Author response: Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.

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Structural Basis for the Secretion of EvpC: A Key Type VI Secretion System Protein from Edwardsiella tarda

PLoS ONE ◽

10.1371/journal.pone.0012910 ◽

2010 ◽

Vol 5 (9) ◽

pp. e12910 ◽

Cited By ~ 43

Author(s):

Chacko Jobichen ◽

Smarajit Chakraborty ◽

Mo Li ◽

Jun Zheng ◽

Lissa Joseph ◽

...

Keyword(s):

Secretion System ◽

Edwardsiella Tarda ◽

Structural Basis ◽

Type Vi Secretion System ◽

Type Vi Secretion

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Structural basis for type VI secreted peptidoglycanDL-endopeptidase function, specificity and neutralization inSerratia marcescens

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913022725 ◽

2013 ◽

Vol 69 (12) ◽

pp. 2468-2482 ◽

Cited By ~ 30

Author(s):

Velupillai Srikannathasan ◽

Grant English ◽

Nhat Khai Bui ◽

Katharina Trunk ◽

Patrick E. F. O'Rourke ◽

...

Keyword(s):

Protein Interactions ◽

Secretion System ◽

Effector Proteins ◽

Diaminopimelic Acid ◽

Structural Basis ◽

Type Vi Secretion System ◽

Conserved Sequence ◽

Functional Assays ◽

Type Vi Secretion ◽

Catalytic Cysteine

Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors fromSerratia marcescensis characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-D-glutamic acid and L-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions.

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Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones

10.1101/2020.10.28.356139 ◽

2020 ◽

Author(s):

Shehryar Ahmad ◽

Kara K. Tsang ◽

Kartik Sachar ◽

Dennis Quentin ◽

Tahmid M. Tashin ◽

...

Keyword(s):

Transmembrane Domain ◽

Effector Proteins ◽

Structural Basis ◽

Gram Negative Bacteria ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Type Vi Secretion ◽

Helical Packing ◽

Target Cell Membrane ◽

Abundance And Distribution

AbstractType VI secretion systems facilitate the delivery of antibacterial effector proteins between neighbouring Gram-negative bacteria. A subset of these effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across the target cell membrane. However, the abundance and distribution of these TMD-containing effectors has remained unknown. Here we report the discovery of prePAAR, a conserved motif found in over 6,000 putative TMD-containing effectors. Based on their differing sizes and number of TMDs these effectors fall into two distinct classes that are unified by their requirement for a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR functions to facilitate proper folding of the downstream PAAR domain, which is required for effector interaction with the T6SS spike. Taken together, our findings define the mechanism of chaperone-assisted secretion of a widespread family of T6SS membrane protein effectors.

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