Secretion System as a Source of Novel Antibacterial Drug Targets

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