ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview

Belyy, Alexander; Mechold, Undine; Renault, Louis; Ladant, Daniel

doi:10.1016/j.toxicon.2017.12.046

Cited by 18 publications

(13 citation statements)

References 64 publications

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“…Interaction with either F- or G-actin could in turn affect the localization of the enzyme within cells and thus the localization of the generated cNMPs. Future investigations will reveal whether the interaction of AA-NC toxins with either F- or G-actin can also induce different alterations of the actin cytoskeleton dynamics in host cells independently from their catalytic activity [ 6 , 26 ].…”

Section: Discussionmentioning

confidence: 99%

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin

et al. 2018

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Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.

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Section: Discussionmentioning

confidence: 99%

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin

et al. 2018

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“…ExoY was identified by Yahr et al (19) as a 378-amino acidlong protein that shares sequence similarity with well-characterized calmodulin-activated adenylyl cyclases from Bordetella pertussis (CyaA) and Bacillus anthracis (edema factor). These toxins share highly conserved regions responsible for catalysis but are different in regions predicted to bind the activator (19,20). Structural models of the CyaA-calmodulin and edema factor-calmodulin complexes, solved by X-ray diffraction and supplemented by numerous biophysical studies and mutagenesis analysis, give insight into the activator recognition and the rearrangements of the active center upon calmodulin binding (21)(22)(23)(24).…”

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confidence: 99%

The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin

Belyy

Santecchia²,

Renault

et al. 2018

Journal of Biological Chemistry

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Edited by Velia M. FowlerBacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 35-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.

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“…Among the identified proteins, several T3SS proteins were co-purified with PscN, including the exoenzymes S, T, Y, the translocators PopB (SctE), PopD (SctB) and their cognate chaperone PcrH, the stator PscL (SctL), the major export apparatus protein PcrD (SctV), the accessory protein PscK (SctK) and the chaperone PscB of the gate-keeper (SctW) complex (Table 1). In addition to structural partners, most of these partner proteins were shown to be secreted by the T3SS (Belyy et al, 2018;Goure et al, 2004;Maresso et al, 2006;Shen et al, 2008;Yahr et al, 1996;Yang et al, 2007) being consistent with a role of PscN in the recognition of exported protein as the first step of the secretion process.…”

Section: Resultsmentioning

confidence: 70%

The PopN gate-keeper complex acts on the ATPase PscN to regulate the T3SS secretion switch from early to middle substrates inPseudomonas aeruginosa

Ngo

Perdu

Jneid

et al. 2020

Preprint

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Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we disclose a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa.HighlightsT3SS substrates are secreted sequentially but information on the switches are missingInteraction of the T3SS ATPase with secreted proteins were investigated by different approachesMicroscale Thermophoresis revealed a lower affinity for chaperones alone compared to complexesThe Gate-keeper complex binds to the ATPase and increases its affinity for the needle complexA new role of the Gate-keeper complex is proposed, directly acting on the T3SS ATPase

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ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview

Cited by 18 publications

References 64 publications

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin

The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin

The PopN gate-keeper complex acts on the ATPase PscN to regulate the T3SS secretion switch from early to middle substrates inPseudomonas aeruginosa

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