scholarly journals YM155 Inhibits NleB and SseK Arginine Glycosyltransferase Activity

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 253
Author(s):  
Congrui Zhu ◽  
Samir El Qaidi ◽  
Peter McDonald ◽  
Anuradha Roy ◽  
Philip R. Hardwidge
Keyword(s):  
Type Iii Secretion ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Cross Reactivity ◽  
Effector Proteins ◽  
Protein Substrates ◽  
Arginine Residues ◽  
Screening Assays ◽  
Bacterial Growth Rates ◽  
Salmonella Survival

The type III secretion system effector proteins NleB and SseK are glycosyltransferases that glycosylate protein substrates on arginine residues. We conducted high-throughput screening assays on 42,498 compounds to identify NleB/SseK inhibitors. Such small molecules may be useful as mechanistic probes and may have utility in the eventual development of anti-virulence therapies against enteric bacterial pathogens. We observed that YM155 (sepantronium bromide) inhibits the activity of Escherichia coli NleB1, Citrobacter rodentium NleB, and both Salmonella enterica SseK1 and SseK2. YM155 was not toxic to mammalian cells, nor did it show cross-reactivity with the mammalian O-linked N-acetylglucosaminyltransferase (OGT). YM155 reduced Salmonella survival in mouse macrophage-like cells but had no direct impact on bacterial growth rates, suggesting YM155 may have utility as a potential anti-virulence inhibitor.

scholarly journals Primary and Secondary Drug Screening Assays for Friedreich Ataxia

2011 ◽  
Vol 17 (3) ◽  
pp. 303-313 ◽  
Author(s):  
M. Grazia Cotticelli ◽  
Lynn Rasmussen ◽  
Nicole L. Kushner ◽  
Sara McKellip ◽  
Melinda Ingrum Sosa ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Mitochondrial Function ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Friedreich Ataxia ◽  
Krebs Cycle ◽  
C2c12 Cells ◽  
Transport Chain ◽  
Screening Assays ◽  
Yeast Mutants

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron–sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease.

scholarly journals A Solvent-Exposed Patch in Chaperone-Bound YopE Is Required for Translocation by the Type III Secretion System

2010 ◽  
Vol 192 (12) ◽  
pp. 3114-3122 ◽  
Author(s):  
Loren Rodgers ◽  
Romila Mukerjea ◽  
Sara Birtalan ◽  
Devorah Friedberg ◽  
Partho Ghosh
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Effector Proteins ◽  
Host Cells ◽  
Type Iii ◽  
Potential Association ◽  
Chaperone Binding ◽  
Bacterial Type ◽  
Characteristic Manner

ABSTRACT Most effector proteins of bacterial type III secretion (T3S) systems require chaperone proteins for translocation into host cells. Such effectors are bound by chaperones in a conserved and characteristic manner, with the chaperone-binding (Cb) region of the effector wound around the chaperone in a highly extended conformation. This conformation has been suggested to serve as a translocation signal in promoting the association between the chaperone-effector complex and a bacterial component required for translocation. We sought to test a prediction of this model by identifying a potential association site for the Yersinia pseudotuberculosis chaperone-effector pair SycE-YopE. We identified a set of residues in the YopE Cb region that are required for translocation but are dispensable for expression, SycE binding, secretion into the extrabacterial milieu, and stability in mammalian cells. These residues form a solvent-exposed patch on the surface of the chaperone-bound Cb region, and thus their effect on translocation is consistent with the structure of the chaperone-bound Cb region serving as a signal for translocation.

scholarly journals Recombinant Yersinia YopT Leads to Uncoupling of RhoA-Effector Interaction

2001 ◽  
Vol 69 (12) ◽  
pp. 7535-7543 ◽  
Author(s):  
Isabel Sorg ◽  
Udo-Michael Goehring ◽  
Klaus Aktories ◽  
Gudula Schmidt
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Actin Cytoskeleton ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Underlying Mechanism ◽  
Effector Proteins ◽  
Lung Cells ◽  
Rho Proteins

ABSTRACT Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis deliver different Yop (Yersinia outer proteins) effector proteins into mammalian cells by a type III secretion mechanism. Recently, it was shown thatYersinia producing YopT leads to disruption of the actin cytoskeleton of HeLa cells (M. Iriarte and G. R. Cornelis, Mol. Microbiol. 29:915–929, 1998). To analyze the molecular mechanism of YopT, we cloned and expressed YopT as a glutathioneS-transferase fusion protein. Recombinant YopT caused rounding up of embryonic bovine lung cells and redistribution of the actin cytoskeleton rapidly after microinjection. The Escherichia coli cytotoxic necrotizing factor (CNF1), which constitutively activates Rho proteins, was not able to inhibit or revert YopT-induced cell rounding. YopT caused release of RhoA from embryonic bovine lung membranes and released recombinant isoprenylated RhoA from artificial PE or PE/PIP2 vesicles. Incubation of lysate or cytosol with YopT caused inhibition of the RhoA-rhotekin interaction but led to increased RhoA-RhoGDI interaction. It is suggested that inhibition of the interaction between RhoA and effectors is the underlying mechanism of the YopT action on the cytoskeleton.

scholarly journals Impact of Host Membrane Pore Formation by the Yersinia pseudotuberculosis Type III Secretion System on the Macrophage Innate Immune Response

2013 ◽  
Vol 81 (3) ◽  
pp. 905-914 ◽  
Author(s):  
Laura Kwuan ◽  
Walter Adams ◽  
Victoria Auerbuch
Keyword(s):  
Cell Death ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Effector Proteins ◽  
Innate Immune ◽  
Host Cells ◽  
Content Type ◽  
Host Cell Death ◽  
Tnf Α

ABSTRACTType III secretion systems (T3SSs) are used by Gram-negative pathogens to form pores in host membranes and deliver virulence-associated effector proteins inside host cells. In pathogenicYersinia, the T3SS pore-forming proteins are YopB and YopD. Mammalian cells recognize theYersiniaT3SS, leading to a host response that includes secretion of the inflammatory cytokine interleukin-1β (IL-1β), Toll-like receptor (TLR)-independent expression of the stress-associated transcription factor Egr1 and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), and host cell death. The knownYersiniaT3SS effector proteins are dispensable for eliciting these responses, but YopB is essential. Three models describe how theYersiniaT3SS might trigger inflammation: (i) mammalian cells sense YopBD-mediated pore formation, (ii) innate immune stimuli gain access to the host cytoplasm through the YopBD pore, and/or (iii) the YopB-YopD translocon itself or its membrane insertion is proinflammatory. To test these models, we constructed aYersinia pseudotuberculosismutant expressing YopD devoid of its predicted transmembrane domain (YopDΔTM) and lacking the T3SS cargo proteins YopHEMOJTN. This mutant formed pores in macrophages, but it could not mediate translocation of effector proteins inside host cells. Importantly, this mutant did not elicit rapid host cell death, IL-1β secretion, or TLR-independent Egr1 and TNF-α expression. These data suggest that YopBD-mediated translocation of unknown T3SS cargo leads to activation of host pathways influencing inflammation, cell death, and response to stress. As the YopDΔTMY. pseudotuberculosismutant formed somewhat smaller pores with delayed kinetics, an alternative model is that the wild-type YopB-YopD translocon is specifically sensed by host cells.

scholarly journals Burkholderia thailandensis as a Model System for the Study of the Virulence-Associated Type III Secretion System of Burkholderia pseudomallei

2008 ◽  
Vol 76 (11) ◽  
pp. 5402-5411 ◽  
Author(s):  
Andrea Haraga ◽  
T. Eoin West ◽  
Mitchell J. Brittnacher ◽  
Shawn J. Skerrett ◽  
Samuel I. Miller
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Burkholderia Pseudomallei ◽  
Clinical Symptoms ◽  
Type Iii Secretion System ◽  
Model System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Type Iii

ABSTRACT Burkholderia pseudomallei is a bacterial pathogen that causes a broad spectrum of clinical symptoms collectively known as melioidosis. Since it can be acquired by inhalation and is difficult to eradicate due to its resistance to a wide group of antibiotics and capacity for latency, work with B. pseudomallei requires a biosafety level 3 (BSL-3) containment facility. The bsa (Burkholderia secretion apparatus)-encoded type III secretion system (TTSS) has been shown to be required for its full virulence in a number of animal models. TTSSs are export devices found in a variety of gram-negative bacteria that translocate bacterial effector proteins across host cell membranes into the cytoplasm of host cells. Although the Bsa TTSS has been shown to play an important role in the ability of B. pseudomallei to survive and replicate in mammalian cells, escape from the endocytic vacuole, and spread from cell to cell, little is known about its effectors mediating these functions. Using bioinformatics, we identified homologs of several known TTSS effectors from other bacteria in the B. pseudomallei genome. In addition, we show that orthologs of these putative effectors exist in the genome of B. thailandensis, a closely related bacterium that is rarely pathogenic to humans. By generating a Bsa TTSS mutant B. thailandensis strain, we also demonstrated that the Bsa TTSS has similar functions in the two species. Therefore, we propose B. thailandensis as a useful BSL-1 model system to study the role of the Bsa TTSS during Burkholderia infection of mammalian cells and animals.

scholarly journals Angiotensin-Converting Enzyme 2 (ACE2) As a Novel Biorecognition Element in A Cell-Based Biosensor for the Ultra-Rapid, Ultra-Sensitive Detection of the SARS-CoV-2 S1 Spike Protein Antigen

Chemosensors ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 341
Author(s):  
Sofia Mavrikou ◽  
Vasileios Tsekouras ◽  
Kyriaki Hatziagapiou ◽  
Asimina Tsalidou ◽  
Petros Bakakos ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Converting Enzyme ◽  
Immobilized Cell ◽  
Angiotensin Converting Enzyme 2 ◽  
Wide Range

Antigen screening for the SARS-CoV-2 S1 spike protein is among the most promising tools for the mass monitoring of asymptomatic carriers of the virus, especially in limited resource environments. Herewith, we report on the possible use of the angiotensin-converting enzyme 2 (ACE2), the natural receptor and entry point of the virus, as a biorecognition element for the detection of the S1 antigen combined with an established bioelectric biosensor based on membrane-engineered cells. The working principle of our approach is based on the measurable change of the electric potential of membrane-engineered mammalian cells bearing ACE2 after attachment of the respective viral protein. We demonstrate that sensitive and selective detection of the S1 antigen is feasible in just three min, with a limit of detection of 20 fg/mL. In a preliminary clinical application, positive patient-derived samples were identified with a 87.9% score compared to RT-PCR. No cross-reactivity was observed against a wide range of nucleocapsid protein concentrations. The novel biosensor is embedded in a commercially ready-to-use testing platform, complete with the consumable immobilized cell–electrode interface and a portable read-out device operable through smartphone or tablet. In addition, the possible application of the system for the high throughput screening of potential pharmacological inhibitors of the ACE2 receptor-S1 RBD interaction is discussed.

Identification of translocation inhibitors targeting the type III secretion system of enteropathogenic Escherichia coli

2021 ◽  
Author(s):  
Sabrina Mühlen ◽  
Viktor A. Zapol'skii ◽  
Ursula Bilitewski ◽  
Petra Dersch
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Immune Recognition ◽  
Type Iii ◽  
Compound Libraries ◽  
High Throughput Screening Assay

Infections with enteropathogenic E. coli (EPEC) cause severe diarrhea in children. The non-invasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir. Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. Additionally, these substances reduced bacterial binding to host cells, effector-dependent cell detachment and abolished A/E lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and EHEC infections.

scholarly journals Targeting Type III Secretion in Yersinia pestis

2008 ◽  
Vol 53 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Ning J. Pan ◽  
Michael J. Brady ◽  
John M. Leong ◽  
Jon D. Goguen
Keyword(s):  
Small Molecules ◽  
Yersinia Pestis ◽  
Type Iii Secretion ◽  
High Throughput Screening ◽  
Screening Method ◽  
Effector Proteins ◽  
Host Cells ◽  
Chemical Libraries ◽  
Type Iii ◽  
Structure Activity

ABSTRACT Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations. Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but also distinct from previously described candidate type III secretion inhibitors.

scholarly journals High-Throughput Fluorescence-Based Screening Assays for Tryptophan-Catabolizing Enzymes

2014 ◽  
Vol 19 (9) ◽  
pp. 1266-1274 ◽  
Author(s):  
Nicole Seegers ◽  
Antoon M. van Doornmalen ◽  
Joost C. M. Uitdehaag ◽  
Jos de Man ◽  
Rogier C. Buijsman ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Target ◽  
Cross Reactivity ◽  
Excitation Wavelength ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Screening Assays ◽  
Green Fluorescent ◽  
Relative Selectivity

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

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