Maf‐dependent bacterial flagellin glycosylation occurs before chaperone binding and flagellar T3SS export

Jennifer L Parker,Graham P Stafford,Phillip C Wright,Narciso A S Couto,Rebecca C Lowry,Jonathan G Shaw

doi:10.1111/mmi.12549

Jennifer L Parker, Graham P Stafford + Show 4 more

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https://doi.org/10.1111/mmi.12549

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Journal: Molecular microbiology	Publication Date: Mar 4, 2014
Citations: 36	License type: CC BY 3.0

Affiliation: University of Sheffield

Abstract

Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non-glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form.

Highlights

Many bacterial species are motile through the action of flagella, a complex nanomachine in which a 10–15 μm helical filament extends from the cell surface and is anchored to a rotating basal body spanning the bacterial cell envelope that is powered by an ion gradient
Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system
We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of pseudaminic acid (Pse) onto flagellin at the later stages of the glycosylation pathway

Summary

Introduction

Many bacterial species are motile through the action of flagella, a complex nanomachine in which a 10–15 μm helical filament extends from the cell surface and is anchored to a rotating basal body spanning the bacterial cell envelope that is powered by an ion gradient. Bacterial flagellins have conserved N and C-terminal D0 and D1 domains that are required for polymerization, chaperone binding and export (Auvray et al, 2001). Secretion of flagellins and other T3SS substrates is controlled by the N-terminal export signal of the T3SS substrate and the presence of specific chaperones. The flagellin-specific chaperone FliS binds to the C-terminal helical domain of flagellin, stabilizing flagellins in the cytoplasm prior to export (Auvray et al, 2001). The importance of a fully functional flagellum and motility in the adherence of pathogenic bacteria to eukaryotic cells and colonization of the host, has been demonstrated in a number of studies on a number of pathogens, with functional flagella systems contributing to virulence (Eaton et al, 1996; Merino et al, 1997; Pratt and Kolter, 1998)

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