Dictyosteliumlacking the single atlastin homolog Sey1 shows aberrantERarchitecture, proteolytic processes and expansion of theLegionella‐containing vacuole

Abstract

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1–3 (ATL1‐3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER‐associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the “Legionella‐containing vacuole” (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.

TheLegionella-driven PtdIns(4)Pgradient at LCV-ER membrane contact sites promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole remodeling

“…4B ). Intriguingly, the LCVs in the Δ osbK mutant strain were even smaller than in the Δ sey1 mutant used as a control [66]. The production of GFP-Sac1_ΔTMD in D. discoideum Ax3 significantly reduced the LCV size at 2 h, 8 h and 16 h post infection compared to amoeba producing GFP- Sac1 or GFP-Sac1_C383S ( Fig.…”

“…Comparative proteomics of LCVs from D. discoideum wild-type and Δsey1 reveals MCS components Given the tight and stable association of the LCV limiting membrane with the ER, MCS are likely formed between the compartments. The ER tubule-resident large GTPase Sey1/Atl3 (DDB_G0279823) promotes the decoration of LCVs with ER, and in absence of the GTPase, significantly less ER accumulates on the pathogen vacuole, while the PtdIns(4)P-positive LCV appears to form normally [59,66]. To gain further insights into the process of LCV formation, and in particular the role of the ER, we analyzed by comparative proteomics LCVs from the L. pneumophila-infected D. discoideum Ax3 wild-type strain or the Δsey1 mutant.…”

Dictyostelium Dynamin Superfamily GTPases Implicated in Vesicle Trafficking and Host-Pathogen Interactions

“…Non-essential genes can be disrupted or replaced by homologous recombination, RNA interference has been described, and ectopic expression of endogenous or foreign genes is straightforward ( Kuspa and Loomis, 1992 ; Faix et al, 2004 ; Kuhlmann et al, 2006 ; Paschke et al, 2018 ). In fact, recent cell biological and infection studies using dually labeled D. discoideum strains and confocal laser scanning microscopy or imaging flow cytometry revealed intriguing mechanistic insights into the virulence of the facultative intracellular bacterium Legionella pneumophila ( Weber et al, 2014 , 2018 ; Bärlocher et al, 2017a ; Steiner et al, 2017 ; Welin et al, 2018 ; Hüsler et al, 2021 ).…”

“…Sey1 localizes to interconnected ER membranes and ER tubules as well as to perinuclear ER membranes. To determine its function, we have recently generated and characterized a sey1 deletion mutant strain (Δ sey1 ) ( Hüsler et al, 2021 ). Cells lacking Sey1 show pleiotropic defects, including aberrant ER morphology and ER tubules dynamics, defective lysosomal enzymes exocytosis and intracellular proteolysis, impaired cell motility, and inability to cope with prolonged ER stress.…”

“…Of note, Sey1 was not required for the formation of the PtdIns(4) P -positive LCVs and initial ER recruitment, but for the DSP-catalyzed pathogen vacuole expansion. Moreover, D. discoideum lacking the sey1 gene was found to be less permissive for intracellular L. pneumophila replication ( Hüsler et al, 2021 ). LCV formation was impaired in this mutant strain, since the ER was less efficiently recruited to the pathogen vacuole, and LCV expansion was retarded at early stages of infection.…”