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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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Frequency of flagellar filament formation of fliHIJ mutants is increased in atpA and flgM  backgrounds.The absence of the flagellar ATPase subunits FliH, FliI and FliJ results in a non-flagellated phenotype (A). Additional deletions in atpA (B) and flgM (C) substantially increase the frequency of flagellar filament formation of fliI, fliHI and fliHIJ mutant strains. Flagellar formation in the flgM  background is further enhanced by combination with the atpA mutation (D). Top: A montage of representative fluorescent microscopy images is shown. Flagellar filaments were stained using anti-FliC immunostaining and detected by FITC-coupled secondary antibodies (green), DNA was stained using Hoechst (blue) and cell membranes using FM-64 (red). Scale bar 2 µm. The percentage of cells with at least one filament is presented in the upper left corner. Bottom: Histogram of counted flagellar filaments per cell body. Number of counted cells and average number of filaments per cell +/− standard deviation based on Gaussian non-linear regression analysis is given in the upper right hand corner.
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pgen-1004800-g003: Frequency of flagellar filament formation of fliHIJ mutants is increased in atpA and flgM backgrounds.The absence of the flagellar ATPase subunits FliH, FliI and FliJ results in a non-flagellated phenotype (A). Additional deletions in atpA (B) and flgM (C) substantially increase the frequency of flagellar filament formation of fliI, fliHI and fliHIJ mutant strains. Flagellar formation in the flgM background is further enhanced by combination with the atpA mutation (D). Top: A montage of representative fluorescent microscopy images is shown. Flagellar filaments were stained using anti-FliC immunostaining and detected by FITC-coupled secondary antibodies (green), DNA was stained using Hoechst (blue) and cell membranes using FM-64 (red). Scale bar 2 µm. The percentage of cells with at least one filament is presented in the upper left corner. Bottom: Histogram of counted flagellar filaments per cell body. Number of counted cells and average number of filaments per cell +/− standard deviation based on Gaussian non-linear regression analysis is given in the upper right hand corner.

Mentions: The type-III secretion apparatus is a PMF-powered protein exporter and we reasoned that increased PMF due to the atpA deletion provided additional energy to overcome the requirement of the flagellar ATPase complex for flagellar assembly. Flagellar filament assembly will only occur in the presence of a functional and efficiently working fT3SS. Deletion mutants of fliI, fliHI and fliHIJ were non-flagellated (Fig. 3A). The atpA deletion resulted in a small increase in the frequency of bacteria that produced functional flagella in a fliI and fliHIJ deletion strain up to approximately 4% of the population. However, the absence of atpA in a fliHI deletion mutant increased the frequency of flagella formation to about 10% of the bacteria (Fig. 3B). There appears to exist an important bottleneck in flagellar assembly that was overcome by the increase in PMF in the atpA deletion mutants. We observed formation of only one, singular filament in cases where flagellar assembly could proceed beyond completion of a hook-basal body complex. It has been shown previously that under wildtype conditions about 90% of detectable HBBs had a filament attached. Thus, virtually every secretion competent HBB had switched to late substrate secretion mode [32]. Since we observed only single flagellar filaments of wildtype lengths in our fliHI mutants (Fig. 3B and Fig. S6), it is a possibility that a localization mechanism exists that preferentially targets late flagellar substrates to a secretion system that is in late-type secretion mode.


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

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

Frequency of flagellar filament formation of fliHIJ mutants is increased in atpA and flgM  backgrounds.The absence of the flagellar ATPase subunits FliH, FliI and FliJ results in a non-flagellated phenotype (A). Additional deletions in atpA (B) and flgM (C) substantially increase the frequency of flagellar filament formation of fliI, fliHI and fliHIJ mutant strains. Flagellar formation in the flgM  background is further enhanced by combination with the atpA mutation (D). Top: A montage of representative fluorescent microscopy images is shown. Flagellar filaments were stained using anti-FliC immunostaining and detected by FITC-coupled secondary antibodies (green), DNA was stained using Hoechst (blue) and cell membranes using FM-64 (red). Scale bar 2 µm. The percentage of cells with at least one filament is presented in the upper left corner. Bottom: Histogram of counted flagellar filaments per cell body. Number of counted cells and average number of filaments per cell +/− standard deviation based on Gaussian non-linear regression analysis is given in the upper right hand corner.
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pgen-1004800-g003: Frequency of flagellar filament formation of fliHIJ mutants is increased in atpA and flgM backgrounds.The absence of the flagellar ATPase subunits FliH, FliI and FliJ results in a non-flagellated phenotype (A). Additional deletions in atpA (B) and flgM (C) substantially increase the frequency of flagellar filament formation of fliI, fliHI and fliHIJ mutant strains. Flagellar formation in the flgM background is further enhanced by combination with the atpA mutation (D). Top: A montage of representative fluorescent microscopy images is shown. Flagellar filaments were stained using anti-FliC immunostaining and detected by FITC-coupled secondary antibodies (green), DNA was stained using Hoechst (blue) and cell membranes using FM-64 (red). Scale bar 2 µm. The percentage of cells with at least one filament is presented in the upper left corner. Bottom: Histogram of counted flagellar filaments per cell body. Number of counted cells and average number of filaments per cell +/− standard deviation based on Gaussian non-linear regression analysis is given in the upper right hand corner.
Mentions: The type-III secretion apparatus is a PMF-powered protein exporter and we reasoned that increased PMF due to the atpA deletion provided additional energy to overcome the requirement of the flagellar ATPase complex for flagellar assembly. Flagellar filament assembly will only occur in the presence of a functional and efficiently working fT3SS. Deletion mutants of fliI, fliHI and fliHIJ were non-flagellated (Fig. 3A). The atpA deletion resulted in a small increase in the frequency of bacteria that produced functional flagella in a fliI and fliHIJ deletion strain up to approximately 4% of the population. However, the absence of atpA in a fliHI deletion mutant increased the frequency of flagella formation to about 10% of the bacteria (Fig. 3B). There appears to exist an important bottleneck in flagellar assembly that was overcome by the increase in PMF in the atpA deletion mutants. We observed formation of only one, singular filament in cases where flagellar assembly could proceed beyond completion of a hook-basal body complex. It has been shown previously that under wildtype conditions about 90% of detectable HBBs had a filament attached. Thus, virtually every secretion competent HBB had switched to late substrate secretion mode [32]. Since we observed only single flagellar filaments of wildtype lengths in our fliHI mutants (Fig. 3B and Fig. S6), it is a possibility that a localization mechanism exists that preferentially targets late flagellar substrates to a secretion system that is in late-type secretion mode.

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