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Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria.

Ates LS, Ummels R, Commandeur S, van de Weerd R, van der Weerd R, Sparrius M, Weerdenburg E, Alber M, Kalscheuer R, Piersma SR, Abdallah AM, Abd El Ghany M, Abdel-Haleem AM, Pain A, Jiménez CR, Bitter W, Houben EN - PLoS Genet. (2015)

Bottom Line: Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly.This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity.Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.

ABSTRACT
Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

No MeSH data available.


Related in: MedlinePlus

Secretion analysis of ESX-5 mutant strains.A) A schematic representation of the ESX-5 region of M. marinum with the different ESX-5 mutations used in this study. Bars above the gene cluster indicate regions deleted by targeted knock-out mutagenesis. Arrows below indicate position and orientation of transposons (named LA1 to LA12) in mutants of the parental strain M. marinum::mspA defective in ESX-5 dependent secretion. B) Secretion analysis of M.marinum::mspA (WT::mspA), a mycP5 transposon mutant (mycP5::tn, corresponding to LA9 in (A)) and the complemented version of this strain (mycP5::tn-C). Secreted proteins (S) were separated from bacterial cells (P) by centrifugation. In addition, surface-associated proteins were enriched from the bacterial cells by extraction with 0.5% Genapol X-080 (GS) and separated from non-solubilized proteins (GP) by centrifugation. All fractions were analyzed for the presence of PE_PGRS proteins by immunoblotting. GroEL2 staining was used as a loading and lysis control. C) Expression of EccB5 and EspG5 was analyzed by immunoblotting of total cell lysates of wild-type M. marinum (WT), the Δesx-5::mspA mutant and the complemented Δesx-5::esx-5tub strain. D) The same strains as under (C) were analyzed for their ability to express and secrete PE_PGRS proteins following the same procedure as under (B).
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pgen.1005190.g003: Secretion analysis of ESX-5 mutant strains.A) A schematic representation of the ESX-5 region of M. marinum with the different ESX-5 mutations used in this study. Bars above the gene cluster indicate regions deleted by targeted knock-out mutagenesis. Arrows below indicate position and orientation of transposons (named LA1 to LA12) in mutants of the parental strain M. marinum::mspA defective in ESX-5 dependent secretion. B) Secretion analysis of M.marinum::mspA (WT::mspA), a mycP5 transposon mutant (mycP5::tn, corresponding to LA9 in (A)) and the complemented version of this strain (mycP5::tn-C). Secreted proteins (S) were separated from bacterial cells (P) by centrifugation. In addition, surface-associated proteins were enriched from the bacterial cells by extraction with 0.5% Genapol X-080 (GS) and separated from non-solubilized proteins (GP) by centrifugation. All fractions were analyzed for the presence of PE_PGRS proteins by immunoblotting. GroEL2 staining was used as a loading and lysis control. C) Expression of EccB5 and EspG5 was analyzed by immunoblotting of total cell lysates of wild-type M. marinum (WT), the Δesx-5::mspA mutant and the complemented Δesx-5::esx-5tub strain. D) The same strains as under (C) were analyzed for their ability to express and secrete PE_PGRS proteins following the same procedure as under (B).

Mentions: We previously failed to isolate transposon mutants in any of the genes encoding ESX-5 membrane components by screening transposon mutant libraries for secretion defects [25,29]. To determine whether we could now isolate such transposon mutants by introduction of MspA, we repeated our original screens using a transposon library created in M. marinum expressing mspA. This transposon library of ~10.000 mutants was screened for the secretion of the ESX-5 dependent PE_PGRS proteins, using the previously described double filter assay [29]. In total, eight transposon mutants were identified that showed completely abolished PE_PGRS secretion. All eight secretion mutants had transposon insertions in the ESX-5 region (Fig 3A), four of which were affected in genes encoding the membrane components eccB5, eccD5 and mycP5. Secretion analysis confirmed that these ESX-5 transposon mutants showed strongly reduced expression and secretion of PE_PGRS proteins and lacked expression of the mutated components (Figs 3B and S3). To show reversibility of the phenotype, the MycP5 transposon mutant (LA9) was complemented (Fig 3B). In conclusion, the introduction of MspA allowed transposon insertions in the ESX-5 locus and only mutants within the ESX-5 gene cluster showed a complete lack of PE_PGRS secretion, underscoring the importance of this locus in this process.


Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria.

Ates LS, Ummels R, Commandeur S, van de Weerd R, van der Weerd R, Sparrius M, Weerdenburg E, Alber M, Kalscheuer R, Piersma SR, Abdallah AM, Abd El Ghany M, Abdel-Haleem AM, Pain A, Jiménez CR, Bitter W, Houben EN - PLoS Genet. (2015)

Secretion analysis of ESX-5 mutant strains.A) A schematic representation of the ESX-5 region of M. marinum with the different ESX-5 mutations used in this study. Bars above the gene cluster indicate regions deleted by targeted knock-out mutagenesis. Arrows below indicate position and orientation of transposons (named LA1 to LA12) in mutants of the parental strain M. marinum::mspA defective in ESX-5 dependent secretion. B) Secretion analysis of M.marinum::mspA (WT::mspA), a mycP5 transposon mutant (mycP5::tn, corresponding to LA9 in (A)) and the complemented version of this strain (mycP5::tn-C). Secreted proteins (S) were separated from bacterial cells (P) by centrifugation. In addition, surface-associated proteins were enriched from the bacterial cells by extraction with 0.5% Genapol X-080 (GS) and separated from non-solubilized proteins (GP) by centrifugation. All fractions were analyzed for the presence of PE_PGRS proteins by immunoblotting. GroEL2 staining was used as a loading and lysis control. C) Expression of EccB5 and EspG5 was analyzed by immunoblotting of total cell lysates of wild-type M. marinum (WT), the Δesx-5::mspA mutant and the complemented Δesx-5::esx-5tub strain. D) The same strains as under (C) were analyzed for their ability to express and secrete PE_PGRS proteins following the same procedure as under (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4418733&req=5

pgen.1005190.g003: Secretion analysis of ESX-5 mutant strains.A) A schematic representation of the ESX-5 region of M. marinum with the different ESX-5 mutations used in this study. Bars above the gene cluster indicate regions deleted by targeted knock-out mutagenesis. Arrows below indicate position and orientation of transposons (named LA1 to LA12) in mutants of the parental strain M. marinum::mspA defective in ESX-5 dependent secretion. B) Secretion analysis of M.marinum::mspA (WT::mspA), a mycP5 transposon mutant (mycP5::tn, corresponding to LA9 in (A)) and the complemented version of this strain (mycP5::tn-C). Secreted proteins (S) were separated from bacterial cells (P) by centrifugation. In addition, surface-associated proteins were enriched from the bacterial cells by extraction with 0.5% Genapol X-080 (GS) and separated from non-solubilized proteins (GP) by centrifugation. All fractions were analyzed for the presence of PE_PGRS proteins by immunoblotting. GroEL2 staining was used as a loading and lysis control. C) Expression of EccB5 and EspG5 was analyzed by immunoblotting of total cell lysates of wild-type M. marinum (WT), the Δesx-5::mspA mutant and the complemented Δesx-5::esx-5tub strain. D) The same strains as under (C) were analyzed for their ability to express and secrete PE_PGRS proteins following the same procedure as under (B).
Mentions: We previously failed to isolate transposon mutants in any of the genes encoding ESX-5 membrane components by screening transposon mutant libraries for secretion defects [25,29]. To determine whether we could now isolate such transposon mutants by introduction of MspA, we repeated our original screens using a transposon library created in M. marinum expressing mspA. This transposon library of ~10.000 mutants was screened for the secretion of the ESX-5 dependent PE_PGRS proteins, using the previously described double filter assay [29]. In total, eight transposon mutants were identified that showed completely abolished PE_PGRS secretion. All eight secretion mutants had transposon insertions in the ESX-5 region (Fig 3A), four of which were affected in genes encoding the membrane components eccB5, eccD5 and mycP5. Secretion analysis confirmed that these ESX-5 transposon mutants showed strongly reduced expression and secretion of PE_PGRS proteins and lacked expression of the mutated components (Figs 3B and S3). To show reversibility of the phenotype, the MycP5 transposon mutant (LA9) was complemented (Fig 3B). In conclusion, the introduction of MspA allowed transposon insertions in the ESX-5 locus and only mutants within the ESX-5 gene cluster showed a complete lack of PE_PGRS secretion, underscoring the importance of this locus in this process.

Bottom Line: Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly.This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity.Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.

ABSTRACT
Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

No MeSH data available.


Related in: MedlinePlus