Development of a Novel Functional High-Throughput Screening System for Pathogen Effectors in the Yeast<i>Saccharomyces cerevisiae</i>

Tabuchi, Mitsuaki; Kawai, Yasuhiro; Nishie-Fujita, Makiko; Akada, Rinji; Izumi, Terumi; Izumi, Yasukatsu; Miyashita, Naoyuki; Ouchi, Kazunobu; Kishi, Fumio

doi:10.1271/bbb.90360

Cited by 12 publications

(16 citation statements)

References 32 publications

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“…Transcriptional reporters responsive to endoplasmic reticulum (ER) stress and vacuolar and endosomal fluorescent dyes have been successfully exploited to monitor interference with vesicle trafficking and endocytosis [ 49 , 50 ]. The recent adaptation to budding yeast of the carboxypeptidase Y-invertase system to monitor vesicle trafficking [ 51 ] holds promise for studies of T3Es modulating this process. Finally, the biological functions of certain T3Es that manipulate Rho GTPase and ADP-ribosyltransferase activities of the host cell have been revealed by measuring bound GTP [ 33 , 41 ] and incorporation of radioactive ADP-ribose [ 34 ], respectively.…”

Section: Methodological Approaches For T3e Characterisation In Yeastmentioning

confidence: 99%

Yeast as a Heterologous Model System to Uncover Type III Effector Function

et al. 2016

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Type III effectors (T3E) are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. “Favourite” targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK) signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure–function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations of S. cerevisiae as a model system and its most promising future applications.

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Section: Methodological Approaches For T3e Characterisation In Yeastmentioning

confidence: 99%

Yeast as a Heterologous Model System to Uncover Type III Effector Function

et al. 2016

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“…1E), we expected that the growth inhibition effect caused by RipAY must be eliminated by simultaneous deletion of its candidate activators, Trx1 and Trx2. RipAY and Gcg1 proteins were expressed under the control of the doxycycline-repressible Tet-off promoter (33) in yeast WT, trx3⌬ single-, trx1/2⌬ double-, and trx1/2/3⌬ triple-mutant cells, and the growth of yeast was scored by a spot assay (Fig. 7A).…”

Section: Inoculation Of R Solanacearum Into Eggplant Leaves Causes Amentioning

confidence: 99%

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

Fujiwara

Kawazoe

Ohnishi

et al. 2016

Journal of Biological Chemistry

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The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits ␥-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ϳ30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other ␥-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.

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“…Lesser and Miller reported in 2001 that the expression of bacterial effector proteins in S. cerevisiae can serve as a model system to study the molecular mechanisms of effector functions (34). Others have reported similar results, and yeast provides significant benefits over mammalian cell culture, given its genetic tractability and wellstudied signaling pathways (7,10,46,51,(53)(54)(55). We focused our studies on strain AM-19226, a clinically isolated O39 serogroup strain of V. cholerae whose genomic sequence analysis led to the identification of the V. cholerae T3SS, and screened a subset of ORFs found within the T3SS island for their ability to induce growth defects when expressed in S. cerevisiae.…”

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confidence: 96%

Identification of Vibrio cholerae Type III Secretion System Effector Proteins

et al. 2011

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AM-19226 is a pathogenic O39 serogroup Vibrio cholerae strain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ϳ49.7-kb T3SS genomic island to identify potential effector proteins. We identified 15 ORFs for their ability to inhibit growth when expressed in yeast and then used a ␤-lactamase (TEM1) fusion reporter system to demonstrate that 11 proteins were bona fide effectors translocated into HeLa cells in vitro in a T3SS-dependent manner. One effector, which we named VopX (A33_1663), is conserved only in V. cholerae and Vibrio parahaemolyticus T3SS-positive strains and has not been previously studied. A vopX deletion reduces the ability of strain AM-19226 to colonize in vivo, and the bile-induced expression of a vopX-lacZ transcriptional fusion in vitro is regulated by the T3SS-encoded transcriptional regulators VttR A and VttR B . An RLM1 yeast deletion strain rescued the growth inhibition induced by VopX expression, suggesting that VopX interacts with components of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway. The collective results show that the V. cholerae T3SS encodes multiple effector proteins, one of which likely has novel activities that contribute to disease via interference with eukaryotic signaling pathways.Vibrio cholerae is the etiologic agent of the severe and potentially lethal diarrheal disease called cholera (31, 49). Although over 200 different serogroups of V. cholerae have been identified, only O1 and O139 serogroup strains cause epidemic and pandemic disease (5, 6, 16). In pathogenic O1 and O139 serogroup isolates, the virulence factors for colonization (toxincoregulated pilus [TCP]) and toxin production (cholera toxin [CT]) are essential, and their expression is controlled by a transcriptional cascade regulated primarily by the transmembrane protein ToxR (36). Strains belonging to other serogroups can also cause disease and are collectively referred to as non-O1/non-O139 serogroup strains. Although they are typically associated with sporadic diarrheal disease, reports of increased incidence of non-O1/non-O139-associated disease suggest that these strains warrant increased attention and may be an emerging threat (17, 47). However, the pathogenic mechanisms of non-O1/non-O139 strains are not as well studied as those of O1 and O139 strains. Recent reports suggest that a type III secretion system (T3SS) is present in a subset of non-O1/non-O139 isolates and represents an important virulence mechanism for some V. cholerae strains (4,8,12,56).T3SSs are commonly found in pathogenic, Gram-negative bacteria, such as Yersinia, Salmonella, Shigella, enteropathogenic/enterohemorrhagic Escherichia coli (EPEC/EHEC), and Pseudomonas. However, T3SS conservation among species is ...

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Development of a Novel Functional High-Throughput Screening System for Pathogen Effectors in the YeastSaccharomyces cerevisiae

Cited by 12 publications

References 32 publications

Yeast as a Heterologous Model System to Uncover Type III Effector Function

Yeast as a Heterologous Model System to Uncover Type III Effector Function

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

Identification of Vibrio cholerae Type III Secretion System Effector Proteins

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