Interbacterial competition mediated by the type VIIb secretion system

Boardman, Eleanor R.; Palmer, Tracy; Alcock, Felicity

doi:10.1099/mic.0.001420

Cited by 6 publications

(2 citation statements)

References 122 publications

(380 reference statements)

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“…The third protein encoded at this locus, SACOL2601, has a DUF4176 domain. Genes encoding DUF4176 domains are frequently associated with T7SS loci [3] and the involvement of a DUF4176 domain-protein in LXG toxin secretion was recently determined in S. intermedius [27].…”

Section: Identification Of Tspa Secretion Partnersmentioning

confidence: 99%

“…It is notorious for developing resistance to frontline antimicrobials, with methicillin resistance now widespread and vancomycin resistance also increasing [1,2]. Like many Gram-positive bacteria, S. aureus carries a specialised protein secretion machinery called the type VII secretion system (T7SS), which it utilises to secrete antibacterial toxins targeting competitor bacteria (reviewed in [3]). T7SS-mediated antagonism is likely to be important for colonisation [4], and the S. aureus T7SS has been shown to be required for virulence in mouse infection models [5][6][7][8][9].…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Identification Of Tspa Secretion Partnersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Yang

Alcock

Kneuper

et al. 2023

Preprint

Self Cite

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High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus

Yang,

Scott,

Kneuper

et al. 2024

Open Biol.

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Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.

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A group B streptococcal type VII secreted LXG toxin mediates interbacterial competition and colonization of the female genital tract

Job,

Doran,

Spencer

2024

Preprint

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Group BStreptococcus(GBS) asymptomatically colonizes the vagina but can opportunistically ascend to the uterus and be transmitted vertically during pregnancy, resulting in neonatal pneumonia, bacteremia and meningitis. GBS is a leading etiologic agent of neonatal infection and understanding the mechanisms by which GBS persists within the polymicrobial female genital mucosa has potential to mitigate subsequent transmission and disease. Type VIIb secretion systems (T7SSb) are encoded by Firmicutes and often mediate interbacterial competition using LXG toxins that contain conserved N-termini important for secretion and variable C-terminal toxin domains that confer diverse biochemical activities. Our recent work characterized a role for the GBS T7SSb in vaginal colonization and ascending infection but the mechanisms by which the T7SSb promotes GBS persistence in this polymicrobial niche remain unknown. Herein, we investigate the GBS T7SS in interbacterial competition and GBS niche establishment in the female genital tract. We demonstrate GBS T7SS-dependent inhibition of mucosal pathobiontEnterococcus faecalisbothin vitrousing predator-prey assays andin vivoin the murine genital tract and found that a GBS LXG protein encoded within the T7SS locus (herein named groupBstreptococcalLXGToxinA) that contributes to these phenotypes. We identify BltA as a T7SS substrate that is toxic toE. coliandS. aureusupon induction of expression along with associated chaperones. Finally, we show that BltA and its chaperones contribute to GBS vaginal colonization. Altogether, these data reveal a role for a novel T7b-secreted toxin in GBS mucosal persistence and competition.ImportanceCompetition between neighboring, non-kin bacteria is essential for microbial niche establishment in mucosal environments. Gram-positive bacteria encoding T7SSb have been shown to engage in competition through export of LXG-motif containing toxins, but these have not been characterized in group BStreptococcus(GBS), an opportunistic colonizer of the polymicrobial female genital tract. Here, we show a role for GBS T7SS in competition with mucosal pathobiontEnterococcus faecalis, bothin vitroandin vivo. We further find that a GBS LXG protein contributing to this antagonism is exported by the T7SS and is intracellularly toxic to other bacteria; therefore, we have named this protein groupBstreptococcalLXGToxinA(BltA). Finally, we show that BltA and its associated chaperones promote persistence within female genital tract tissuesin vivo.These data reveal previously unrecognized mechanisms by which GBS may compete with other mucosal opportunistic pathogens to persist within the female genital tract.

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Interbacterial competition mediated by the type VIIb secretion system

Cited by 6 publications

References 122 publications

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

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

High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus

A group B streptococcal type VII secreted LXG toxin mediates interbacterial competition and colonization of the female genital tract

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