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Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in Serratia marcescens.

Srikannathasan V, English G, Bui NK, Trunk K, O'Rourke PE, Rao VA, Vollmer W, Coulthurst SJ, Hunter WN - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized.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.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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Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.

ABSTRACT
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 from Serratia marcescens is 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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Subgroups of Tae4 family proteins and their associated immunity proteins. Neighbour-joining tree calculated from a multiple sequence alignment of Ssp1, Ssp2 and other Tae4 homologues from different bacterial species (a full alignment is shown in Supplementary Fig. S6). For each Tae4 protein, the adjacently encoded candidate immunity protein was identified by genomic analysis and the S. marcescens Rap protein to which it was most closely related was determined. Tae4 homologues with associated immunity proteins similar to Rap1a (Tai4a) are shown by red circles, whereas those with immunity proteins of the Rap1b/Rap2a/Rap2b (Tai4) type are shown by squares (in light blue, green or dark blue for most similarity to Rap1, Rap2a or Rap2b, respectively). Apart from Ssp1 and Ssp2 from S. marcescens, the Tae4 homologues are labelled by organism and their identities are as follows (UniProt or genomic identifiers): Acinetobacter baumannii, B0VVE3_ACIBS; Agrobacterium tumefaciens, Atu4347; Burkholderia cenocepacia, Bcen_4030; Cronobacter sakazakii, ESA_03935; Enterobacter cloacae, ECL_01542; Entero­bacter hormaechei, F5RYK9_9ENTR; Erwinia amylovora, EAMY_3018; Erwinia tasmaniensis, ETA_06210; Escherichia coli, ECEG_03250; Pantoea sp., S7A_11480; Pseudomonas syringae, Psyr_4040; Salmonella enterica serovar Newport, SNSL254_A0303; S. enterica serovar Typhimurium, STM0277; S. enterica serovar Typhi, STY0307; Serratia odorifera, D4E4R6_SEROD. Asterisks indicate the Tae4–Tai4 homologues for which structures have previously been reported (Zhang et al., 2013 ▶) and the open square indicates that this candidate immunity protein showed only weak similarity to Rap2a. Details of the Tae4 homologues and associated immunity proteins are given in Supplementary Table S1.
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fig8: Subgroups of Tae4 family proteins and their associated immunity proteins. Neighbour-joining tree calculated from a multiple sequence alignment of Ssp1, Ssp2 and other Tae4 homologues from different bacterial species (a full alignment is shown in Supplementary Fig. S6). For each Tae4 protein, the adjacently encoded candidate immunity protein was identified by genomic analysis and the S. marcescens Rap protein to which it was most closely related was determined. Tae4 homologues with associated immunity proteins similar to Rap1a (Tai4a) are shown by red circles, whereas those with immunity proteins of the Rap1b/Rap2a/Rap2b (Tai4) type are shown by squares (in light blue, green or dark blue for most similarity to Rap1, Rap2a or Rap2b, respectively). Apart from Ssp1 and Ssp2 from S. marcescens, the Tae4 homologues are labelled by organism and their identities are as follows (UniProt or genomic identifiers): Acinetobacter baumannii, B0VVE3_ACIBS; Agrobacterium tumefaciens, Atu4347; Burkholderia cenocepacia, Bcen_4030; Cronobacter sakazakii, ESA_03935; Enterobacter cloacae, ECL_01542; Entero­bacter hormaechei, F5RYK9_9ENTR; Erwinia amylovora, EAMY_3018; Erwinia tasmaniensis, ETA_06210; Escherichia coli, ECEG_03250; Pantoea sp., S7A_11480; Pseudomonas syringae, Psyr_4040; Salmonella enterica serovar Newport, SNSL254_A0303; S. enterica serovar Typhimurium, STM0277; S. enterica serovar Typhi, STY0307; Serratia odorifera, D4E4R6_SEROD. Asterisks indicate the Tae4–Tai4 homologues for which structures have previously been reported (Zhang et al., 2013 ▶) and the open square indicates that this candidate immunity protein showed only weak similarity to Rap2a. Details of the Tae4 homologues and associated immunity proteins are given in Supplementary Table S1.

Mentions: A comparison of the amino-acid sequences of Ssp1, Ssp2 and Tae4 homologues showed that Ssp1 and several other Tae4 homologues form a distinct grouping (Fig. 8 ▶). This is consistent with several structural differences being observed in Ssp1 compared with EcTae4 (Supplementary Fig. S2). When the adjacently encoded known or putative immunity protein was identified for all of these effectors and compared with the four S. marcescens Rap proteins, a pattern became evident. The Ssp1-like proteins all co-occur with immunity proteins of the Rap1a type. The other Tae4 homologues are less separated from each other, but there is a clustering of Tae4 proteins sharing related immunity proteins. So, for example, the three Tae4 proteins whose immunity proteins are most closely related to Rap1b all cluster together (Fig. 8 ▶) and the Tae4 homologues most similar to Ssp2 all have associated Rap2a-like immunity proteins (Fig. 8 ▶). Hence, effector and immunity proteins appear to have co-evolved within the Tae4 and Tai4 family. In particular, the Ssp1-like proteins appear to form a subgroup distinct enough to utilize a structurally unrelated immunity protein (‘Tai4a’), exemplified by Rap1a. The reason for this divergence is unclear, although it is consistent with the distinct biological phenotypes associated with the two effectors. Having both Ssp1 and Ssp2 is likely to confer an evolutionary advantage on the secreting organism, perhaps with each being more efficient against different target species or under different growth conditions than the other. Additionally, having Ssp1 may allow attack on a close relative with Ssp2/Rap2a and vice versa, maximizing the ability to distinguish ‘self’ from competitors.


Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in Serratia marcescens.

Srikannathasan V, English G, Bui NK, Trunk K, O'Rourke PE, Rao VA, Vollmer W, Coulthurst SJ, Hunter WN - Acta Crystallogr. D Biol. Crystallogr. (2013)

Subgroups of Tae4 family proteins and their associated immunity proteins. Neighbour-joining tree calculated from a multiple sequence alignment of Ssp1, Ssp2 and other Tae4 homologues from different bacterial species (a full alignment is shown in Supplementary Fig. S6). For each Tae4 protein, the adjacently encoded candidate immunity protein was identified by genomic analysis and the S. marcescens Rap protein to which it was most closely related was determined. Tae4 homologues with associated immunity proteins similar to Rap1a (Tai4a) are shown by red circles, whereas those with immunity proteins of the Rap1b/Rap2a/Rap2b (Tai4) type are shown by squares (in light blue, green or dark blue for most similarity to Rap1, Rap2a or Rap2b, respectively). Apart from Ssp1 and Ssp2 from S. marcescens, the Tae4 homologues are labelled by organism and their identities are as follows (UniProt or genomic identifiers): Acinetobacter baumannii, B0VVE3_ACIBS; Agrobacterium tumefaciens, Atu4347; Burkholderia cenocepacia, Bcen_4030; Cronobacter sakazakii, ESA_03935; Enterobacter cloacae, ECL_01542; Entero­bacter hormaechei, F5RYK9_9ENTR; Erwinia amylovora, EAMY_3018; Erwinia tasmaniensis, ETA_06210; Escherichia coli, ECEG_03250; Pantoea sp., S7A_11480; Pseudomonas syringae, Psyr_4040; Salmonella enterica serovar Newport, SNSL254_A0303; S. enterica serovar Typhimurium, STM0277; S. enterica serovar Typhi, STY0307; Serratia odorifera, D4E4R6_SEROD. Asterisks indicate the Tae4–Tai4 homologues for which structures have previously been reported (Zhang et al., 2013 ▶) and the open square indicates that this candidate immunity protein showed only weak similarity to Rap2a. Details of the Tae4 homologues and associated immunity proteins are given in Supplementary Table S1.
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Related In: Results  -  Collection

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Show All Figures
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fig8: Subgroups of Tae4 family proteins and their associated immunity proteins. Neighbour-joining tree calculated from a multiple sequence alignment of Ssp1, Ssp2 and other Tae4 homologues from different bacterial species (a full alignment is shown in Supplementary Fig. S6). For each Tae4 protein, the adjacently encoded candidate immunity protein was identified by genomic analysis and the S. marcescens Rap protein to which it was most closely related was determined. Tae4 homologues with associated immunity proteins similar to Rap1a (Tai4a) are shown by red circles, whereas those with immunity proteins of the Rap1b/Rap2a/Rap2b (Tai4) type are shown by squares (in light blue, green or dark blue for most similarity to Rap1, Rap2a or Rap2b, respectively). Apart from Ssp1 and Ssp2 from S. marcescens, the Tae4 homologues are labelled by organism and their identities are as follows (UniProt or genomic identifiers): Acinetobacter baumannii, B0VVE3_ACIBS; Agrobacterium tumefaciens, Atu4347; Burkholderia cenocepacia, Bcen_4030; Cronobacter sakazakii, ESA_03935; Enterobacter cloacae, ECL_01542; Entero­bacter hormaechei, F5RYK9_9ENTR; Erwinia amylovora, EAMY_3018; Erwinia tasmaniensis, ETA_06210; Escherichia coli, ECEG_03250; Pantoea sp., S7A_11480; Pseudomonas syringae, Psyr_4040; Salmonella enterica serovar Newport, SNSL254_A0303; S. enterica serovar Typhimurium, STM0277; S. enterica serovar Typhi, STY0307; Serratia odorifera, D4E4R6_SEROD. Asterisks indicate the Tae4–Tai4 homologues for which structures have previously been reported (Zhang et al., 2013 ▶) and the open square indicates that this candidate immunity protein showed only weak similarity to Rap2a. Details of the Tae4 homologues and associated immunity proteins are given in Supplementary Table S1.
Mentions: A comparison of the amino-acid sequences of Ssp1, Ssp2 and Tae4 homologues showed that Ssp1 and several other Tae4 homologues form a distinct grouping (Fig. 8 ▶). This is consistent with several structural differences being observed in Ssp1 compared with EcTae4 (Supplementary Fig. S2). When the adjacently encoded known or putative immunity protein was identified for all of these effectors and compared with the four S. marcescens Rap proteins, a pattern became evident. The Ssp1-like proteins all co-occur with immunity proteins of the Rap1a type. The other Tae4 homologues are less separated from each other, but there is a clustering of Tae4 proteins sharing related immunity proteins. So, for example, the three Tae4 proteins whose immunity proteins are most closely related to Rap1b all cluster together (Fig. 8 ▶) and the Tae4 homologues most similar to Ssp2 all have associated Rap2a-like immunity proteins (Fig. 8 ▶). Hence, effector and immunity proteins appear to have co-evolved within the Tae4 and Tai4 family. In particular, the Ssp1-like proteins appear to form a subgroup distinct enough to utilize a structurally unrelated immunity protein (‘Tai4a’), exemplified by Rap1a. The reason for this divergence is unclear, although it is consistent with the distinct biological phenotypes associated with the two effectors. Having both Ssp1 and Ssp2 is likely to confer an evolutionary advantage on the secreting organism, perhaps with each being more efficient against different target species or under different growth conditions than the other. Additionally, having Ssp1 may allow attack on a close relative with Ssp2/Rap2a and vice versa, maximizing the ability to distinguish ‘self’ from competitors.

Bottom Line: Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.

ABSTRACT
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 from Serratia marcescens is 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.

Show MeSH
Related in: MedlinePlus