Dataset Information

Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates.

ABSTRACT: A remarkable feature of the flagellar-specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook-basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N-terminal secretion signal, mRNA secretion signals within the 5'-untranslated region (5'-UTR) and for late substrates, piloting proteins known as the T3S chaperones. Here, we utilized translational ?-lactamase fusions to probe the secretion efficacy of the N-terminal secretion signal of fourteen secreted flagellar substrates in Salmonella enterica. We observed a surprising variety in secretion capability between flagellar proteins of the same secretory class. The peptide secretion signals of the early-type substrates FlgD, FlgF, FlgE and the late-type substrate FlgL were analysed in detail. Analysing the role of the 5'-UTR in secretion of flgB and flgE revealed that the native 5'-UTR substantially enhanced protein translation and secretion. Based on our data, we propose a multicomponent signal that drives secretion via the flagellar T3SS. Both mRNA and peptide signals are recognized by the export apparatus and together with substrate-specific chaperones allowing for targeted secretion of flagellar substrates.

SUBMITTER: Singer HM

PROVIDER: S-EPMC5076378 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates.

Singer Hanna M HM Erhardt Marc M Hughes Kelly T KT

Molecular microbiology 20140706 3

A remarkable feature of the flagellar-specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook-basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N-terminal secretion signal, mRNA secretion signals within the 5'-untranslated region (5'-U ...[more]

PMID: 24946091

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Project description:The 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.The 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

Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates.

Publications

Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates.

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