Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier

Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development. Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Maroeska J. Burggraaf [verfasserIn] Louis S. Ates [verfasserIn] Alexander Speer [verfasserIn] Kim van der Kuij [verfasserIn] Coen Kuijl [verfasserIn] Wilbert Bitter [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2019

Schlagwörter:	Heterologous secretion Mycobacterium Vaccine BCG LipY ESX

Übergeordnetes Werk:	In: Microbial Cell Factories - BMC, 2003, 18(2019), 1, Seite 14
Übergeordnetes Werk:	volume:18 ; year:2019 ; number:1 ; pages:14

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1186/s12934-019-1093-1

Katalog-ID:	DOAJ050459864

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520			\|a Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.
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author_variant	m j b mjb l s a lsa a s as k v d k kvdk c k ck w b wb
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allfields	10.1186/s12934-019-1093-1 doi (DE-627)DOAJ050459864 (DE-599)DOAJf8114a9246ae4caf9e492790b9690434 DE-627 ger DE-627 rakwb eng QR1-502 Maroeska J. Burggraaf verfasserin aut Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development. Heterologous secretion Mycobacterium Vaccine BCG LipY ESX Microbiology Louis S. Ates verfasserin aut Alexander Speer verfasserin aut Kim van der Kuij verfasserin aut Coen Kuijl verfasserin aut Wilbert Bitter verfasserin aut In Microbial Cell Factories BMC, 2003 18(2019), 1, Seite 14 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:18 year:2019 number:1 pages:14 https://doi.org/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/article/f8114a9246ae4caf9e492790b9690434 kostenfrei http://link.springer.com/article/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 18 2019 1 14
spelling	10.1186/s12934-019-1093-1 doi (DE-627)DOAJ050459864 (DE-599)DOAJf8114a9246ae4caf9e492790b9690434 DE-627 ger DE-627 rakwb eng QR1-502 Maroeska J. Burggraaf verfasserin aut Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development. Heterologous secretion Mycobacterium Vaccine BCG LipY ESX Microbiology Louis S. Ates verfasserin aut Alexander Speer verfasserin aut Kim van der Kuij verfasserin aut Coen Kuijl verfasserin aut Wilbert Bitter verfasserin aut In Microbial Cell Factories BMC, 2003 18(2019), 1, Seite 14 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:18 year:2019 number:1 pages:14 https://doi.org/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/article/f8114a9246ae4caf9e492790b9690434 kostenfrei http://link.springer.com/article/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 18 2019 1 14
allfields_unstemmed	10.1186/s12934-019-1093-1 doi (DE-627)DOAJ050459864 (DE-599)DOAJf8114a9246ae4caf9e492790b9690434 DE-627 ger DE-627 rakwb eng QR1-502 Maroeska J. Burggraaf verfasserin aut Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development. Heterologous secretion Mycobacterium Vaccine BCG LipY ESX Microbiology Louis S. Ates verfasserin aut Alexander Speer verfasserin aut Kim van der Kuij verfasserin aut Coen Kuijl verfasserin aut Wilbert Bitter verfasserin aut In Microbial Cell Factories BMC, 2003 18(2019), 1, Seite 14 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:18 year:2019 number:1 pages:14 https://doi.org/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/article/f8114a9246ae4caf9e492790b9690434 kostenfrei http://link.springer.com/article/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 18 2019 1 14
allfieldsGer	10.1186/s12934-019-1093-1 doi (DE-627)DOAJ050459864 (DE-599)DOAJf8114a9246ae4caf9e492790b9690434 DE-627 ger DE-627 rakwb eng QR1-502 Maroeska J. Burggraaf verfasserin aut Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development. Heterologous secretion Mycobacterium Vaccine BCG LipY ESX Microbiology Louis S. Ates verfasserin aut Alexander Speer verfasserin aut Kim van der Kuij verfasserin aut Coen Kuijl verfasserin aut Wilbert Bitter verfasserin aut In Microbial Cell Factories BMC, 2003 18(2019), 1, Seite 14 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:18 year:2019 number:1 pages:14 https://doi.org/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/article/f8114a9246ae4caf9e492790b9690434 kostenfrei http://link.springer.com/article/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 18 2019 1 14
allfieldsSound	10.1186/s12934-019-1093-1 doi (DE-627)DOAJ050459864 (DE-599)DOAJf8114a9246ae4caf9e492790b9690434 DE-627 ger DE-627 rakwb eng QR1-502 Maroeska J. Burggraaf verfasserin aut Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development. Heterologous secretion Mycobacterium Vaccine BCG LipY ESX Microbiology Louis S. Ates verfasserin aut Alexander Speer verfasserin aut Kim van der Kuij verfasserin aut Coen Kuijl verfasserin aut Wilbert Bitter verfasserin aut In Microbial Cell Factories BMC, 2003 18(2019), 1, Seite 14 (DE-627)355987651 (DE-600)2091377-1 14752859 nnns volume:18 year:2019 number:1 pages:14 https://doi.org/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/article/f8114a9246ae4caf9e492790b9690434 kostenfrei http://link.springer.com/article/10.1186/s12934-019-1093-1 kostenfrei https://doaj.org/toc/1475-2859 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 18 2019 1 14
language	English
source	In Microbial Cell Factories 18(2019), 1, Seite 14 volume:18 year:2019 number:1 pages:14
sourceStr	In Microbial Cell Factories 18(2019), 1, Seite 14 volume:18 year:2019 number:1 pages:14
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Heterologous secretion Mycobacterium Vaccine BCG LipY ESX Microbiology
isfreeaccess_bool	true
container_title	Microbial Cell Factories
authorswithroles_txt_mv	Maroeska J. Burggraaf @@aut@@ Louis S. Ates @@aut@@ Alexander Speer @@aut@@ Kim van der Kuij @@aut@@ Coen Kuijl @@aut@@ Wilbert Bitter @@aut@@
publishDateDaySort_date	2019-01-01T00:00:00Z
hierarchy_top_id	355987651
id	DOAJ050459864
language_de	englisch
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Burggraaf</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2019</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. 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callnumber-first	Q - Science
author	Maroeska J. Burggraaf
spellingShingle	Maroeska J. Burggraaf misc QR1-502 misc Heterologous secretion misc Mycobacterium misc Vaccine misc BCG misc LipY misc ESX misc Microbiology Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier
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topic_title	QR1-502 Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier Heterologous secretion Mycobacterium Vaccine BCG LipY ESX
topic	misc QR1-502 misc Heterologous secretion misc Mycobacterium misc Vaccine misc BCG misc LipY misc ESX misc Microbiology
topic_unstemmed	misc QR1-502 misc Heterologous secretion misc Mycobacterium misc Vaccine misc BCG misc LipY misc ESX misc Microbiology
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title	Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier
ctrlnum	(DE-627)DOAJ050459864 (DE-599)DOAJf8114a9246ae4caf9e492790b9690434
title_full	Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier
author_sort	Maroeska J. Burggraaf
journal	Microbial Cell Factories
journalStr	Microbial Cell Factories
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lang_code	eng
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publishDateSort	2019
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author_browse	Maroeska J. Burggraaf Louis S. Ates Alexander Speer Kim van der Kuij Coen Kuijl Wilbert Bitter
container_volume	18
class	QR1-502
format_se	Elektronische Aufsätze
author-letter	Maroeska J. Burggraaf
doi_str_mv	10.1186/s12934-019-1093-1
author2-role	verfasserin
title_sort	optimization of secretion and surface localization of heterologous ova protein in mycobacteria by using lipy as a carrier
callnumber	QR1-502
title_auth	Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier
abstract	Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.
abstractGer	Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.
abstract_unstemmed	Abstract Background Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens. Results We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association. Conclusion We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.
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container_issue	1
title_short	Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier
url	https://doi.org/10.1186/s12934-019-1093-1 https://doaj.org/article/f8114a9246ae4caf9e492790b9690434 http://link.springer.com/article/10.1186/s12934-019-1093-1 https://doaj.org/toc/1475-2859
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author2	Louis S. Ates Alexander Speer Kim van der Kuij Coen Kuijl Wilbert Bitter
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up_date	2024-07-03T14:58:39.872Z
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Optimization of secretion and surface localization of heterologous OVA protein in mycobacteria by using LipY as a carrier

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