Acting on Actin: Rac and Rho Played by Yersinia

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery—also named injectisome—assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.

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Section: Resultsmentioning

confidence: 99%

Visualization of translocons in Yersinia type III protein secretion machines during host cell infection

et al. 2018

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“…To enhance Yop effector translocation into HeLa cells, cells were infected with the YopE-deficient strain WA-314ΔYopE or cells expressing the constitutively active Rho GTP-binding protein Rac1Q61L were infected with wild type Yersinia. Under these conditions Yop-translocation rates increase 5-10-fold [35] which is a consequence of the elevated Rac activity in the host cells [35, 59, 60]. In the case of WA-314ΔYopE the elevated Rac activity is caused by the diminished Rac inhibition that is normally imposed by the Rho GTPase-activatingprotein YopE [61, 62], Notably, the fraction of YopB positive bacteria increased from around 2% in wild type infected cells to 30% in WA-314ΔYopE infected cells and also reached around 30% in the Rac1Q61L expressing and wild type infected cells at 1 h post infection (Fig 4A).…”

Section: Resultsmentioning

confidence: 99%

Visualization and regulation of translocons inYersiniatype III protein secretion machines during host cell infection

Nauth

Huschka

Schweizer

et al. 2018

Preprint

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Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery - also named injectisome - assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their assembly have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the putatively operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology we provide novel information about the spatiotemporal organisation of T3SS translocons and their regulation by host cell factors. Author Summary Many human, animal and plant pathogenic bacteria employ a molecular machine termed injectisome to inject their toxins into host cells. Because injectisomes are crucial for these bacteria’s infectious potential they have been considered as targets for antiinfective drugs. Injectisomes are highly similar between the different bacterial pathogens and most of their overall structure is well established at the molecular level. However, only little information is available for a central part of the injectisome named the translocon. This pore-like assembly integrates into host cell membranes and thereby serves as an entry gate for the bacterial toxins. We used state of the art fluorescence microscopy to watch translocons of the diarrheagenic pathogen Yersinia enterocolitica during infection of human host cells. Thereby we could for the first time - with fluorescence microscopy - visualize translocons connected to other parts of the injectisome. Furthermore, because translocons mark functional injectisomes we could obtain evidence that injectisomes only become active when the bacteria are almost completely enclosed by host cells. These findings provide a novel view on the organisation and regulation of bacteri...

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“…Despite important differences in the pathogenesis of plants and animals, which are caused, among others, by the presences of a rigid plant cell wall as a physical barrier for pathogen invasion and the fact that plant-pathogenic bacteria do not enter intact host cells, there are conceptual and mechanistic similarities between the roles of plant and animal Rho family proteins in disease and immunity. In fact, specifically for infectious diseases of mammals, Rho GTPases have been extensively documented to be exploited to promote either invasive or antiphagocytic activities of bacterial pathogens [ 93 , 94 ]. Both activities require the manipulation of host cell processes that govern cytoskeleton rearrangements.…”

Section: An Excursion Into Mammalian Rho Signaling In Bacterial Pamentioning

confidence: 99%

“…Furthermore, the lipid phosphatase activity of SopB results in the recruitment of RhoB, RhoH and RhoJ to bacterial invasion sites, where the activity of these GTPases have been shown to contribute to bacterial invasion [ 102 ]. For Yersinia bacteria, in the antiphagocytic part of the life cycle, the inhibition of phagocytosis is achieved by targeting the same Rho GTPases involved in actin modeling which are required for bacterial invasion [ 94 ]. The type III secreted effector protein YopE was shown to harbor a strong GAP activity toward Rac1 and RhoG [ 103 , 104 ], while the cysteine protease activity of YopT leads to the removal of Rho proteins from the plasma membrane upon proteolytical cleavage of the lipid-modified C-terminal cysteine [ 105 ], indicating a tightly controlled activity of the effector protein repertoire in both invasive and antiphagocytic life phases of the bacteria.…”

Section: An Excursion Into Mammalian Rho Signaling In Bacterial Pamentioning

confidence: 99%

Regulation and Functions of ROP GTPases in Plant–Microbe Interactions

Engelhardt

Trutzenberg

Hückelhoven

2020

Cells

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Rho proteins of plants (ROPs) form a specific clade of Rho GTPases, which are involved in either plant immunity or susceptibility to diseases. They are intensively studied in grass host plants, in which ROPs are signaling hubs downstream of both cell surface immune receptor kinases and intracellular nucleotide-binding leucine-rich repeat receptors, which activate major branches of plant immune signaling. Additionally, invasive fungal pathogens may co-opt the function of ROPs for manipulation of the cytoskeleton, cell invasion and host cell developmental reprogramming, which promote pathogenic colonization. Strikingly, mammalian bacterial pathogens also initiate both effector-triggered susceptibility for cell invasion and effector-triggered immunity via Rho GTPases. In this review, we summarize central concepts of Rho signaling in disease and immunity of plants and briefly compare them to important findings in the mammalian research field. We focus on Rho activation, downstream signaling and cellular reorganization under control of Rho proteins involved in disease progression and pathogen resistance.

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Acting on Actin: Rac and Rho Played by Yersinia

Cited by 10 publications

References 104 publications

Visualization of translocons in Yersinia type III protein secretion machines during host cell infection

Visualization of translocons in Yersinia type III protein secretion machines during host cell infection

Visualization and regulation of translocons inYersiniatype III protein secretion machines during host cell infection

Regulation and Functions of ROP GTPases in Plant–Microbe Interactions

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