Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants
Staphylococcus aureus small-colony variants (SCVs) are associated with chronic, persistent, and relapsing courses of infection and are characterized by slow growth combined with other phenotypic and molecular traits. Although certain mechanisms have been described, the genetic basis of clinical SCVs...
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| Format: | Article |
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Frontiers Media S.A.
2019-09-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2019.02044/full |
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| author | Nina Schleimer Ursula Kaspar Britta Ballhausen Sarah A. Fotiadis Jessica M. Streu André Kriegeskorte Richard A. Proctor Karsten Becker |
| author_facet | Nina Schleimer Ursula Kaspar Britta Ballhausen Sarah A. Fotiadis Jessica M. Streu André Kriegeskorte Richard A. Proctor Karsten Becker |
| author_sort | Nina Schleimer |
| collection | DOAJ |
| description | Staphylococcus aureus small-colony variants (SCVs) are associated with chronic, persistent, and relapsing courses of infection and are characterized by slow growth combined with other phenotypic and molecular traits. Although certain mechanisms have been described, the genetic basis of clinical SCVs remains often unknown. Hence, we adapted an episomal tool for rapid identification and investigation of putative SCV phenotype-associated genes via antisense gene silencing based on previously described Tnl0-encoded tet-regulatory elements. Targeting the SCV phenotype-inducing enoyl-acyl-carrier-protein reductase gene (fabI), plasmid pSN1-AS‘fabI’ was generated leading to antisense silencing, which was proven by pronounced growth retardation in liquid cultures, phenotype switch on solid medium, and 200-fold increase of antisense ‘fabI’ expression. A crucial role of TetR repression in effective regulation of the system was demonstrated. Based on the use of anhydrotetracycline as effector, an easy-to-handle one-plasmid setup was set that may be applicable to different S. aureus backgrounds and cell culture studies. However, selection of the appropriate antisense fragment of the target gene remains a critical factor for effectiveness of silencing. This inducible gene expression system may help to identify SCV phenotype-inducing genes, which is prerequisite for the development of new antistaphylococcal agents and future alternative strategies to improve treatment of therapy-refractory SCV-related infections by iatrogenically induced phenotypic switch. Moreover, it can be used as controllable phenotype switcher to examine important aspects of SCV biology in cell culture as well as in vivo. |
| first_indexed | 2024-12-20T15:34:55Z |
| format | Article |
| id | doaj.art-9a74fe5bf8434a21ae94504943ab00bb |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-20T15:34:55Z |
| publishDate | 2019-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-9a74fe5bf8434a21ae94504943ab00bb2022-12-21T19:35:27ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-09-011010.3389/fmicb.2019.02044477176Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony VariantsNina Schleimer0Ursula Kaspar1Britta Ballhausen2Sarah A. Fotiadis3Jessica M. Streu4André Kriegeskorte5Richard A. Proctor6Karsten Becker7Institute of Medical Microbiology, University Hospital Münster, Münster, GermanyInstitute of Medical Microbiology, University Hospital Münster, Münster, GermanyInstitute of Medical Microbiology, University Hospital Münster, Münster, GermanyInstitute of Medical Microbiology, University Hospital Münster, Münster, GermanyInstitute of Medical Microbiology, University Hospital Münster, Münster, GermanyInstitute of Medical Microbiology, University Hospital Münster, Münster, GermanyDepartments of Medical Microbiology/Immunology and Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, United StatesInstitute of Medical Microbiology, University Hospital Münster, Münster, GermanyStaphylococcus aureus small-colony variants (SCVs) are associated with chronic, persistent, and relapsing courses of infection and are characterized by slow growth combined with other phenotypic and molecular traits. Although certain mechanisms have been described, the genetic basis of clinical SCVs remains often unknown. Hence, we adapted an episomal tool for rapid identification and investigation of putative SCV phenotype-associated genes via antisense gene silencing based on previously described Tnl0-encoded tet-regulatory elements. Targeting the SCV phenotype-inducing enoyl-acyl-carrier-protein reductase gene (fabI), plasmid pSN1-AS‘fabI’ was generated leading to antisense silencing, which was proven by pronounced growth retardation in liquid cultures, phenotype switch on solid medium, and 200-fold increase of antisense ‘fabI’ expression. A crucial role of TetR repression in effective regulation of the system was demonstrated. Based on the use of anhydrotetracycline as effector, an easy-to-handle one-plasmid setup was set that may be applicable to different S. aureus backgrounds and cell culture studies. However, selection of the appropriate antisense fragment of the target gene remains a critical factor for effectiveness of silencing. This inducible gene expression system may help to identify SCV phenotype-inducing genes, which is prerequisite for the development of new antistaphylococcal agents and future alternative strategies to improve treatment of therapy-refractory SCV-related infections by iatrogenically induced phenotypic switch. Moreover, it can be used as controllable phenotype switcher to examine important aspects of SCV biology in cell culture as well as in vivo.https://www.frontiersin.org/article/10.3389/fmicb.2019.02044/fullStaphylococcus aureussmall-colony variant (SCV)antisense silencingphenotype switchenoyl-acyl-carrier-protein reductase gene (fabI)tet-regulatory expression system |
| spellingShingle | Nina Schleimer Ursula Kaspar Britta Ballhausen Sarah A. Fotiadis Jessica M. Streu André Kriegeskorte Richard A. Proctor Karsten Becker Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants Frontiers in Microbiology Staphylococcus aureus small-colony variant (SCV) antisense silencing phenotype switch enoyl-acyl-carrier-protein reductase gene (fabI) tet-regulatory expression system |
| title | Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants |
| title_full | Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants |
| title_fullStr | Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants |
| title_full_unstemmed | Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants |
| title_short | Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants |
| title_sort | adaption of an episomal antisense silencing approach for investigation of the phenotype switch of staphylococcus aureus small colony variants |
| topic | Staphylococcus aureus small-colony variant (SCV) antisense silencing phenotype switch enoyl-acyl-carrier-protein reductase gene (fabI) tet-regulatory expression system |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2019.02044/full |
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