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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.

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Lengths of flagellar filaments in fliHIJ mutants.Plot showing the lengths of individual flagellar filaments of the fliHIJ mutants visualized by anti-FliC immunostaining in Figure 3 and Figure 4. The average lengths of flagellar filaments +/− standard deviation and the number of measured filaments are presented in the upper part of the graph.
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pgen-1004800-g006: Lengths of flagellar filaments in fliHIJ mutants.Plot showing the lengths of individual flagellar filaments of the fliHIJ mutants visualized by anti-FliC immunostaining in Figure 3 and Figure 4. The average lengths of flagellar filaments +/− standard deviation and the number of measured filaments are presented in the upper part of the graph.

Mentions: The cytoplasmic C-ring of the flagellum (composed of FliG, FliM, FliN) is essential for efficient localization of flagellar secretion substrates to the vicinity of the export gate by functioning as an affinity site for the localization of substrate/ATPase complexes. We have previously shown that a defect in the C-ring structure can be bypassed by providing an excess of secretion substrate [28]. Mutations in flgM, clpX and the fliAH14D (FlgM-bypass) allele result in increased secretion substrate levels. We tested the effects of increased substrate levels on secretion via the flagellar type-III secretion apparatus in the absence of the FliHIJ ATPase complex. In various fliHIJ mutant backgrounds mutations of flgM, clpX or fliAH14D increased the frequency of bacteria able to produce at least one flagellum up to 35% of the population (Fig. 3C, Fig. 4). The deletion of flgM and atpA enhanced the frequency of flagellar assembly to a maximum of 74% of the population in the fliHI mutant background (Figs. 3C and 3D). The absence of the ATP synthase increased flagellar filament formation in the clpX or fliAH14D backgrounds to 55% and 36%, respectively (Figs. 4A and 4B). A significant increase in secreted flagellin was also observed in the flgM, clpX or fliAH14D mutant backgrounds under conditions when the PMF was apparently elevated by the atpA deletion (Fig. 5). We additionally measured the lengths of flagellar filaments visualized by flagellin immunostaining (Figs. 3 and 4) and observed that the average lengths of flagellar filaments of the various fliHIJ mutants is as long or longer than the wildtype filament lengths. The average lengths of filaments were also increased in an otherwise wildtype background if an excess of secretion substrates (e.g. by deleting the negative regulator flgM) was provided or the PMF was increased by deletion of the atpA subunit (Fig. 6).


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

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

Lengths of flagellar filaments in fliHIJ mutants.Plot showing the lengths of individual flagellar filaments of the fliHIJ mutants visualized by anti-FliC immunostaining in Figure 3 and Figure 4. The average lengths of flagellar filaments +/− standard deviation and the number of measured filaments are presented in the upper part of the graph.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004800-g006: Lengths of flagellar filaments in fliHIJ mutants.Plot showing the lengths of individual flagellar filaments of the fliHIJ mutants visualized by anti-FliC immunostaining in Figure 3 and Figure 4. The average lengths of flagellar filaments +/− standard deviation and the number of measured filaments are presented in the upper part of the graph.
Mentions: The cytoplasmic C-ring of the flagellum (composed of FliG, FliM, FliN) is essential for efficient localization of flagellar secretion substrates to the vicinity of the export gate by functioning as an affinity site for the localization of substrate/ATPase complexes. We have previously shown that a defect in the C-ring structure can be bypassed by providing an excess of secretion substrate [28]. Mutations in flgM, clpX and the fliAH14D (FlgM-bypass) allele result in increased secretion substrate levels. We tested the effects of increased substrate levels on secretion via the flagellar type-III secretion apparatus in the absence of the FliHIJ ATPase complex. In various fliHIJ mutant backgrounds mutations of flgM, clpX or fliAH14D increased the frequency of bacteria able to produce at least one flagellum up to 35% of the population (Fig. 3C, Fig. 4). The deletion of flgM and atpA enhanced the frequency of flagellar assembly to a maximum of 74% of the population in the fliHI mutant background (Figs. 3C and 3D). The absence of the ATP synthase increased flagellar filament formation in the clpX or fliAH14D backgrounds to 55% and 36%, respectively (Figs. 4A and 4B). A significant increase in secreted flagellin was also observed in the flgM, clpX or fliAH14D mutant backgrounds under conditions when the PMF was apparently elevated by the atpA deletion (Fig. 5). We additionally measured the lengths of flagellar filaments visualized by flagellin immunostaining (Figs. 3 and 4) and observed that the average lengths of flagellar filaments of the various fliHIJ mutants is as long or longer than the wildtype filament lengths. The average lengths of filaments were also increased in an otherwise wildtype background if an excess of secretion substrates (e.g. by deleting the negative regulator flgM) was provided or the PMF was increased by deletion of the atpA subunit (Fig. 6).

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