Limits...
ATPase-independent type-III protein secretion in Salmonella enterica.

Erhardt M, Mertens ME, Fabiani FD, Hughes KT - PLoS Genet. (2014)

Bottom Line: Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex.We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion.Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.

View Article: PubMed Central - PubMed

Affiliation: Junior Research Group Infection Biology of Salmonella, Helmholtz Centre for Infection Research, Braunschweig, Germany.

ABSTRACT
Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.

Show MeSH

Related in: MedlinePlus

Protein secretion via the vT3SS in a catalytically-inactive ATPase mutant strain is rescued by deletion of atpA.Secretion of the Spi1 vT3SS substrate InvJ or a 3×HA tagged InvJ variant. (A) Secreted InvJ protein in the wildtype, ΔinvJ, ΔatpA, the catalytically-inactive Spi1 ATPase invCK165E, invCK165E ΔatpA, invCK165E ΔflgM, and invCK165E ΔflgM ΔfliHIJ ΔssaN ΔatpA mutant strains. Detection of DnaK protein was included as a cell lysis control. (B) Levels of secreted InvJ::3×HA protein in the wildtype, ΔinvJ::3×HA, ΔinvJ::3×HA ΔatpA, ΔinvJ::3×HA invCK165E, and ΔinvJ::3×HA invCK165E ΔatpA mutant strains. 288 ng BSA was added to each supernatant fractions (except for lane 2) and served as a precipitation control. DnaK protein served as a cell lysis control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230889&req=5

pgen-1004800-g008: Protein secretion via the vT3SS in a catalytically-inactive ATPase mutant strain is rescued by deletion of atpA.Secretion of the Spi1 vT3SS substrate InvJ or a 3×HA tagged InvJ variant. (A) Secreted InvJ protein in the wildtype, ΔinvJ, ΔatpA, the catalytically-inactive Spi1 ATPase invCK165E, invCK165E ΔatpA, invCK165E ΔflgM, and invCK165E ΔflgM ΔfliHIJ ΔssaN ΔatpA mutant strains. Detection of DnaK protein was included as a cell lysis control. (B) Levels of secreted InvJ::3×HA protein in the wildtype, ΔinvJ::3×HA, ΔinvJ::3×HA ΔatpA, ΔinvJ::3×HA invCK165E, and ΔinvJ::3×HA invCK165E ΔatpA mutant strains. 288 ng BSA was added to each supernatant fractions (except for lane 2) and served as a precipitation control. DnaK protein served as a cell lysis control.

Mentions: The ATPase InvC is associated with the vT3SS encoded by Spi1 and essential for Spi1 vT3SS function and virulence [17], [36]. Previously, loss-of-function mutations in InvC have been isolated that specifically target membrane association, oligomerization and catalytic activity of the ATPase [37]. Here, we employed the catalytically-inactive InvCK165E mutant to elucidate the role of ATPase activity in protein secretion via the Spi1 vT3SS. The InvCK165E mutant strain secreted significantly less InvJ substrate protein, however the defect in secretion was bypassed by a mutation in the atpA subunit of ATP synthase (Fig. 8A). Importantly, the fT3SS-associated ATPase FliI did not complement InvC function, as a fliHIJ deletion in the InvCK165E ΔatpA mutant strain did not abolish InvJ secretion. Similar results were obtained using a 3×HA-tagged InvJ substrate protein (Fig. 8B).


ATPase-independent type-III protein secretion in Salmonella enterica.

Erhardt M, Mertens ME, Fabiani FD, Hughes KT - PLoS Genet. (2014)

Protein secretion via the vT3SS in a catalytically-inactive ATPase mutant strain is rescued by deletion of atpA.Secretion of the Spi1 vT3SS substrate InvJ or a 3×HA tagged InvJ variant. (A) Secreted InvJ protein in the wildtype, ΔinvJ, ΔatpA, the catalytically-inactive Spi1 ATPase invCK165E, invCK165E ΔatpA, invCK165E ΔflgM, and invCK165E ΔflgM ΔfliHIJ ΔssaN ΔatpA mutant strains. Detection of DnaK protein was included as a cell lysis control. (B) Levels of secreted InvJ::3×HA protein in the wildtype, ΔinvJ::3×HA, ΔinvJ::3×HA ΔatpA, ΔinvJ::3×HA invCK165E, and ΔinvJ::3×HA invCK165E ΔatpA mutant strains. 288 ng BSA was added to each supernatant fractions (except for lane 2) and served as a precipitation control. DnaK protein served as a cell lysis control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230889&req=5

pgen-1004800-g008: Protein secretion via the vT3SS in a catalytically-inactive ATPase mutant strain is rescued by deletion of atpA.Secretion of the Spi1 vT3SS substrate InvJ or a 3×HA tagged InvJ variant. (A) Secreted InvJ protein in the wildtype, ΔinvJ, ΔatpA, the catalytically-inactive Spi1 ATPase invCK165E, invCK165E ΔatpA, invCK165E ΔflgM, and invCK165E ΔflgM ΔfliHIJ ΔssaN ΔatpA mutant strains. Detection of DnaK protein was included as a cell lysis control. (B) Levels of secreted InvJ::3×HA protein in the wildtype, ΔinvJ::3×HA, ΔinvJ::3×HA ΔatpA, ΔinvJ::3×HA invCK165E, and ΔinvJ::3×HA invCK165E ΔatpA mutant strains. 288 ng BSA was added to each supernatant fractions (except for lane 2) and served as a precipitation control. DnaK protein served as a cell lysis control.
Mentions: The ATPase InvC is associated with the vT3SS encoded by Spi1 and essential for Spi1 vT3SS function and virulence [17], [36]. Previously, loss-of-function mutations in InvC have been isolated that specifically target membrane association, oligomerization and catalytic activity of the ATPase [37]. Here, we employed the catalytically-inactive InvCK165E mutant to elucidate the role of ATPase activity in protein secretion via the Spi1 vT3SS. The InvCK165E mutant strain secreted significantly less InvJ substrate protein, however the defect in secretion was bypassed by a mutation in the atpA subunit of ATP synthase (Fig. 8A). Importantly, the fT3SS-associated ATPase FliI did not complement InvC function, as a fliHIJ deletion in the InvCK165E ΔatpA mutant strain did not abolish InvJ secretion. Similar results were obtained using a 3×HA-tagged InvJ substrate protein (Fig. 8B).

Bottom Line: Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex.We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion.Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.

View Article: PubMed Central - PubMed

Affiliation: Junior Research Group Infection Biology of Salmonella, Helmholtz Centre for Infection Research, Braunschweig, Germany.

ABSTRACT
Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.

Show MeSH
Related in: MedlinePlus