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The facultative intracellular bacteriumLegionella pneumophilaemploys the Icm/Dot type IV secretion system (T4SS) to replicate in a unique membrane-bound compartment, theLegionella-containing vacuole (LCV). The endoplasmic reticulum (ER)-resident large fusion GTPase Sey1/atlastin promotes remodeling and expansion of LCVs, and the GTPase is also implicated in the formation of ER-derived lipid droplets (LDs). Here we show that LCVs intimately interact with palmitate-induced LDs inDictyostelium discoideumamoeba. Comparative proteomics of LDs isolated from theD. discoideumparental strain Ax3 or ⊗sey1revealed 144 differentially produced proteins, of which 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or ⊗sey1, respectively. Using dually fluorescence-labeled amoeba producing the LCV marker P4C-GFP or AmtA-GFP and the LD marker mCherry-perilipin, we discovered that Sey1 and theL. pneumophilaIcm/Dot T4SS as well as the effector LegG1 promote LCV-LD interactions.In vitroreconstitution of the LCV-LD interactions using purified LCVs and LDs fromD. discoideumAx3 or ⊗sey1revealed that Sey1 and GTP promote this process. The LCV-LD interactions were impaired for ⊗sey1-derived LDs, suggesting that Sey1 regulates LD composition. Palmitate promoted the growth of (i)L. pneumophilawild-type inD. discoideumAx3 but not in ⊗sey1mutant amoeba and (ii)L. pneumophilawild-type but not ⊗fadLmutant bacteria lacking a homologue of theE. colifatty acid transporter FadL. Finally, isotopologue profiling indicated that intracellularL. pneumophilametabolizes13C-palmitate, and its catabolism was reduced inD. discoideum⊗sey1andL. pneumophila⊗fadL. Taken together, our results reveal that Sey1 mediates LD- and FadL-dependent fatty acid metabolism of intracellularL. pneumophila.

Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in

Platelets are currently thought to harbor antimicrobial functions and might therefore play a crucial role in infections, e.g., those caused by Aspergillus or mucormycetes. The incidence of invasive fungal infections is increasing, particularly during the coronavirus disease 2019 (COVID-19) pandemic, and such infections continue to be life-threatening in immunocompromised patients. For this reason, the interaction of antimycotics with platelets is a key issue to evaluate modern therapeutic regimens. Amphotericin B (AmB) is widely used for the therapy of invasive fungal infections either as deoxycholate (AmB-D) or as a liposomal formulation (L-AmB). We showed that AmB strongly activates platelets within a few minutes. AmB concentrations commonly measured in the blood of patients were sufficient to stimulate platelets, indicating that this effect is highly relevant

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.

The Gram‐negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella‐containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non‐virulent and a non‐replicating, virulent/transmissive phase. Here, we show on a single‐cell level that at late stages of infection, individual motile (PflaA‐GFP‐positive) and virulent (PralF‐ and PsidC‐GFP‐positive) L. pneumophila emerge in the cluster of non‐growing bacteria within an LCV. Comparative proteomics of PflaA‐GFP‐positive and PflaA‐GFP‐negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the PflaA‐GFP‐positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi‐phasic life cycle of L. pneumophila., Legionella forms a distinct vacuole in phagocytes, wherein the pathogen adopts a bi‐phasic life cycle and phenotypic heterogeneity. Quorum sensing elicits a transmissive subpopulation at the vacuole periphery, which spearheads compartment exit and host cell lysis.

The water-borne bacterium Legionella pneumophila is the causative agent of Legionnaires' disease. In the environment, the opportunistic pathogen colonizes different niches, including free-living protozoa and biofilms. The physiological state(s) of sessile Legionella in biofilms and their functional consequences are not well understood. Using single-cell techniques and fluorescent growth rate probes as well as promoter reporters, we show here that sessile L. pneumophila exhibits phenotypic heterogeneity and adopts growing and nongrowing ("dormant") states in biofilms and microcolonies. Phenotypic heterogeneity is controlled by the Legionella quorum sensing (Lqs) system, the transcription factor LvbR, and the temperature. The Lqs system and LvbR determine the ratio between growing and nongrowing sessile subpopulations, as well as the frequency of growth resumption ("resuscitation") and microcolony formation of individual bacteria. Nongrowing L. pneumophila cells are metabolically active, express virulence genes and show tolerance toward antibiotics. Therefore, these sessile nongrowers are persisters. Taken together, the Lqs system, LvbR and the temperature control the phenotypic heterogeneity of sessile L. pneumophila, and these factors regulate the formation of a distinct subpopulation of nongrowing, antibiotic tolerant, virulent persisters. Hence, the biofilm niche of L. pneumophila has a profound impact on the ecology and virulence of this opportunistic pathogen.

Legionella pneumophila is a water-borne opportunistic pathogen causing a life-threatening pneumonia called 'Legionnaires' disease'. The Legionella quorum sensing (Lqs) system produces and responds to the α-hydroxyketone signaling molecule 3-hydroxypentadecane-4-one (Legionella autoinducer-1, LAI-1). The Lqs system controls the switch between the replicative/non-virulent and the transmissive/virulent phase of L. pneumophila, and it is a major regulator of natural competence, motility and virulence of the pathogen. Yet, beyond gene regulation, LAI-1 also directly affects pathogen-host interactions, since the signaling molecule modulates the migration of eukaryotic cells. Genes encoding Lqs homologues are present in many environmental bacteria, suggesting that α-hydroxyketone signaling is widely used for inter-bacterial as well as inter-kingdom signaling. In this review we summarize recent advances on the characterization of the Lqs system and its role in L. pneumophila-host cell interactions.

Legionella pneumophila resides in multispecies biofilms, where it infects and replicates in environmental protozoa such as Acanthamoeba castellanii. Studies on L. pneumophila physiology and host-pathogen interactions are frequently conducted using clonal bacterial populations and population level analysis, overlooking the remarkable differences in single cell behavior. The fastidious nutrient requirements of extracellular L. pneumophila and the extraordinary motility of Acanthamoeba castellanii hamper an analysis at single cell resolution. In this chapter, we describe a method to study L. pneumophila and its natural host A. castellanii at single cell level by using an agarose embedment assay. Agarose-embedded bacteria and infected cells can be monitored over several hours up to several days. Using properly adapted flow chambers, agarose-embedded specimens can be subjected to a wide range of fluctuating conditions.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection. In addition to their role in thrombosis and hemostasis, platelets can also migrate to sites of infection to help trap bacteria and clear the vascular surface.