Three small partner proteins facilitate the type VII-dependent secretion export of an antibacterial nuclease

Palmer, Tracy; Yang, Yaping; Boardman, Eleanor; Deme, Justin C.; Alcock, Felicity; Lea, Susan M.

doi:10.1101/2023.04.01.535202

Cited by 4 publications

(3 citation statements)

References 47 publications

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“…By contrast, we were able to generate high confidence models of full-length TelC because an X-ray structure is available for its toxin domain, and of full-length YxiD toxin from B. subtilis because it lacks a middle linker region and possesses a confidently predicted nuclease toxin (4,5). Based on these results and preliminary structural data described by others, we propose that full-length toxin-Lap-Lap complexes adopt a tomahawk shaped structure with the metaphorical blade and shaft subunits being formed by the toxin domain and LXG-Lap-Lap complex, respectively (24). Given that there are currently over 4000 annotated LXG toxins in publicly available sequence databases, we expect that our structure will allow for the high confidence modelling of the vast majority of tripartite LXG secretion complexes found in diverse species of T7SS-containing Firmicutes.…”

Section: Lxg Targeting Complexes Adopt a Conserved Architecturementioning

confidence: 61%

Structure of a tripartite protein complex that targets toxins to the type VII secretion system

Klein,

Shah,

Gkragkopoulou

et al. 2023

Preprint

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Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Angstrom structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated alpha-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.

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Section: Lxg Targeting Complexes Adopt a Conserved Architecturementioning

confidence: 61%

Structure of a tripartite protein complex that targets toxins to the type VII secretion system

Klein,

Shah,

Gkragkopoulou

et al. 2023

Preprint

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show abstract

“…While these were originally identified as effector proteins e.g. 18 , it is becoming increasingly clear that at least some of them also serve as stabilising and/or targeting factors for larger T7SS substrates, with which they are cosecreted 10,19,20,21 . The LXG proteins are large antibacterial toxins secreted by the T7SSb, which form complexes with two or three WXG100-like partners.…”

Section: Introductionmentioning

confidence: 99%

“…The LXG proteins are large antibacterial toxins secreted by the T7SSb, which form complexes with two or three WXG100-like partners. These small partners, which have been designated Lap (LXG-associated -helical protein) interact with the N-terminal helical LXG domain and are predicted to form a helical stack 19,20,21 . Larger substrates of the T7SSa include the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) proteins.…”

Section: Introductionmentioning

confidence: 99%

An interbacterial lipase toxin with an unprecedented reverse domain arrangement defines a new class of type VII secretion system effector

Garrett

Mietrach

Deme³

et al. 2023

Preprint

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The type VII protein secretion system (T7SS) is found in many Gram-positive bacteria and in pathogenic mycobacteria. All T7SS substrate proteins described to date share a common helical domain architecture at the N-terminus that typically interacts with other helical partner proteins, forming a composite signal sequence for targeting to the T7SS. The C-terminal domains are functionally diverse and in Gram-positive bacteria such as Staphylococcus aureus often specify toxic anti-bacterial activity. Here we describe the first example of a new class of T7 substrate, TslA, that has an unexpected reverse domain organisation. TslA is widely found across Bacillota including Staphylococcus, Enterococcus and Listeria. We show that the S. aureus TslA N-terminal domain is a phospholipase A with anti-staphylococcal activity that is neutralised by the immunity lipoprotein TilA. Two small helical partner proteins, TlaA1 and TlaA2 are essential for T7-dependent secretion of TslA and at least one of these interacts with the TslA C-terminal domain to form a helical stack. Cryo-EM analysis of purified TslA complexes indicate that they share structural similarity with canonical T7 substrates. Our findings suggest that the T7SS has the extraordinary feature of recognising a secretion signal present at either end of a substrate.

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A high throughput assay provides novel insight into type VII secretion inStaphylococcus aureus

Yang

Alcock

Kneuper

et al. 2023

Preprint

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Successful colonisation by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. S. aureus strains harbour a T7b-subtype type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Firmicutes which functions in bacterial antagonism and virulence. Traditional assays for monitoring protein secretion by the T7SSb rely on concentrating cell culture supernatants for immunoblot analysis, a procedure which is time consuming, low throughput, non-quantitative and has low sensitivity. To overcome some of these disadvantages we have utilised NanoLuc Binary Technology to develop a simple assay to monitor protein secretion via detection of bioluminescence in 384-well plates. High-throughput analysis of secretion in replicate cultures reveals overlapping distributions for T7+ and T7- populations highlighting a significant limitation of lower-throughput methods. We also demonstrate the utility of the NanoLuc assay in comparing T7-dependent protein secretion across different strains and find differences in the temperature dependence of T7 secretion across S. aureus isolates.

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Three small partner proteins facilitate the type VII-dependent secretion export of an antibacterial nuclease

Cited by 4 publications

References 47 publications

Structure of a tripartite protein complex that targets toxins to the type VII secretion system

Structure of a tripartite protein complex that targets toxins to the type VII secretion system

An interbacterial lipase toxin with an unprecedented reverse domain arrangement defines a new class of type VII secretion system effector

A high throughput assay provides novel insight into type VII secretion inStaphylococcus aureus

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