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C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation.

ABSTRACT: The cytoplasmic C-ring of the flagellum consists of FliG, FliM and FliN and acts as an affinity cup to localize secretion substrates for protein translocation via the flagellar-specific type III secretion system. Random T-POP transposon mutagenesis was employed to screen for insertion mutants that allowed flagellar type III secretion in the absence of the C-ring using the flagellar type III secretion system-specific hook-beta-lactamase reporter (Lee and Hughes, 2006). Any condition resulting in at least a twofold increase in flhDC expression was sufficient to overcome the requirement for the C-ring and the ATPase complex FliHIJ in flagellar type III secretion. Insertions in known and unknown flagellar regulatory loci were isolated as well as chromosomal duplications of the flhDC region. The twofold increased flhDC mRNA level coincided in a twofold increase in the number of hook-basal bodies per cell as analysed by fluorescent microscopy. These results indicate that the C-ring functions as a nonessential affinity cup-like structure during flagellar type III secretion to enhance the specificity and efficiency of the secretion process.

SUBMITTER: Erhardt M

PROVIDER: S-EPMC3194100 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation.

Erhardt Marc M Hughes Kelly T KT

Molecular microbiology 20091117 2

The cytoplasmic C-ring of the flagellum consists of FliG, FliM and FliN and acts as an affinity cup to localize secretion substrates for protein translocation via the flagellar-specific type III secretion system. Random T-POP transposon mutagenesis was employed to screen for insertion mutants that allowed flagellar type III secretion in the absence of the C-ring using the flagellar type III secretion system-specific hook-beta-lactamase reporter (Lee and Hughes, 2006). Any condition resulting in ...[more]

PMID: 19919668

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Project description:UnlabelledThe opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa.ImportanceThe inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

Project description:BACKGROUND:Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant. RESULTS:We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5'UTR and first 47 amino acidsof FliC from E. coli (but no 3'UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L-1, in low cell density culture. CONCLUSIONS:We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.

Dataset Information

C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation.

Publications

C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation.

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