Dual Role for DsbA in Attacking and Targeted Bacterial Cells during Type VI Secretion System-Mediated Competition

Mariano, Giuseppina; Monlezun, Laura; Coulthurst, Sarah J.

doi:10.1016/j.celrep.2017.12.075

Cited by 32 publications

(32 citation statements)

References 45 publications

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“…To confirm this interaction in the physiologically-relevant context, we generated strains expressing PpkA fused with a C-terminal haemagglutinin (HA) tag (PpkA-HA) and/or RtkS fused with a C-terminal hexahistidine tag (RtkS-His), with the fusion proteins being expressed from the normal chromosomal locations. Strains carrying either or both fusions exhibited normal T6SS-dependent anti-bacterial activity, confirming that the fusions did not impair function ( S7 Fig , [ 32 ]). Using membrane fractions prepared from these strains, specific co-immunoprecipitation of RtkS-His with PpkA-HA, and, conversely, co-purification of PpkA-HA with RtkS-His could be observed ( Fig 7B ).…”

Section: Resultsmentioning

confidence: 62%

Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system

et al. 2018

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The Type VI secretion system (T6SS) is widely used by bacterial pathogens as an effective weapon against bacterial competitors and is also deployed against host eukaryotic cells in some cases. It is a contractile nanomachine which delivers toxic effector proteins directly into target cells by dynamic cycles of assembly and firing. Bacterial cells adopt distinct post-translational regulatory strategies for deployment of the T6SS. ‘Defensive’ T6SSs assemble and fire in response to incoming attacks from aggressive neighbouring cells, and can utilise the Threonine Protein Phosphorylation (TPP) regulatory pathway to achieve this control. However, many T6SSs are ‘offensive’, firing at all-comers without the need for incoming attack or other cell contact-dependent signal. Post-translational control of the offensive mode has been less well defined but can utilise components of the same TPP pathway. Here, we used the anti-bacterial T6SS of Serratia marcescens to elucidate post-translational regulation of offensive T6SS deployment, using single-cell microscopy and genetic analyses. We show that the integration of the TPP pathway with the negative regulator TagF to control core T6SS machine assembly is conserved between offensive and defensive T6SSs. Signal-dependent PpkA-mediated phosphorylation of Fha is required to overcome inhibition of membrane complex assembly by TagF, whilst PppA-mediated dephosphorylation promotes spatial reorientation and efficient killing. In contrast, the upstream input of the TPP pathway defines regulatory strategy, with a new periplasmic regulator, RtkS, shown to interact with the PpkA kinase in S. marcescens. We propose a model whereby the opposing actions of the TPP pathway and TagF impose a delay on T6SS re-assembly after firing, providing an opportunity for spatial re-orientation of the T6SS in order to maximise the efficiency of competitor cell targeting. Our findings provide a better understanding of how bacterial cells deploy competitive weapons effectively, with implications for the structure and dynamics of varied polymicrobial communities.

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Section: Resultsmentioning

confidence: 62%

Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system

et al. 2018

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“…Hijacking a highly conserved and essential molecule of the recipient cell to improve attacker fitness is not uncommon. The examples are CdiA-CT EC93 hijacking essential proteins BamA and AcrB, CdiA-CT EC536 hijacking the recipient CysK, and Ssp2 and Ssp4 hijacking recipient DsbA (Aoki et al, 2008;Diner et al, 2012;Mariano et al, 2018). The ClpP protease, on the other hand, is highly conserved in both prokaryotes and eukaryotic organelles like plastid and mitochondria (Culp and Wright, 2016;Moreno et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…The role of the recipient EF-Tu involved in an interbacterial competition of Tse6 remains elusive. A T6SS study in Serratia marcescens demonstrated that the recipient protein DsbA plays a role in activating S. marcescens T6SS effectors Ssp2 and Ssp4, but not Rhs2 (Mariano et al, 2018). The S. marcescens T6SS kills its Ssp2-sensitive siblings only when the recipient cells harbor dsbA homologs (dsbA1 + dsbA2 + ).…”

Section: Introductionmentioning

confidence: 99%

A High-Throughput Interbacterial Competition Screen Identifies ClpAP in Enhancing Recipient Susceptibility to Type VI Secretion System-Mediated Attack by Agrobacterium tumefaciens

Lin

Sriramoju

et al. 2020

Front. Microbiol.

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The type VI secretion system (T6SS) is an effector delivery system used by Gramnegative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli. We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the clpP encoding ClpP protease, which is universal and highly conserved in both prokaryotes and eukaryotic organelles, was selected for further characterizations. We demonstrated that ClpP is responsible for enhancing susceptibility to the T6SS killing. Because ClpP protease depends on other adapter proteins such as ClpA and ClpX for substrate recognition, further mutant studies followed by complementation tests were carried out to reveal that ClpP-associated AAA + ATPase ClpA, but not ClpX, is involved in enhancing susceptibility to A. tumefaciens T6SS killing. Moreover, functional and biochemical studies of various ClpP amino acid substitution variants provided evidence that ClpA-ClpP interaction is critical in enhancing susceptibility to the T6SS killing. This study highlights the importance of recipient factors in determining the outcome of the T6SS killing and shows the universal ClpP protease as a novel recipient factor hijacked by the T6SS of A. tumefaciens.

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“…The situation seems to be exceptionally complicated in microorganisms possessing complex Dsb system with redundant Dsb proteins. An EcdsbA mutation can be easily complemented in trans, whereas expression from plasmid Serratia marescens dsbA1 (but not dsbA2) or dsbA1, dsbA2 or c8j_1298 of C. jejuni have a deleterious impact on cell growth or results in genetic rearrangements [80].…”

Section: Plos Onementioning

confidence: 99%

C8J_1298, a bifunctional thiol oxidoreductase of Campylobacter jejuni, affects Dsb (disulfide bond) network functioning

et al. 2020

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Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner.

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Dual Role for DsbA in Attacking and Targeted Bacterial Cells during Type VI Secretion System-Mediated Competition

Cited by 32 publications

References 45 publications

Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system

Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system

A High-Throughput Interbacterial Competition Screen Identifies ClpAP in Enhancing Recipient Susceptibility to Type VI Secretion System-Mediated Attack by Agrobacterium tumefaciens

C8J_1298, a bifunctional thiol oxidoreductase of Campylobacter jejuni, affects Dsb (disulfide bond) network functioning

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