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On the road to structure-based development of anti-virulence therapeutics targeting the type III secretion system injectisome.

ABSTRACT: The type III secretion system injectisome is a syringe-like multimembrane spanning nanomachine that is essential to the pathogenicity but not viability of many clinically relevant Gram-negative bacteria, such as enteropathogenic Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa. Due to the rise in antibiotic resistance, new strategies must be developed to treat the growing spectre of drug resistant infections. Targeting the injectisome via an 'anti-virulence strategy' is a promising avenue to pursue as an alternative to the more commonly used bactericidal therapeutics, which have a high propensity for resulting resistance development and often more broad killing profile, including unwanted side effects in eliminating favourable members of the microbiome. Building on more than a decade of crystallographic work of truncated or isolated forms of the more than two dozen components of the secretion apparatus, recent advances in the field of single-particle cryo-electron microscopy have allowed for the elucidation of atomic resolution structures for many of the type III secretion system components in their assembled, oligomerized state including the needle complex, export apparatus and ATPase. Cryo-electron tomography studies have also advanced our understanding of the direct pathogen-host interaction between the type III secretion system translocon and host cell membrane. These new structural works that further our understanding of the myriad of protein-protein interactions that promote injectisome function will be highlighted in this review, with a focus on those that yield promise for future anti-virulence drug discovery and design. Recently developed inhibitors, including both synthetic, natural product and peptide inhibitors, as well as promising new developments of immunotherapeutics will be discussed. As our understanding of this intricate molecular machinery advances, the development of anti-virulence inhibitors can be enhanced through structure-guided drug design.

SUBMITTER: Lyons BJE

PROVIDER: S-EPMC6748289 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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On the road to structure-based development of anti-virulence therapeutics targeting the type III secretion system injectisome.

Lyons Bronwyn J E BJE Strynadka Natalie C J NCJ

MedChemComm 20190620 8

The type III secretion system injectisome is a syringe-like multimembrane spanning nanomachine that is essential to the pathogenicity but not viability of many clinically relevant Gram-negative bacteria, such as enteropathogenic Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa. Due to the rise in antibiotic resistance, new strategies must be developed to treat the growing spectre of drug resistant infections. Targeting the injectisome via an 'anti-viru ...[more]

PMID: 31534650

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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:Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.

Dataset Information

On the road to structure-based development of anti-virulence therapeutics targeting the type III secretion system injectisome.

Publications

On the road to structure-based development of anti-virulence therapeutics targeting the type III secretion system injectisome.

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