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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

ESX-5 is involved in fatty acid uptake.A) Growth of indicated M. marinum strains on Tween-80 as a sole carbon source was assessed by measuring optical density at different time points. Depicted is the average of three biological replicates. Error bars indicate standard deviations. B) Uptake of a fluorescently labeled fatty acid after 72 hours of hypoxic growth was measured by FACS analysis. 20.000 events gated for similar size were acquired for WT::mspA (black), ΔmycP5::mspA (light grey) or ΔmycP5-C::mspA (dark grey). C) Quantification of FACS analysis. Mean fluorescent intensity of three experiments per strain was acquired by FACS. Background staining, quantified by adding the fluorescent fatty acid to an unstained culture one hour before washing the cells, was deducted from the measured values. Error bars indicate the standard deviations and One-way ANOVA showed a statistical difference between the samples of p = 0.010. D) Uptake of the fluorescently labeled fatty acid and formation of lipid bodies was confirmed by confocal microscopy.
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pgen.1005190.g005: ESX-5 is involved in fatty acid uptake.A) Growth of indicated M. marinum strains on Tween-80 as a sole carbon source was assessed by measuring optical density at different time points. Depicted is the average of three biological replicates. Error bars indicate standard deviations. B) Uptake of a fluorescently labeled fatty acid after 72 hours of hypoxic growth was measured by FACS analysis. 20.000 events gated for similar size were acquired for WT::mspA (black), ΔmycP5::mspA (light grey) or ΔmycP5-C::mspA (dark grey). C) Quantification of FACS analysis. Mean fluorescent intensity of three experiments per strain was acquired by FACS. Background staining, quantified by adding the fluorescent fatty acid to an unstained culture one hour before washing the cells, was deducted from the measured values. Error bars indicate the standard deviations and One-way ANOVA showed a statistical difference between the samples of p = 0.010. D) Uptake of the fluorescently labeled fatty acid and formation of lipid bodies was confirmed by confocal microscopy.

Mentions: To test whether the ESX-5 system is involved in the uptake of nutrients, M. marinum-ΔmycP5::mspA and several control strains were grown in a modified 7H9 medium supplemented with different single carbon sources [51]. The mycP5 mutant grew almost as fast as the control strains in the presence of small hydrophilic carbon sources such as glucose, glycerol or acetate (S4 Fig). This was expected, as these strains contained the hydrophilic pore-forming MspA protein [37,38]. However, when the strains were grown on medium with Tween-80 (Fig 5A) or Tween-40 (S4 Fig) as sole carbon source, only the mycP5 deletion strain showed strongly reduced growth. Mycobacteria are able to hydrolyze Tween and use the fatty acid components as carbon source. However, it is also known that free fatty acids can be toxic for mycobacteria. To discriminate between these two possibilities we added 0.2% glucose to the cultures after 8 days of growth on Tween-80. This resulted in normal outgrowth of the mycP5 mutant, indicating that the Tween-80 present in the medium did not specifically hamper growth of this strain, but that these cells were still viable and therefore probably starved (S4 Fig). This result indicates a role for the ESX-5 system in either the (extracellular) hydrolysis of Tween-80 or the uptake of released oleic acid. To examine whether ESX-5 secreted substrates are involved in the breakdown of Tween-80, co-culture experiments using wild-type M. marinum and M. marinum-ΔmycP5::mspA were performed. Growth of the mycP5 mutant strain was not rescued by the presence of wild-type bacteria (S4 Fig), indicating that factors secreted to the culture filtrate do not play a role in the observed growth defect. This was further confirmed by testing the role of the ESX-5 dependent lipase LipY in the ability of M. marinum to grow on Tween-80. LipY is the most active and abundant lipase secreted via ESX-5 [52] and therefore a prime candidate for hydrolyzing Tween-80. However, an M. marinum lipY deletion mutant grew to a similar extent as the wild-type strain on medium with Tween-80 as a sole carbon source (S4 Fig), showing that LipY is not responsible for the ESX-5 dependent growth on Tween-80.


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)

ESX-5 is involved in fatty acid uptake.A) Growth of indicated M. marinum strains on Tween-80 as a sole carbon source was assessed by measuring optical density at different time points. Depicted is the average of three biological replicates. Error bars indicate standard deviations. B) Uptake of a fluorescently labeled fatty acid after 72 hours of hypoxic growth was measured by FACS analysis. 20.000 events gated for similar size were acquired for WT::mspA (black), ΔmycP5::mspA (light grey) or ΔmycP5-C::mspA (dark grey). C) Quantification of FACS analysis. Mean fluorescent intensity of three experiments per strain was acquired by FACS. Background staining, quantified by adding the fluorescent fatty acid to an unstained culture one hour before washing the cells, was deducted from the measured values. Error bars indicate the standard deviations and One-way ANOVA showed a statistical difference between the samples of p = 0.010. D) Uptake of the fluorescently labeled fatty acid and formation of lipid bodies was confirmed by confocal microscopy.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4418733&req=5

pgen.1005190.g005: ESX-5 is involved in fatty acid uptake.A) Growth of indicated M. marinum strains on Tween-80 as a sole carbon source was assessed by measuring optical density at different time points. Depicted is the average of three biological replicates. Error bars indicate standard deviations. B) Uptake of a fluorescently labeled fatty acid after 72 hours of hypoxic growth was measured by FACS analysis. 20.000 events gated for similar size were acquired for WT::mspA (black), ΔmycP5::mspA (light grey) or ΔmycP5-C::mspA (dark grey). C) Quantification of FACS analysis. Mean fluorescent intensity of three experiments per strain was acquired by FACS. Background staining, quantified by adding the fluorescent fatty acid to an unstained culture one hour before washing the cells, was deducted from the measured values. Error bars indicate the standard deviations and One-way ANOVA showed a statistical difference between the samples of p = 0.010. D) Uptake of the fluorescently labeled fatty acid and formation of lipid bodies was confirmed by confocal microscopy.
Mentions: To test whether the ESX-5 system is involved in the uptake of nutrients, M. marinum-ΔmycP5::mspA and several control strains were grown in a modified 7H9 medium supplemented with different single carbon sources [51]. The mycP5 mutant grew almost as fast as the control strains in the presence of small hydrophilic carbon sources such as glucose, glycerol or acetate (S4 Fig). This was expected, as these strains contained the hydrophilic pore-forming MspA protein [37,38]. However, when the strains were grown on medium with Tween-80 (Fig 5A) or Tween-40 (S4 Fig) as sole carbon source, only the mycP5 deletion strain showed strongly reduced growth. Mycobacteria are able to hydrolyze Tween and use the fatty acid components as carbon source. However, it is also known that free fatty acids can be toxic for mycobacteria. To discriminate between these two possibilities we added 0.2% glucose to the cultures after 8 days of growth on Tween-80. This resulted in normal outgrowth of the mycP5 mutant, indicating that the Tween-80 present in the medium did not specifically hamper growth of this strain, but that these cells were still viable and therefore probably starved (S4 Fig). This result indicates a role for the ESX-5 system in either the (extracellular) hydrolysis of Tween-80 or the uptake of released oleic acid. To examine whether ESX-5 secreted substrates are involved in the breakdown of Tween-80, co-culture experiments using wild-type M. marinum and M. marinum-ΔmycP5::mspA were performed. Growth of the mycP5 mutant strain was not rescued by the presence of wild-type bacteria (S4 Fig), indicating that factors secreted to the culture filtrate do not play a role in the observed growth defect. This was further confirmed by testing the role of the ESX-5 dependent lipase LipY in the ability of M. marinum to grow on Tween-80. LipY is the most active and abundant lipase secreted via ESX-5 [52] and therefore a prime candidate for hydrolyzing Tween-80. However, an M. marinum lipY deletion mutant grew to a similar extent as the wild-type strain on medium with Tween-80 as a sole carbon source (S4 Fig), showing that LipY is not responsible for the ESX-5 dependent growth on Tween-80.

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