Dataset Information

Stoichiometry of the bacterial type III secretion export apparatus

ABSTRACT: Bacterial type III secretion systems are cell envelope-spanning effector protein-delivery machines essential for colonization and survival of many Gram-negative pathogens and symbionts. The membrane-embedded core unit of these secretion systems is termed needle complex. The needle complex is composed of a base that anchors the machinery to the inner and outer membranes, a hollow filament formed by inner rod and needle subunits that serves as conduit for substrate proteins, and a membrane-embedded export apparatus facilitating substrate translocation. While the stoichiometry of the base and of the major export apparatus protein have been revealed by structural analyses, the stoichiometries of the other export apparatus components and of the inner rod remain unknown. We employed peptide concatenated standard and absolute quantification-based strategies to analyze the stoichiometry of the entire needle complex by mass spectrometry. Here we provide evidence that the export apparatus of the type III secretion system encoded on Salmonella pathogenicity island 1 contains 5 SpaP, 1 SpaQ, 1 SpaR, and 1 SpaS. We have corroborated the previously suggested stoichiometry of 9 InvA per needle complex and describe a loose association of InvA with other needle complex components that may reflect its function. These numbers indicate that the inner membrane patch of the needle complex base houses 104 transmembrane domains in total, a dense assembly whose function in the secretion process we merely understand. Furthermore, we present evidence that not more than 6 PrgJ form the inner rod of the needle complex.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Typhimurium Str. Sl1344

SUBMITTER: Nicolas Nalpas

LAB HEAD: Samuel Wagner

PROVIDER: PXD003113 | Pride | 2016-02-25

REPOSITORIES: Pride

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Publications

Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach.

Zilkenat Susann S Franz-Wachtel Mirita M Stierhof York-Dieter YD Galán Jorge E JE Macek Boris B Wagner Samuel S

Molecular & cellular proteomics : MCP 20160221 5

Precisely knowing the stoichiometry of their components is critical for investigating structure, assembly, and function of macromolecular machines. This has remained a technical challenge in particular for large, hydrophobic membrane-spanning protein complexes. Here, we determined the stoichiometry of a type III secretion system of Salmonella enterica serovar Typhimurium using two complementary protocols of gentle complex purification combined with peptide concatenated standard and synthetic sta ...[more]

PMID: 26900162

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Project description:Spirochetes are long, thin, motile, helical or flat wave bacteria that are unique among the prokaryotes by having flagella or axial filaments confined to an internal periplasmic space. These flagella are complex organelles that can play major roles in bacterial pathogenicity and are used as propellers allowing bacteria to move through liquids, viscous environments or along surfaces. While most bacteria species use transcriptional regulatory cascades to regulate the synthesis and assembly of their flagella, spirochetes employ an unusual post-transcriptional mechanism. Using next generation sequencing, we characterized a natural mutant of the relapsing fever spirochete Borrelia hermsii lacking the flagellar export apparatus protein FliH. The mutant was non-motile, uncoiled and straight compared to the wild-type spirochetes. The B. hermsii fliH mutant produced only a residual amount of the major flagellin protein FlaB, which was correlated with a reduced number of periplasmic flagella. The amounts of flaB transcript were comparable in the fliH mutant and the wild-type strain. The synthesis of FlaB, the motility and the infectivity of the fliH mutant were rescued by trans-complementation. This report reveals a new function for FliH, and we propose that this regulator of the flagellar export apparatus influences the post-transcriptional processing of the flagella, motility and virulence of the relapsing fever spirochete Borrelia hermsii. Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their flat-wave morphology and motility. While other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ posttranscriptional mechanism(s) that are only partially known. In the present study, we characterize a non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in flagella. We used next generation DNA sequencing of the mutant’s genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcriptome analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major flagellar filament core protein FlaB. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete while the amount of FlaA was similar to the wild-type level. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the posttranscriptional level by influencing the synthesis of FlaB. Borrelia hermsii wild type vs. motility mutant

Dataset Information

Stoichiometry of the bacterial type III secretion export apparatus

Publications

Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach.

Similar Datasets

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