Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems

Poole, Stephen J.; Diner, Elie J.; Aoki, Stephanie K.; Braaten, Bruce A.; Roodenbeke, Claire T'Kint de; Low, David A.; Hayes, Christopher S.

doi:10.1371/journal.pgen.1002217

Cited by 166 publications

(275 citation statements)

References 41 publications

(66 reference statements)

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“…The CdiA-CT II Bp1026b /CdiI II Bp1026b proteins are derived from the CDI locus on chromosome II of Bp1026b (5), and the CdiA-CT o11 EC869 /CdiI o11 EC869 complex is encoded by the 11th "orphan" (o11) module of E. coli EC869. Orphan cdiA-CT/cdiI modules are toxin/immunity gene pairs that have been displaced from fulllength cdiA genes (8). Tandem arrays of these modules are often associated with CDI systems and are thought to represent reservoirs of toxin/immunity diversity.…”

Section: Resultsmentioning

confidence: 99%

“…2A) is required for DNase activity. This toxin-encoding sequence could not be cloned in the absence of the cognate immunity gene; therefore, we used controlled proteolysis of CdiI o11 EC869 to activate the CdiA-CT o11 EC869 toxin inside E. coli cells (8,12). Briefly, the C terminus of CdiI o11 EC869 was tagged with the ssrA(DAS) peptide, which targets the immunity protein for degradation by the ClpXP protease, thereby liberating the CdiA-CT to exert its toxic activity.…”

Section: Cdia-ct O11mentioning

confidence: 99%

“…CdiI proteins are also highly variable and bind their cognate CdiA-CTs to block toxin activity. Because CdiA-CT/CdiI binding interactions are highly specific, immunity proteins provide no protection from the toxins deployed by other CDI systems (1,5,8). Thus, intercellular competition is thought to drive the diversification of CDI toxin/immunity pairs.…”

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confidence: 99%

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Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems

Morse¹,

Nikolakakis²,

Willett

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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168

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Contact-dependent growth inhibition (CDI) systems encode polymorphic toxin/immunity proteins that mediate competition between neighboring bacterial cells. We present crystal structures of CDI toxin/ immunity complexes from Escherichia coli EC869 and Burkholderia pseudomallei 1026b. Despite sharing little sequence identity, the toxin domains are structurally similar and have homology to endonucleases. The EC869 toxin is a Zn 2+ -dependent DNase capable of completely degrading the genomes of target cells, whereas the Bp1026b toxin cleaves the aminoacyl acceptor stems of tRNA molecules. Each immunity protein binds and inactivates its cognate toxin in a unique manner. The EC869 toxin/immunity complex is stabilized through an unusual β-augmentation interaction. In contrast, the Bp1026b immunity protein exploits shape and charge complementarity to occlude the toxin active site. These structures represent the initial glimpse into the CDI toxin/immunity network, illustrating how sequence-diverse toxins adopt convergent folds yet retain distinct binding interactions with cognate immunity proteins. Moreover, we present visual demonstration of CDI toxin delivery into a target cell.structural biology | bacterial competition | β-complementation | tRNase activity

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Section: Resultsmentioning

confidence: 99%

Section: Cdia-ct O11mentioning

confidence: 99%

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Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems

Morse¹,

Nikolakakis²,

Willett

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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168

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“…Although CysK is critical for CdiA-CT EC536 nuclease activity, related toxins from Gram-positive bacteria probably do not require extrinsic activation because purified Tox28 domain from R. lactaris has tRNase activity in vitro. Ntox28 domains are typically found at the C terminus of proteins that mediate interbacterial competition (4,(29)(30)(31). For example, the R. lactaris Tox28 Rlac domain is part of a larger EC536 were treated with formaldehyde, and cross-linked nucleoprotein complexes were purified by Ni 2+ -affinity chromatography.…”

Section: Discussionmentioning

confidence: 99%

Unraveling the essential role of CysK in CDI toxin activation

Johnson

Beck

Morse

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI + bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT EC536 deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT EC536 binary complex and the neutralized ternary complex of CysK/CdiA-CT/ CdiI EC536 . CdiA-CT EC536 inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT EC536 exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT EC536 thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT EC536 , and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.bacterial competition | structural biology | toxin chaperone | tRNase activity

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“…We disrupted sciW, rhs1, and rhs2 and found that disruption of rhs2 produced the same defect in systemic dissemination in mice as disruption of core T6SS genes and the entire SPI-6 locus (19). This Rhs element is located with sciX in a bicistronic operon and may also function as a toxin/immunity gene pair participating in contact-dependent growth inhibition (40).…”

Section: Figmentioning

confidence: 97%

Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium

2012

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ABSTRACTThe enteropathogenSalmonella entericaserovar Typhimurium employs a suite of tightly regulated virulence factors within the intracellular compartment of phagocytic host cells resulting in systemic dissemination in mice. A type VI secretion system (T6SS) withinSalmonellapathogenicity island 6 (SPI-6) has been implicated in this process; however, the regulatory inputs and the roles of noncore genes in this system are not well understood. Here we describe four clusters of noncore T6SS genes in SPI-6 based on a comparative relationship with the T6SS-3 ofBurkholderia malleiand report that the disruption of these genes results in defects in intracellular replication and systemic dissemination in mice. In addition, we show that the expression of the SPI-6-encoded Hcp and VgrG orthologs is enhanced during late stages of macrophage infection. We identify six regions that are transcriptionally active during cell infections and that have regulatory contributions from the regulators of virulence SsrB, PhoP, and SlyA. We show that levels of protein expression are very weak underin vitroconditions and that expression is not enhanced upon the deletion ofssrB,phoP,slyA,qseC,ompR, orhfq, suggesting an unknown activating factor. These data suggest that the SPI-6 T6SS has been integrated into theSalmonellaTyphimurium virulence network and customized for host-pathogen interactions through the action of noncore genes.

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Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems

Cited by 166 publications

References 41 publications

Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems

Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems

Unraveling the essential role of CysK in CDI toxin activation

Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium

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