Localization of the flagellum-specific secretion signal in Salmonella flagellin

Végh, Barbara M.; Gál, Péter; Dobó, József; Závodszky, Péter; Vonderviszt, Ferenc

doi:10.1016/j.bbrc.2006.04.055

Cited by 40 publications

(51 citation statements)

References 18 publications

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“…The secretion signal for substrates of the injectisomeand flagellum-T3SSs is typically located within the first 15 to 20 amino-terminal residues (35)(36)(37). To examine the requirements for FliC secretion, we fused full-length FliC (FliC 1-394 -CyaA) or the first 20 amino-terminal residues of FliC (FliC 1-20 -CyaA) to a truncated form of the calmodulin-dependent adenylate cyclase domain (ϳ50 kDa) from Bordetella pertussis CyaA.…”

Section: Resultsmentioning

confidence: 99%

Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System

2015

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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. 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. Innate immunity constitutes the first line of defense against invading microorganisms and functions, in part, by detecting pathogen-and danger-associated molecular patterns (PAMPs and DAMPs, respectively) and activating host defense mechanisms (1, 2). PAMPs and DAMPs are detected through a diverse array of pattern recognition receptors (PRRs) that may be secreted, surface exposed, or localized to various intracellular compartments. The nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) represent a large family of intracellular PRRs (3). NLR receptors detect a variety of endogenous and exogenous signals that include double-stranded DNA (dsDNA), extracellular ATP, uric acid crystals, peptidoglycan, and bacterial flagellin...

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Section: Resultsmentioning

confidence: 99%

Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System

2015

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“…Successful secretion of flagellin through the flagellar basal body requires the ND0 domain, which contains a secretion signal (51), and the CD0 region, which binds chaperone FliS to inhibit cytosolic flagellin polymerization (52,53). None of the chimeric flagellins constructed in this study have alterations in either the putative secretion signal in domain ND0 or in domain CD0, and the chimeras are, thus, predicted to bind C. jejuni FliS in a similar fashion as the wild type flagellin.…”

Section: Discussionmentioning

confidence: 95%

Reconstitution of a Functional Toll-like Receptor 5 Binding Site in Campylobacter jejuni Flagellin

Zoete

Keestra

Wagenaar

et al. 2010

Journal of Biological Chemistry

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Bacterial flagellin is important for intestinal immune homeostasis. Flagellins from most species activate Toll-like receptor 5 (TLR5). The principal bacterial food-borne pathogen Campylobacter jejuni escapes TLR5 recognition, probably due to an alternate flagellin subunit structure. We investigated the molecular basis of TLR5 evasion by aiming to reconstitute TLR5 stimulating activity in live C. jejuni. Both native glycosylated C. jejuni flagellins (FlaA and FlaB) and recombinant proteins purified from Escherichia coli failed to activate NF-B in HEK293 cells expressing TLR5. Introduction of multiple defined regions from Salmonella flagellin into C. jejuni FlaA via a recombinatorial approach revealed three regions critical for the activation of human and mouse TLR5, including a ␤-hairpin structure not previously implicated in TLR5 recognition. Surprisingly, this domain was not required for the activation of chicken TLR5, indicating a selective requirement for the ␤-hairpin in the recognition of mammalian TLR5. Expression of the active chimeric protein in C. jejuni resulted in secreted glycosylated flagellin that induced a potent TLR5 response. Overall, our results reveal a novel structural requirement for TLR5 recognition of bacterial flagellin and exclude flagellin glycosylation as an additional mechanism of bacterial evasion of the TLR5 response.Flagellin, the monomeric subunit of the bacterial motility apparatus, is the natural ligand of the innate immune sensor Toll-like receptor 5 (TLR5) 3 (1). Activation of TLR5 by flagellin initiates a powerful host response that provides crucial signals for maintaining intestinal immune homeostasis (2, 3). The immunostimulatory properties make flagellin an attractive vaccine carrier protein and potent vaccine adjuvant. Its intrinsic adjuvant activity is currently being employed in experimental recombinant vaccines against human influenza, West Nile fever, malaria, tuberculosis, and plague (4 -9). In addition, flagellin-induced immune activation protects the intestine and other tissues against lethal irradiation due to potent TLR5-mediated anti-apoptotic effects (10, 11).The immunological impact of flagellin stimulation has driven several bacterial pathogens to evolve mechanisms to escape the effective TLR5-mediated host defense. In Salmonella enterica serotype Typhi, this is achieved by repression of flagellin expression and secretion (12), whereas Listeria shuts off flagellin expression at the host temperature of 37°C (13). The flagellins of the ␣-and ⑀-proteobacteria, which include the major food-borne pathogen Campylobacter jejuni and the gastric pathogen Helicobacter pylori, fail to activate TLR5 altogether (14 -18). For these organisms the consequences of TLR5 evasion for infection are currently unknown. It has been demonstrated that purified H. pylori flagellin induces severely impaired adaptive immune responses in comparison to TLR5-activating flagellins (19).The flagellin protein of C. jejuni clearly differs from bacterial flagellins that do activate TLR5. Electro...

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“…The first 48 amino acids are sufficient for secretion of FliC into Salmonella culture supernatants (30). We therefore tested whether the FliC secretion domain is sufficient for its translocation into infected host cells.…”

Section: Resultsmentioning

confidence: 99%

Injection of Flagellin into the Host Cell Cytosol by Salmonella enterica Serotype Typhimurium

Sun¹,

Rolán²,

Tsolis³

2007

Journal of Biological Chemistry

156

150

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Bacterial flagella are filamentous appendages on the cell surface that mediate bacterial motility. The filament of the flagellum is composed of ϳ20,000 flagellin subunits (1). Flagellin monomers of many bacterial species function as pathogen-associated molecular patterns and can be detected by host cells using both surface-localized toll-like receptor 5 (TLR5) 2 and cytosolic Nod-like receptors (2-6). The interaction of flagellin with TLR5 at the cell surface has been characterized in detail, identifying the structural basis for the TLR5-flagellin interaction (7,8). TLR5 sensing has been shown to be important in Legionnaires disease, a respiratory tract infection caused by the bacterium Legionella pneumophila, since human TLR5 polymorphisms correlated with disease susceptibility (7,8). In the intestine, TLR5 localizes to the basolateral surface of enterocytes and to CD11c ϩ lamina propria dendritic cells (9, 10). In these locations, TLR5 is thought to be involved in sensing a breach of the intestinal mucosa by enteroinvasive pathogens, triggering an inflammatory response that limits systemic spread of the bacteria (11).In addition to sensing by TLRs, flagellin was recently shown to be sensed by two different Nod-like receptors, Ipaf and Birc1e (also known as Naip5) (2, 4 -6, 12). Ipaf transmits a proinflammatory signal in response to flagellin by activating the inflammasome, resulting in caspase 1-dependent activation of Il-1␤, which is accompanied by cell death (pyroptosis) (13-17). Birc1e/Naip5 signals in response to flagellin, restricting intracellular growth of L. pneumophila by antagonizing the ability of the bacterium to avoid fusion with lysosomes and form a replicative phagosome (18). Several lines of evidence suggest that pathogens residing in a membrane-bound compartment of the cell, such as L. pneumophila and S. Typhimurium (Salmonella enterica serotype Typhimurium), may release flagellins from their vacuole into the host cytosol, which triggers signaling via Ipaf and Birc1e. First, S. Typhimurium and L. pneumophila mutants lacking flagellin elicit reduced caspase activation, IL-1␤ secretion, and cytotoxicity in macrophages (2, 4 -6, 19). Second, mutants of S. Typhimurium lacking the SPI-1 T3SS and L. pneumophila mutants lacking a functional Type IV secretion system (T4SS) fail to trigger Ipaf-dependent signaling (4,5,19). Third, introduction of purified flagellins into the cytosol of macrophages using pore-forming toxins or detergents or by heterologous expression in Escherichia coli expressing listeriolysin O triggers caspase-1 activation and IL-1␤ activation by an Ipaf-dependent mechanism (2, 4 -6, 20).Although injection of effector proteins into host cells by the S. Typhimurium SPI-1 T3SS has been visualized microscopically (21, 22), it has not yet been demonstrated that in infected cells, bacteria are able to translocate flagellin from a membrane-bound compartment into the cytosol. In this report, we show that S. Typhimurium translocates flagellin into the cytosol of infected cells by a process t...

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Localization of the flagellum-specific secretion signal in Salmonella flagellin

Cited by 40 publications

References 18 publications

Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System

Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System

Reconstitution of a Functional Toll-like Receptor 5 Binding Site in Campylobacter jejuni Flagellin

Injection of Flagellin into the Host Cell Cytosol by Salmonella enterica Serotype Typhimurium

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