High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus
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 f...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
The Royal Society
2024-08-01
|
| Series: | Open Biology |
| Subjects: | |
| Online Access: | https://royalsocietypublishing.org/doi/10.1098/rsob.240060 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850191731799097344 |
|---|---|
| author | Yaping Yang Aaron A. Scott Holger Kneuper Felicity Alcock Tracy Palmer |
| author_facet | Yaping Yang Aaron A. Scott Holger Kneuper Felicity Alcock Tracy Palmer |
| author_sort | Yaping Yang |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-e7e60f5eb285483daf48771a66a32a2c |
| institution | OA Journals |
| issn | 2046-2441 |
| language | English |
| publishDate | 2024-08-01 |
| publisher | The Royal Society |
| record_format | Article |
| series | Open Biology |
| spelling | doaj-art-e7e60f5eb285483daf48771a66a32a2c2025-08-20T02:14:49ZengThe Royal SocietyOpen Biology2046-24412024-08-0114810.1098/rsob.240060High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureusYaping Yang0Aaron A. Scott1Holger Kneuper2Felicity Alcock3Tracy Palmer4Newcastle University Biosciences Institute, Newcastle University , Newcastle upon Tyne NE2 4HH, UKNewcastle University Biosciences Institute, Newcastle University , Newcastle upon Tyne NE2 4HH, UKNewcastle University Biosciences Institute, Newcastle University , Newcastle upon Tyne NE2 4HH, UKNewcastle University Biosciences Institute, Newcastle University , Newcastle upon Tyne NE2 4HH, UKNewcastle University Biosciences Institute, Newcastle University , Newcastle upon Tyne NE2 4HH, UKSuccessful 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.https://royalsocietypublishing.org/doi/10.1098/rsob.240060type VII secretion systemStaphylococcus aureusbioluminescenceNanoBittoxinaccessory factors |
| spellingShingle | Yaping Yang Aaron A. Scott Holger Kneuper Felicity Alcock Tracy Palmer High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus Open Biology type VII secretion system Staphylococcus aureus bioluminescence NanoBit toxin accessory factors |
| title | High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus |
| title_full | High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus |
| title_fullStr | High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus |
| title_full_unstemmed | High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus |
| title_short | High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus |
| title_sort | high throughput functional analysis provides novel insight into type vii secretion in staphylococcus aureus |
| topic | type VII secretion system Staphylococcus aureus bioluminescence NanoBit toxin accessory factors |
| url | https://royalsocietypublishing.org/doi/10.1098/rsob.240060 |
| work_keys_str_mv | AT yapingyang highthroughputfunctionalanalysisprovidesnovelinsightintotypeviisecretioninstaphylococcusaureus AT aaronascott highthroughputfunctionalanalysisprovidesnovelinsightintotypeviisecretioninstaphylococcusaureus AT holgerkneuper highthroughputfunctionalanalysisprovidesnovelinsightintotypeviisecretioninstaphylococcusaureus AT felicityalcock highthroughputfunctionalanalysisprovidesnovelinsightintotypeviisecretioninstaphylococcusaureus AT tracypalmer highthroughputfunctionalanalysisprovidesnovelinsightintotypeviisecretioninstaphylococcusaureus |