Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase

Hersch, Steven J.; Lam, Linh; Dong, Tao

doi:10.1128/mbio.01115-21

Cited by 20 publications

(28 citation statements)

References 54 publications

(76 reference statements)

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“…To test how effectors are specifically recognized by cognate VgrGs in SSU, we swapped the VgrG3 tail sequence (P650 to end) with the ones from VgrG1 (K650 to end) and VgrG2 (E650 to end) to test whether these chimeric VgrG3 variants can functionally complement the deletions of the corresponding vgrGs ( Next, we tested whether VgrG-tail swapping could deliver an effector of another species. This is different from our recent report that an effector from A. dhakensis was delivered by the T6SS of V. cholerae as a hybrid fusion to the PAAR2 protein [43]. We expressed VasX, a T6SS effector in V. cholerae, and its associated chaperone VasW and immunity protein TsiV2 in SSU (Fig 2C).…”

Section: Vgrg C-terminal Divergent Tail Dictates Effector Delivery Specificitycontrasting

confidence: 80%

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VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system

Liang

Pei

et al. 2021

PLoS Pathog

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The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly.

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Section: Vgrg C-terminal Divergent Tail Dictates Effector Delivery Specificitycontrasting

confidence: 80%

“…dhakensis was delivered by the T6SS of V . cholerae as a hybrid fusion to the PAAR2 protein [ 43 ]. We expressed VasX, a T6SS effector in V .…”

Section: Resultsmentioning

confidence: 99%

VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system

Liang

Pei

et al. 2021

PLoS Pathog

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“…For example, T3SSs from diverse pathogenic bacteria were used as translocation tools to deliver proteins into mammalian cells ( 4 , 5 ). T6SS, another widespread injectisome in pathogens, was developed as delivery vector in biotechnological applications ( 30 , 31 ). Many other protein complexes, such as Tc toxins ( 10 , 11 ), type IV pilins ( 32 , 33 ) and pore-forming proteins ( 34 , 35 ), were also used to accomplish similar assignments toward eukaryotic cells, particularly killing cancer cells ( 36 ).…”

Section: Discussionmentioning

confidence: 99%

N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system

et al. 2022

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Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, Photorhabdus virulence cassette (PVC) was proposed to inject toxins into eukaryotic cells by puncturing the cell membrane from outside. This makes it an ideal tool for protein delivery in biomedical research. However, how to manipulate this nanocomplex as a molecular syringe is still undetermined. Here, we identify that one group of N-terminal signal peptide (SP) sequences are crucial for the effector loading into the inner tube of PVC complex. By application of genetic operation, cryo–electron microscopy, in vitro translocation assays, and animal experiments, we show that, under the guidance of the SP, numerous prokaryotic and eukaryotic proteins can be loaded into PVC to exert their functions across cell membranes. We therefore might customize PVC as a potent protein delivery nanosyringe for biotherapy by selecting cargo proteins in a broad spectrum, regardless of their species, sizes, and charges.

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“…Data are shown as either Log 10 c.f.u recovered on LB plates with appropriate antibiotics or as recombination efficiency, calculated as Gen R c.f.u/Carb R c.f.u. If no colonies were observed, samples were listed as the detection limit (DL), shown as if 0.5 colonies were counted 22 . Error bars show the mean ± standard deviation of three biological replicates.…”

Section: Cre Delivery Assaymentioning

confidence: 99%

“…These effectors may not only dictate functions but also directly contribute to T6SS assembly because deletion of multiple effector genes abolishes T6SS assembly in several species 9,18,20 . Similar to the spike complex proteins, VgrG and PAAR, some effectors can also serve as carriers when fused with cargo proteins 8,9,21,22 . Therefore, rather than deleting effector genes, an effective strategy has been developed in Vibrio cholerae and Aeromonas dhakensis to use catalytic mutations of effectors to detoxify the T6SS while maintaining the delivery function 9,20,22 .…”

Section: Introductionmentioning

confidence: 99%

A dueling-competent signal-sensing module guides precise delivery of cargo proteins into target cells by engineered Pseudomonas aeruginosa

Dong

Yan

et al. 2022

Preprint

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To recognize and manipulate a specific microbe of a crowded community is a highly challenging task in synthetic biology. Here, we introduce a highly-selective protein delivery platform, termed DUEC, which responds to direct contact of attacking cells by engineering the tit-for-tat/dueling response of H1-T6SS (type VI secretion system) in Pseudomonas aeruginosa. Using a Cre-recombinase-dependent reporter, we screened H1-T6SS secreted substrates and developed Tse6N as the most effective secretion tag for Cre delivery. DUEC cells can discriminately deliver the Tse6N-Cre cargo into the cytosol of T6SS+ but not T6SS- Vibrio cholerae cells in a mixed population. These data demonstrate that the DUEC cell is not only a prototypical physical-contact sensor and delivery platform but also may be coupled with recombination-based circuits with the potential for complex tasks in mixed microbial communities.

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Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase

Abstract: Delivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function.

Cited by 20 publications

References 54 publications

VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system

VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system

N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system

A dueling-competent signal-sensing module guides precise delivery of cargo proteins into target cells by engineered Pseudomonas aeruginosa

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