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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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FliH functions as a negative regulator of type-III protein translocation.Export of FlgE-Bla fusion protein into the periplasm was analyzed in various fliHIJ deletion strains. All strains additionally harbored a deletion of the proximal rod genes (ΔflgBC6557) and the FlgE-Bla fusion protein under its native promoter (flgE6569::bla). (A) Minimal inhibitory concentration (MIC) values with flagellar genes expressed at normal levels. (B) Summary of MIC values with flagellar genes expressed at elevated levels due to a PflhD promoter-up mutation (PflhD* = (P1+P4 -10 TATAAT)). The error bars represent the standard error of the mean (SEM) and biological replicates are shown as individual data points.
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pgen-1004800-g007: FliH functions as a negative regulator of type-III protein translocation.Export of FlgE-Bla fusion protein into the periplasm was analyzed in various fliHIJ deletion strains. All strains additionally harbored a deletion of the proximal rod genes (ΔflgBC6557) and the FlgE-Bla fusion protein under its native promoter (flgE6569::bla). (A) Minimal inhibitory concentration (MIC) values with flagellar genes expressed at normal levels. (B) Summary of MIC values with flagellar genes expressed at elevated levels due to a PflhD promoter-up mutation (PflhD* = (P1+P4 -10 TATAAT)). The error bars represent the standard error of the mean (SEM) and biological replicates are shown as individual data points.

Mentions: It has been suggested that the regulatory subunit of the ATPase complex, FliH, blocks the entrance gate of the secretion channel in the absence of its cognate FliI ATPase subunit [15], [16], [34]. In order to dissect the contribution of individual components of the FliHIJ complex to the type-III export process across the cytoplasmic membrane, we utilized a hook protein - β-lactamase based reporter system (FlgE-Bla). In the absence of the proximal rod subunits FlgB and FlgC, the FlgE-Bla fusion protein is secreted into the periplasm [28], [35], conferring quantifiable resistance to Ampicillin. We analyzed the export capability of various single and combination mutants of fliH, fliI and fliJ by determining the minimal inhibitory concentration (MIC) against Ampicillin. A deletion of the inner membrane scaffold protein, FliF, was used as a negative control and the MIC of the fliF mutant represents the basal level of Ampicillin resistance in this assay. Single deletion mutants of fliH and fliJ substantially increased the MIC about 3.5-fold compared to the fliF control strain (Fig. 7A). This is consistent with the previously reported leaky-motility phenotype of fliH and fliJ mutants [22], [26]. A deletion mutant of fliI also displayed increased MIC values, albeit significantly less than either fliH or fliJ strains. Interestingly, if the fliI mutation was combined with either the fliH or fliHJ, the MIC values reached the level of the single fliH or fliJ strains. Importantly, if only fliH was retained and both fliI and fliJ were deleted, the export capability was reduced to the level of the single fliI deletion. Under excess FlgE-Bla reporter construct conditions, secretion was also restored in the fliI single deletion mutant as MIC levels were 3.5-fold higher compared to the fliF control (Fig. 7B). The MIC values of the fliH and fliJ single mutants were 11 and 6.5-fold increased, respectively. Similar as under physiological FlgE-Bla substrate conditions, the MIC levels were increased 9 and 6-fold in fliHI or fliHIJ combinatory mutations to the levels of the single fliH and fliJ mutants. If only FliH was retained (ΔfliIJ), the MIC values were decreased to the level of the single fliI mutant. These results demonstrate that FliH functions as an inhibitor of the type-III export process. However, significant export can occur in the absence of both the negative regulator FliH and the ATPase FliI if excess substrate is provided, further adding evidence to our results that fT3SS can efficiently occur without ATP hydrolysis by FliI.


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

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

FliH functions as a negative regulator of type-III protein translocation.Export of FlgE-Bla fusion protein into the periplasm was analyzed in various fliHIJ deletion strains. All strains additionally harbored a deletion of the proximal rod genes (ΔflgBC6557) and the FlgE-Bla fusion protein under its native promoter (flgE6569::bla). (A) Minimal inhibitory concentration (MIC) values with flagellar genes expressed at normal levels. (B) Summary of MIC values with flagellar genes expressed at elevated levels due to a PflhD promoter-up mutation (PflhD* = (P1+P4 -10 TATAAT)). The error bars represent the standard error of the mean (SEM) and biological replicates are shown as individual data points.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004800-g007: FliH functions as a negative regulator of type-III protein translocation.Export of FlgE-Bla fusion protein into the periplasm was analyzed in various fliHIJ deletion strains. All strains additionally harbored a deletion of the proximal rod genes (ΔflgBC6557) and the FlgE-Bla fusion protein under its native promoter (flgE6569::bla). (A) Minimal inhibitory concentration (MIC) values with flagellar genes expressed at normal levels. (B) Summary of MIC values with flagellar genes expressed at elevated levels due to a PflhD promoter-up mutation (PflhD* = (P1+P4 -10 TATAAT)). The error bars represent the standard error of the mean (SEM) and biological replicates are shown as individual data points.
Mentions: It has been suggested that the regulatory subunit of the ATPase complex, FliH, blocks the entrance gate of the secretion channel in the absence of its cognate FliI ATPase subunit [15], [16], [34]. In order to dissect the contribution of individual components of the FliHIJ complex to the type-III export process across the cytoplasmic membrane, we utilized a hook protein - β-lactamase based reporter system (FlgE-Bla). In the absence of the proximal rod subunits FlgB and FlgC, the FlgE-Bla fusion protein is secreted into the periplasm [28], [35], conferring quantifiable resistance to Ampicillin. We analyzed the export capability of various single and combination mutants of fliH, fliI and fliJ by determining the minimal inhibitory concentration (MIC) against Ampicillin. A deletion of the inner membrane scaffold protein, FliF, was used as a negative control and the MIC of the fliF mutant represents the basal level of Ampicillin resistance in this assay. Single deletion mutants of fliH and fliJ substantially increased the MIC about 3.5-fold compared to the fliF control strain (Fig. 7A). This is consistent with the previously reported leaky-motility phenotype of fliH and fliJ mutants [22], [26]. A deletion mutant of fliI also displayed increased MIC values, albeit significantly less than either fliH or fliJ strains. Interestingly, if the fliI mutation was combined with either the fliH or fliHJ, the MIC values reached the level of the single fliH or fliJ strains. Importantly, if only fliH was retained and both fliI and fliJ were deleted, the export capability was reduced to the level of the single fliI deletion. Under excess FlgE-Bla reporter construct conditions, secretion was also restored in the fliI single deletion mutant as MIC levels were 3.5-fold higher compared to the fliF control (Fig. 7B). The MIC values of the fliH and fliJ single mutants were 11 and 6.5-fold increased, respectively. Similar as under physiological FlgE-Bla substrate conditions, the MIC levels were increased 9 and 6-fold in fliHI or fliHIJ combinatory mutations to the levels of the single fliH and fliJ mutants. If only FliH was retained (ΔfliIJ), the MIC values were decreased to the level of the single fliI mutant. These results demonstrate that FliH functions as an inhibitor of the type-III export process. However, significant export can occur in the absence of both the negative regulator FliH and the ATPase FliI if excess substrate is provided, further adding evidence to our results that fT3SS can efficiently occur without ATP hydrolysis by FliI.

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