Molecular characterization of the type VI secretion system effector Tlde1a reveals a structurally altered LD-transpeptidase fold

Cobo, Neil Lorente; Sibinelli-Sousa, Stephanie; Biboy, Jacob; Vollmer, Waldemar; Bayer-Santos, Ethel; Prehna, Gerd

doi:10.1016/j.jbc.2022.102556

Cited by 6 publications

(11 citation statements)

References 77 publications

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“…However, all previous experiments used a Prx expression construct that included a non-cleavable C-terminal 6His-tag. It is not uncommon for cloning artifacts such as a 6His-tag to affect protein stability 29 or even stabilize protein conformations as crystal packing artifacts 20, 30-31 . In fact, the Prx crystal dimer (PDBid: 6CKA) is stabilized by the C-terminal 6His-tag of a symmetry mate 14 .…”

Section: Resultsmentioning

confidence: 99%

TheStreptococcusphage protein paratox is an intrinsically disordered protein.

Asakereh,

Rutbeek,

Singh

et al. 2024

Preprint

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The bacteriophage protein paratox blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces ability ofStreptococcusto uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the paratox:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that paratox may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of paratox in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions paratox is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces paratox folding. However, only the addition of ComR was able to induce paratox to adopt its previously characterized globular fold. Furthermore, as we can induce different paratox folding-states we characterize Prx folding thermodynamics and folding kinetics using stopped flow measurements. Based upon the kinetic results, paratox is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein paratox is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized paratox fold is likely the predominant form of paratox in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing inStreptococcus, by a combination of conformational selection and induced-fit binding mechanisms.

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Section: Resultsmentioning

confidence: 99%

TheStreptococcusphage protein paratox is an intrinsically disordered protein.

Asakereh,

Rutbeek,

Singh

et al. 2024

Preprint

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“…In these studies, we used a Prx expression construct that included a non‐cleavable C‐terminal 6His‐tag (Prx‐6His) (Mashburn‐Warren et al, 2018 ; Rutbeek et al, 2021 ). It is not uncommon for cloning artifacts such as a 6His‐tag to affect protein stability (Booth et al, 2018 ) or even stabilize protein conformations as crystal packing artifacts (Lorente Cobo et al, 2022 ; Lovering et al, 2011 ; Shanker et al, 2016 ). In fact, the Prx‐6His crystal dimer is stabilized by the C‐terminal 6His‐tag of a symmetry mate (Mashburn‐Warren et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

The Streptococcus phage protein paratox is an intrinsically disordered protein

Asakereh,

Rutbeek,

Singh

et al. 2024

Protein Science

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The bacteriophage protein paratox (Prx) blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces the ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the Prx:ComR molecular interaction revealed that paratox adopts a well‐ordered globular fold when bound to ComR. However, solution‐state biophysical measurements suggested that Prx may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of Prx in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence‐based protein folding assays. Our work shows that under dilute buffer conditions Prx is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces Prx folding. However, the solute stabilized fold is different from the conformation Prx adopts when it is bound to ComR. Furthermore, we have characterized Prx folding thermodynamics and folding kinetics through steady‐state fluorescence and stopped flow kinetic measurements. Our results show that Prx is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein Prx is an intrinsically disordered protein in a two‐state equilibrium with a solute‐stabilized folded form. Furthermore, the solute‐stabilized fold is likely the predominant form of Prx in a solute‐crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced‐fit binding mechanisms.

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“…In S. Typhimurium and S . Dublin, 9 SPI-6 T6SS effector proteins have been described to date ( Russell et al, 2012 ; Benz et al, 2013 ; Whitney et al, 2013 ; Koskiniemi et al, 2014 ; Sana et al, 2016 ; Sibinelli-Sousa et al, 2020 ; Amaya et al, 2022 ; Jurėnas et al, 2022 ; Lorente-Cobo et al, 2022 ), most of which are encoded within these variable regions ( Figure 1 ; Table 1 ).…”

Section: Introductionmentioning

confidence: 99%

“…Tae2 and Tae4 are peptidoglycan hydrolases able to cleave the DD-crosslinks between D-mDAP and D-alanine or the covalent link between D-Glu and mDAP of the tetrapeptide stem, respectively, thus contributing to interbacterial competition and mice colonization ( Russell et al, 2012 ; Sana et al, 2016 ). VR2 is located downstream of gene tssM and encodes many proteins of unknown function and two E/I modules with peptidoglycan hydrolase activity: Tge2/Tgi2P is predicted to have N-acetylglucosaminidase activity ( Whitney et al, 2013 ), while Tlde1/Tldi shows L,D carboxypeptidase activity against the peptide stems of the peptidoglycan layer ( Sibinelli-Sousa et al, 2020 ; Lorente-Cobo et al, 2022 ). Finally, the VR3 is located downstream of gene tssI and encodes a variable number of Rhs elements, some of them harboring endonuclease domains such as HNHc (DNase) and Ntox47 (RNase), and an ART domain (ADP-ribosyltransferase) linked to the C-terminal of these Rhs proteins ( Koskiniemi et al, 2014 ; Amaya et al, 2022 ; Jurėnas et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Identification and distribution of new candidate T6SS effectors encoded in Salmonella Pathogenicity Island 6

Blondel,

Amaya,

Bustamante

et al. 2023

Front. Microbiol.

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The type VI secretion system (T6SS) is a contact-dependent contractile multiprotein apparatus widely distributed in Gram-negative bacteria. These systems can deliver different effector proteins into target bacterial and/or eukaryotic cells, contributing to the environmental fitness and virulence of many bacterial pathogens. Salmonella harbors five different T6SSs encoded in different genomic islands. The T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, information regarding the total number of effector proteins encoded within SPI-6 and its distribution among different Salmonella enterica serotypes is limited. In this work, we performed bioinformatic and comparative genomics analyses of the SPI-6 T6SS gene cluster to expand our knowledge regarding the T6SS effector repertoire and the global distribution of these effectors in Salmonella. The analysis of a curated dataset of 60 Salmonella enterica genomes from the Secret6 database revealed the presence of 23 new putative T6SS effector/immunity protein (E/I) modules. These effectors were concentrated in the variable regions 1 to 3 (VR1-3) of the SPI-6 T6SS gene cluster. VR1-2 were enriched in candidate effectors with predicted peptidoglycan hydrolase activity, while VR3 was enriched in candidate effectors of the Rhs family with C-terminal extensions with predicted DNase, RNase, deaminase, or ADP-ribosyltransferase activity. A global analysis of known and candidate effector proteins in Salmonella enterica genomes from the NCBI database revealed that T6SS effector proteins are differentially distributed among Salmonella serotypes. While some effectors are present in over 200 serotypes, others are found in less than a dozen. A hierarchical clustering analysis identified Salmonella serotypes with distinct profiles of T6SS effectors and candidate effectors, highlighting the diversity of T6SS effector repertoires in Salmonella enterica. The existence of different repertoires of effector proteins suggests that different effector protein combinations may have a differential impact on the environmental fitness and pathogenic potential of these strains.

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Molecular characterization of the type VI secretion system effector Tlde1a reveals a structurally altered LD-transpeptidase fold

Cited by 6 publications

References 77 publications

TheStreptococcusphage protein paratox is an intrinsically disordered protein.

TheStreptococcusphage protein paratox is an intrinsically disordered protein.

The Streptococcus phage protein paratox is an intrinsically disordered protein

Identification and distribution of new candidate T6SS effectors encoded in Salmonella Pathogenicity Island 6

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