An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

Duncan, Miles C.; Wong, Weng Ruh; Dupzyk, Allison J.; Bray, Walter M.; Linington, Roger G.; Auerbuch, Victoria

doi:10.1128/aac.02025-13

Cited by 34 publications

(38 citation statements)

References 38 publications

(62 reference statements)

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“…As several cyclic peptomers were found to perturb the host actin cytoskeleton and a subset of these disrupted mammalian cell metabolism, further derivatization is warranted to mitigate such off-target effects while improving activity. Importantly, the activities displayed by the cyclic peptomers are comparable to those of other known T3SS inhibitors, such as piericidin A1 (active at 71 M) (26), N-hydroxybenzimidazole derivatives (50% inhibitory concentration [IC 50 ] in the range of 3 to 70 M) (57), various T3SS ATPase inhibitors (IC 50 in the range of 17 to 70 M) (58), and the salicylidene acylhydrazide INP0010 (active at 40 M), for which three molecular targets have been identified (59).…”

Section: Figmentioning

confidence: 58%

“…A promoter containing five repeats of an NF-B enhancer element (5=-TGGGGACTTTCCGC-3=) (26) was cloned upstream of the GFP gene, followed by integration of the construct into the HEK293 chromosome. The primers 5=-ATTAGAGCTCTGCAATTGTTGTTAACTTGTTT ATT-3= (forward primer; SacI restriction site is underlined) and 5=-ATTAAAGCTTTATATACCCTCTAGAGTC TCCGCG-3= (reverse primer; HindIII restriction site is underlined) were used to clone the NF-B reporter construct (26) into the multicloning site of peTurboGFP-PRL-dest1, containing a gene encoding a destabilized variant of green fluorescent protein (TurboGFP; Evrogen, Russia). The plasmid was linearized with BsaI and transfected into HEK293 cells by use of Lipofectamine 2000 (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…The compounds were screened for the ability to inhibit NF-B activation during Y. pseudotuberculosis Δyop6 infection of HEK293-GFP cells. We used 2 standard deviations below the mean for the dimethyl sulfoxide (DMSO) control as the threshold for hit calling (26). The following 8 of the 20 peptomers reduced NF-B-driven GFP expression at the two highest concentrations tested: EpD-1N, EpD-1=N, EpD-2N, EpD-3N, EpD-3=N, EpD-4=N, EpD-6N, and EpD-6=N ( Fig.…”

Section: Development and Use Of An Nf-b-gfp Stable Cell Line For Idenmentioning

confidence: 99%

“…2C; see Fig. S1 in the supplemental material) (26). Y. pseudotuberculosis lacking one of the T3SS structural operons (ΔyscNU) was used as a negative control.…”

Section: Development and Use Of An Nf-b-gfp Stable Cell Line For Idenmentioning

confidence: 99%

“…Yersinia lacking the YopHEMOJT T3SS effector proteins but expressing an otherwise functional Ysc T3SS triggers NF-B activation in HEK293T cells, while Yersinia lacking the YopB translocator protein essential for translocation of T3SS cargo inside host cells does not activate NF-B (25). Previously, we used host NF-B-driven luciferase in HEK293T cells as a readout for Yersinia T3SS activity in a pilot T3SS inhibitor screen and identified a family of T3SS inhibitors called piericidins (26). In order to improve our screening strategy, we generated a HEK293 stable cell line expressing an NF-B-driven green fluorescent protein (GFP) reporter gene.…”

mentioning

confidence: 99%

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Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors

Lam

Schwochert

Lao

et al. 2017

Antimicrob Agents Chemother

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Antibiotic-resistant bacteria are an emerging threat to global public health. New classes of antibiotics and tools for antimicrobial discovery are urgently needed. Type III secretion systems (T3SS), which are required by dozens of Gramnegative bacteria for virulence but largely absent from nonpathogenic bacteria, are promising virulence blocker targets. The ability of mammalian cells to recognize the presence of a functional T3SS and trigger NF-B activation provides a rapid and sensitive method for identifying chemical inhibitors of T3SS activity. In this study, we generated a HEK293 stable cell line expressing green fluorescent protein (GFP) driven by a promoter containing NF-B enhancer elements to serve as a readout of T3SS function. We identified a family of synthetic cyclic peptide-peptoid hybrid molecules (peptomers) that exhibited dose-dependent inhibition of T3SS effector secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa without affecting bacterial growth or motility. Among these inhibitors, EpD-3=N, EpD-1,2N, EpD-1,3=N, EpD-1,2,3=N, and EpD-1,2,4=N exhibited strong inhibitory effects on translocation of the Yersinia YopM effector protein into mammalian cells (Ͼ40% translocation inhibition at 7.5 M) and showed no toxicity to mammalian cells at 240 M. In addition, EpD-3=N and EpD-1,2,4=N reduced the rounding of HeLa cells caused by the activity of Yersinia effector proteins that target the actin cytoskeleton. In summary, we have discovered a family of novel cyclic peptomers that inhibit the injectisome T3SS but not the flagellar T3SS.

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Section: Figmentioning

confidence: 58%

Section: Methodsmentioning

confidence: 99%

Section: Development and Use Of An Nf-b-gfp Stable Cell Line For Idenmentioning

confidence: 99%

“…2C; see Fig. S1 in the supplemental material) (26). Y. pseudotuberculosis lacking one of the T3SS structural operons (ΔyscNU) was used as a negative control.…”

Section: Development and Use Of An Nf-b-gfp Stable Cell Line For Idenmentioning

confidence: 99%

mentioning

confidence: 99%

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Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors

Lam

Schwochert

Lao

et al. 2017

Antimicrob Agents Chemother

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Anti-virulence Strategies to Target Bacterial Infections

Mühlen

Dersch

2015

Current Topics in Microbiology and Immunology

141

139

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Resistance of important bacterial pathogens to common antimicrobial therapies and the emergence of multidrug-resistant bacteria is increasing at an alarming rate and constitutes one of our greatest challenges in the combat of bacterial infection and accompanied diseases. Given the current shortage of effective drugs, lack of successful prevention measures and only a few new antibiotics in the clinical pipeline demands the development of novel treatment options and alternative antimicrobial therapies. Our increasing understanding of bacterial virulence strategies and the induced molecular pathways of the infectious disease provide novel opportunities to target and interfere with crucial pathogenicity factors or virulence-associated traits of the bacteria while bypassing the evolutionary pressure on the bacterium to develop resistance. In the past decade, numerous new bacterial targets for anti-virulence therapies have been identified, and structure-based tailoring of intervention strategies as well as screening assays for small molecule inhibitors of such pathways were successfully established. In this chapter, we will take a closer look at the bacterial virulence-related factors and processes that present promising targets for antivirulence therapies, recently discovered inhibitory substances and their promises and discuss the challenges and problems that have to be faced.

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Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using β-Lactamase Fusions

Nguyen¹,

McCabe

Fasciano³

et al. 2019

Methods in Molecular Biology

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Development of the TEM-CCF2/4-AM FRET based system has enabled investigators to track translocation of effector proteins into mammalian cells during infection. This allows for separation of translocated and non-translocated cell populations for further study. Yersinia strains expressing translational Yop-TEM fusions, containing the secretion and translocation signals of a Yop with the TEM-1 portion of β-lactamase, are used to infect mice, tissues isolated from mice, or mammalian cells in culture. Infected and harvested mammalian cells are treated with either CCF2-AM or CCF4-AM, and cleavage of this fluorescent compound by TEM is detected by FACS analysis. A shift from green to blue emission spectra of individual cells is indicative of translocation of a given Yop-TEM fusion protein into the host cell during Yersinia infection due to a disruption in FRET between the two fluors of the compound. In Yersinia, this method has been used to understand Type III secretion dynamics and Yop functions in cells translocated by effectors during infection. Here, we describe how to generate Yop-TEM constructs, and how to detect, quantify, isolate, and study Yop-TEM containing cells in murine tissues during infection and in ex vivo tissues by cell sorting and flow cytometry analysis. In addition, we provide guidance for analyzing TEM-positive cells via a plate reader and fluorescent microscopy.

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An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

Cited by 34 publications

References 38 publications

Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors

Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors

Anti-virulence Strategies to Target Bacterial Infections

Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using β-Lactamase Fusions

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