Dynamic relocalization of the cytosolic type III secretion system components prevents premature protein secretion at low external pH

Wimmi, Stephan; Balinovic, Alexander; Jeckel, Hannah; Selinger, Lisa; Lampaki, Dimitrios; Eisemann, Emma; Meuskens, Ina; Linke, Dirk; Drescher, Knut; Endesfelder, Ulrike; Diepold, Andreas

doi:10.1101/869214

Cited by 6 publications

(4 citation statements)

References 72 publications

(28 reference statements)

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“…Since all cytosolic T3SS components, including SctL, are required for protein export under normal conditions, we speculate that transient interactions of SctL and the other cytosolic T3SS components with basal body components at the poles might allow the limited export of the needle subunit, which would then stay stably associated with the bacteria despite the disassembly of the cytosolic sorting platform. In agreement with this hypothesis, we had found earlier that the cytosolic T3SS sorting platform components do exchange between the injectisomebound sorting platform and a cytosolic pool [43,49] and that the sorting platform components temporarily dissociate from the remaining injectisomes under certain conditions, such as low external pH [50]. The presence of a low number of needles had previously been reported for the closely related P. aeruginosa T3SS [19].…”

Section: Pilotins Differentially Affect Major Steps In T3ss Assemblysupporting

confidence: 84%

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Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

Wimmi

Fleck

Helbig

et al. 2022

Preprint

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In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics and live-cell microscopy, we determined the contribution of the pilotin to assembly, function and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In the absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. In line, the majority of export apparatus subcomplexes is mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, the export of late T3SS substrates including the virulence effectors is hardly affected in these bacteria, whereas early T3SS substrates, such as the needle subunits, are only exported to a very low degree. We found that pilotins transiently interact with the secretin and the large export apparatus component SctV, but mostly localize throughout the bacterial membrane, where they form transient mobile clusters, which do not colocalize with assembled injectisomes. In addition, pilotins interact with non-T3SS components, including sugar transporters. Pilotins therefore have additional functions downstream injectisome assembly, which include the regulation of type III secretion and a potential new link to the cellular energy metabolism.

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Section: Pilotins Differentially Affect Major Steps In T3ss Assemblysupporting

confidence: 84%

“…Maleimide-based staining to visualize the injectisome needles was performed as described in [50]. Briefly, expression of SctF S5C from the pBAD-His/B plasmid was induced by addition of 1.0% L-arabinose at the time of the temperature shift in strains expressing the native SctF at the same time.…”

Section: Methodsmentioning

confidence: 99%

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

Wimmi

Fleck

Helbig

et al. 2022

Preprint

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“…Images were next analyzed with ThunderSTORM 67 . To reconstruct the tracks, SWIFT vsn 0.3.1 was used 68 . Only tracks with more than 10 localizations were kept for further analysis.…”

Section: Methodsmentioning

confidence: 99%

Nanoscale imaging of CD47 informs how plasma membrane modifications shape apoptotic cell recognition

et al. 2023

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CD47 recognized by its macrophage receptor SIRPα serves as a “don’t eat-me” signal protecting viable cells from phagocytosis. How this is abrogated by apoptosis-induced changes in the plasma membrane, concomitantly with exposure of phosphatidylserine and calreticulin “eat-me“ signals, is not well understood. Using STORM imaging and single-particle tracking, we interrogate how the distribution of these molecules on the cell surface correlates with plasma membrane alteration, SIRPα binding, and cell engulfment by macrophages. Apoptosis induces calreticulin clustering into blebs and CD47 mobility. Modulation of integrin affinity impacts CD47 mobility on the plasma membrane but not the SIRPα binding, whereas CD47/SIRPα interaction is suppressed by cholesterol destabilization. SIRPα no longer recognizes CD47 localized on apoptotic blebs. Overall, the data suggest that disorganization of the lipid bilayer at the plasma membrane, by inducing inaccessibility of CD47 possibly due to a conformational change, is central to the phagocytosis process.

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“…Specifically, a pool of freely diffusing complexes of injectisome proteins that are available for injectisomes binding would provide a mechanism for timely reactivation of secretion. Recent work has shown that an extracellular pH drop from 7 to 4 results in disassociation of Ye SctN, L, Q, and K from injectisomes, an effect that correlated with loss of secretion 41 . Notably, the intracellular pH decreased only slightly (pH=7→6.3) in acidic extracellular environments.…”

Section: Discussionmentioning

confidence: 99%

Distinct cytosolic complexes containing the type III secretion system ATPase resolved by 3D single-molecule tracking in liveYersinia enterocolitica

Prindle

Rocha

Wang

et al. 2022

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The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations as well as freely-diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilize 3D single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the eYFP-labeled ATPase, YeSctN, and its regulator, YeSctL, reveals that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed in both in wild type cells and in ΔsctD mutants, which cannot assemble injectisomes. In ΔsctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding - a feature that could contribute to injectisome functional regulation.

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Dynamic relocalization of the cytosolic type III secretion system components prevents premature protein secretion at low external pH

Cited by 6 publications

References 72 publications

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

Nanoscale imaging of CD47 informs how plasma membrane modifications shape apoptotic cell recognition

Distinct cytosolic complexes containing the type III secretion system ATPase resolved by 3D single-molecule tracking in liveYersinia enterocolitica

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