https://orcid.org/0000-0002-5462-9301
Figures
Abstract
The causative agent of Legionnaires’ disease, Legionella pneumophila, is an amoebae-resistant environmental bacterium, which replicates intracellularly in a distinct compartment, the “Legionella-containing vacuole” (LCV). L. pneumophila employs the α-hydroxyketone compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. LAI-1 promotes intracellular replication and inhibits the migration of mammalian cells and Dictyostelium discoideum. In this study, we revealed that LAI-1 and “clickable” azido-LAI-1 derivatives inhibit the migration of D. discoideum and localize to LCVs. Azido-LAI-1 colocalizes with the LCV markers calnexin, P4C, and AmtA, but not with mitochondrial or lipid droplet markers. Intriguingly, LAI-1-dependent inhibition of D. discoideum migration involves the single guanylate-binding protein (GBP), a member of the GBP family of large GTPases, which in metazoan organisms promote cell autonomous immunity. D. discoideum lacking GBP (Δgnbp) allows more efficient intracellular replication of L. pneumophila, without apparently compromising LCV formation or integrity, and GBP-GFP localizes to the ER at LCV-ER membrane contact sites (MCS). However, the peri-LCV localization of LAI-1 and GBP is not mutually dependent. Synthetic LAI-1 inhibits the expansion/remodeling of LCVs (but not vacuoles harboring avirulent L. pneumophila) in a GBP-dependent manner. Taken together, the work shows that LAI-1 localizes to LCVs, and LAI-1-dependent inter-kingdom signaling involves D. discoideum GBP, which localizes to LCV-ER MCS and acts as an antimicrobial factor by restricting the intracellular growth of L. pneumophila.
Author summary
Small molecule inter-kingdom signaling between pathogens and host cells represents a crucial but only partly understood aspect of microbial virulence. The amoebae-resistant opportunistic pathogen Legionella pneumophila employs the compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. In metazoan cells, the conserved and wide-spread family of guanylate-binding protein (GBP) large GTP-ases usually comprises several distinct paralogues, which are implicated in pathogen detection, inflammation, cell death pathways, and cell autonomous immunity. In the social amoeba Dictyostelium discoideum, only a single gbp gene of unknown function is present. Using approaches from organic chemistry, genetics, cell biology and infection biology, we reveal that GBP is involved in the inhibition of D. discoideum migration and pathogen vacuole expansion/remodeling by LAI-1 as well as in intracellular growth of L. pneumophila. This study provides a novel link between small molecule inter-kingdom signaling and GBP-dependent cell autonomous immunity.
Citation: Solger F, Rauch J, Vormittag S, Fan M, Raykov L, Charki P, et al. (2025) Inter-kingdom signaling by the Legionella autoinducer LAI-1 involves the antimicrobial guanylate binding protein GBP. PLoS Pathog 21(4): e1013026. https://doi.org/10.1371/journal.ppat.1013026
Editor: David Skurnik, Universite Paris Descartes Faculte de Medecine, FRANCE
Received: December 2, 2024; Accepted: March 7, 2025; Published: April 29, 2025
Copyright: © 2025 Solger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are contained in the manuscript and the supplementary information.
Funding: This work was supported by Swiss National Science Foundation (SNF) project grants to HH (31003A_175557, 310030_200706) and TS (310030_169386, 310030_188813) as well as a SNF Sinergia grant to TS (CRSII5_189921). Work in the group of JS was supported by the Deutsche Forschungsgemeinschaft (DFG) within the research training group RTG2581. The salary of FS and MF was provided by the SNF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: Icm/Dot, intracellular multiplication/defective organelle trafficking; c-di-GMP, cyclic di-guanosine monophosphate; DBCO, dibenzocyclooctyne; human/mouse guanylate binding protein, h/mGBP; interferon-γ, IFN-γ; LAI-1, Legionella autoinducer-1; LCV, Legionella-containing vacuole; Lqs, Legionella quorum sensing; LvbR, Legionella virulence and biofilm regulator; GBP, guanylate-binding protein; GFP, green fluorescent protein; OMVs, outer membrane vesicles; T4SS, type IV secretion system
Introduction
Small molecule inter-kingdom communication between bacterial pathogens and eukaryotic target cells represents a crucial aspect of microbial pathogenesis [1–5]. Zoonotic enteropathogenic bacteria such as Salmonella enterica or pathogenic Escherichia coli, as well as environmental bacteria such as Vibrio cholerae or Legionella pneumophila employ small molecule inter-kingdom signaling as a virulence strategy.
In the environment, the opportunistic pathogen L. pneumophila replicates in free-living protozoa, including the amoebae Acanthamoeba castellanii and Dictyostelium discoideum [6–8]. Upon inhalation of L. pneumophila-contaminated aerosols, the bacteria grow within and destroy lung macrophages, thereby causing a severe pneumonia termed Legionnaires’ disease [9–11]. To govern the interactions with these evolutionarily distant phagocytes, amoebae and macrophages, L. pneumophila employs the Icm/Dot type IV secretion system (T4SS), which translocates more than 300 “effector proteins” into the host cells [12–16]. Some of these effectors have been described to target crucial host processes and to promote the formation of a replication-permissive compartment termed the Legionella-containing vacuole (LCV) [17–20].
L. pneumophila employs the Legionella quorum sensing (Lqs) system for small molecule signaling, which produces, detects and responds to an organic α-hydroxyketone compound called Legionella autoinducer-1 (LAI-1, 3-hydroxypentadecane-4-one) [5,21–23]. LAI-1 is secreted and delivered to prokaryotic and eukaryotic cells by bacterial outer membrane vesicles [24]. The Lqs system comprises the autoinducer synthase LqsA [25], the homologous membrane-bound sensor histidine kinases LqsS [26] and LqsT [27], and the cognate cytosolic response regulator LqsR [28,29], which dimerizes upon phosphorylation and harbors an output domain resembling nucleotide-binding domains [30,31].
The Lqs system is linked through the pleiotropic transcription factor LvbR to signaling pathways involving the inorganic gas nitric oxide (NO) [32]. These signaling pathways comprise three distinct NO receptors upstream of two-component systems converging on c-di-GMP metabolism [33]. NO signaling regulates virulence, motility, biofilm formation and dispersal, as well as phenotypic heterogeneity of L. pneumophila. Taken together, LAI-1-dependent quorum sensing is linked to NO and c-di-GMP signaling jointly regulating a plethora of L. pneumophila traits [23,34,35].
LAI-1 and the Lqs-LvbR signaling network promote intra-species communication and regulate various features of L. pneumophila, including virulence [5,36], motility and flagellum production [37], growth phase switch and temperature-dependent cell density [28,38], expression of a 133 kb genomic “fitness island” and natural competence for DNA uptake [5,36]. Moreover, the Lqs-LvbR network also regulates phenotypic heterogeneity and the occurrence of functionally different L. pneumophila subpopulations (e.g., “persisters”) upon infection of amoebae and macrophages [39,40] as well as in biofilms and under sessile conditions [41]. Finally, LAI-1 and the Lqs-LvbR signaling network govern inter-kingdom communication by modulating the motility of eukaryotic cells, including D. discoideum, macrophages, or epithelial cells [42], intracellular replication [24], and migration of A. castellanii through L. pneumophila biofilms [43]. Specifically, LAI-1 inhibits epithelial cell migration through a pathway requiring the scaffold protein IQGAP1, the small GTPase Cdc42 (but not RhoA or Rac1), as well as the Cdc42-specific guanine nucleotide exchange factor (GEF) ARHGEF9 (Fig 1A).
(A) LAI-1-dependent inter-kingdom signaling of L. pneumophila comprises cell migration inhibition and cytoskeleton disintegration. LAI-1 is detected and/or taken up by eukaryotic host cells by unknown mechanisms. The single D. discoideum large GTPase guanylate-binding protein, DdGBP, restricts intracellular growth of L. pneumophila, localizes to LCV-ER membrane contact sites and is required for LAI-1-dependent LCV size remodeling. Thus, LAI-1 links small molecule inter-kingdom signaling and GBP-dependent cell autonomous immunity. Created in BioRender. Solger, F. (2025) https://BioRender.com/b17h436. (B) Azido-(S)-LAI-1 can be attached to various conjugation partners (e.g., dyes) using bioorthogonal strain-promoted alkyne-azide cycloadditions (SPAAC). (C) The diazirine function of azido-diazirine-LAI-1 is stimulated by UV light and forms a carbene by releasing nitrogen. The highly reactive carbene can interact with various chemical moieties and thus covalently binds to its biological environment. The covalently fixed azido-LAI-1-derivative can then be attached to various conjugation partners (e.g., dyes, biotin) using SPAAC.
To counteract microbial assaults, eukaryotic cells have evolved a sophisticated array of cell-autonomous defense mechanisms. Guanylate-binding protein (GBP) large GTPases comprise a conserved and wide-spread family of antimicrobial nanomachines [44–46]. In mammalian cells, several paralogues of interferon-γ (IFN-γ)-induced GBPs are usually present, e.g., 7 human and 11 mouse GBPs have been identified [47]. Increasing evidence suggests that these GBPs represent cytosolic pattern recognition receptors (PRRs), which bind conserved microbial structures like lipopolysaccharide (LPS) of Gram-negative bacteria and directly kill microorganisms [48,49]. Furthermore, GBPs also disrupt pathogen vacuoles and/or activate the inflammasome and pyroptotic cell death to protect eukaryotic cells from invading microorganisms. In human macrophages, GBP1 is required for IFN-γ-induced inflammasome responses against L. pneumophila and co-localizes with LCVs to promote the rupture of the pathogen vacuole [50,51]. In mouse macrophages, GBPs are also required for inflammasome-dependent clearance of L. pneumophila but dispensable for LCV rupture [52,53]. GBPs comprise a globular N-terminal GTPase domain followed by an extended C-terminal α-helical domain [54,55]. Upon GTP binding, GBPs form homodimers, which further assemble into large, multimeric complexes on microbial or host membranes. However, the target specificity and mode of action of GBPs are incompletely understood at present.
The D. discoideum genome encodes a single GBP homologue termed DdGBP [56,57], which is most closely related to human GBP3 (26% protein identity, e-value 3 × 10-38). The corresponding gnbp gene (Q54TN9/ DDB_G0281639), encodes a protein of 796 amino acids (92,707 Da). DdGBP is predicted to harbor an N-terminal signal peptide with a basic double lysine motif and a C-terminal transmembrane domain; yet, the protein/gene has not been characterized. Intriguingly, however, GBP was identified by mass spectrometry in the proteome of purified macropinosomes [58] and of purified LCVs isolated from L. pneumophila-infected D. discoideum [59].
In this study, we reveal that (i) LAI-1 and GBP-GFP localize to LCVs, (ii) GBP restricts intracellular growth of L. pneumophila, and (iii) LAI-1-dependent inhibition of D. discoideum migration and LCV size expansion/remodeling involves GBP. Hence, LAI-1-dependent inter-kingdom signaling involves the single antimicrobial D. discoideum GBP.
Results
LAI-1 and azido-derivatives inhibit amoebae migration and localize to LCVs
Synthetic LAI-1 inhibits the migration of D. discoideum in a dose-dependent manner [42]. To further characterize LAI-1-dependent inter-kingdom signaling, we sought to introduce functional LAI-1 derivatives, “clickable” azido-LAI-1 and UV-activatable azido-diazirine-LAI-1 (termed diazirine-LAI-1 in this study; see Materials and Methods section for synthesis details). Previously, we have shown that functionalized clickable azido-sphingolipids are incorporated into cellular membranes similar to control sphingolipids without modification allowing the visualization of sphingomyelin distribution and sphingomyelinase activity in infection processes [60–62].
LAI-1 was synthetically equipped with an azide group, which enables the spontaneous click reaction between the azide and dibenzocyclooctyne (DBCO) dyes (Fig 1B). The terminal azide promotes minimally invasive bioorthogonal strain-promoted alkyne-azide cycloadditions (SPAAC), allowing the conjugation with fluorescent dyes (e.g., DIBO594). Since conjugation with large molecules has a major influence on the substance properties and biological targets might diffuse in the cell, covalent photo-crosslinking with bifunctional LAI-1 is desirable. Accordingly, we further synthesized UV-activatable azido-diazirine-LAI-1, allowing the spontaneous cross-linking of LAI-1 with membranes and proteins by photo-activation (Fig 1C). Diazirine-LAI-1 can be excited by UV radiation, whereupon a highly reactive carbene is formed, which crosslinks with functional groups in close proximity. This covalent modification prevents diffusion from the target, and the additional azide group enables coupling with various conjugation partners via SPAAC.
Treatment of D. discoideum Ax3 with 10 µM LAI-1, azido-LAI-1, or diazirine-LAI-1 inhibited the migration of single amoebae (Fig 2A). Compared to the solvent control, the velocity of D. discoideum treated with LAI-1, azido-LAI-1, or diazirine-LAI-1 was significantly reduced by ca. 30% (Fig 2B). LAI-1 and the derivatives reduced amoebae velocity to a similar extent, indicating that the inter-kingdom signaling activity of the derivatives is not compromised. Analogously, azido-LAI-1 triggered the luminescence of a Vibrio cholerae α-hydroxyketone reporter strain in a concentration-dependent manner (S1A Fig), similarly to what has been previously found for LAI-1 [24]. Diazirine-LAI-1 triggered luminescence of the V. cholerae reporter strain less effectively but still above background level. In summary, azido- and diazirine-azido-LAI-1 derivatives are biologically active in inter-kingdom as well as inter-bacterial signaling, and hence, the derivatives are valid tools to study the effects of LAI-1 on eukaryotic and prokaryotic cells.
(A) D. discoideum Ax3 producing GFP (pSW102) was treated (10 µM, 1 h) with (S)-LAI-1, (S)-azido-LAI-1 (termed “azido-LAI-1)”, (S)-azido-diazirine-LAI-1 (termed “diazirine-LAI-1”) or DMSO (solvent control), and single cell migration was recorded continuously for 2 h with 2 min time interval (n ≈ 15 cells per sample). (B) Amoebae velocity was quantified using the ImageJ manual tracker and Ibidi chemotaxis software. Data shown are means and standard deviations of at least biological triplicates (Student’s t-test; *, p ≤ 0.05; **, p ≤ 0.01; n = 45-60 cells per sample). (C) D. discoideum Ax2 producing calnexin (CnxA)-GFP (pAW016) or P4C-GFP (pWS034) was treated with azido-LAI-1 or diazirine-LAI-1 (10 µM, 1 h), infected (MOI 5, 8 h) with mCerulean-producing L. pneumophila JR32 (pNP99), exposed to UV light (5 min), clicked with DIBO594 dye, fixed (24 h p.i.) and analyzed by confocal laser scanning microscopy. Scale bars, 3 μm. The colocalization of azido-LAI-1 (DIBO594 dye) or diazirine-LAI-1 (DIBO594 dye) with CnxA-GFP or P4C-GFP was quantified by Pearson’s correlation coefficient for (D) LCVs or (E) entire cells. Data shown are (C) representative of three biological replicates, or (D, E) means and standard deviations of biological triplicates (Student’s t-test; **, p ≤ 0.01; 45 cells per sample).
The clickable LAI-1 derivatives were then used to assess the intracellular localization of LAI-1. Upon addition of clickable DIBO594 dye to D. discoideum pre-treated with 10 µM azido-LAI-1 or LAI-1 as a negative control, the clickable LAI-1 derivative but not the LAI-1 control labeled the amoebae (S1B Fig). To test the intracellular localization of LAI-1, D. discoideum amoebae were treated with 10 µM azido-LAI-1 or diazirine-LAI-1 and infected with mCerulean‐producing L. pneumophila JR32. At 8 h post infection (p.i.), some of the infected amoebae were UV-irradiated, further incubated for 24 h (with clickable DIBO594 dye for the last 30 min), fixed, and imaged by confocal microscopy (Fig 2C). This approach revealed that LAI-1 localizes to LCVs as judged from the co-localization with LCV-associated ER (calnexin/CnxA) as well as with the PtdIns(4)P-positive LCV membrane (P4C) (Fig 2D). In addition to LCVs, LAI-1 also localizes to other membrane-bound cellular compartments throughout the cells (Fig 2E). Upon treatment of the samples with UV, diazirine-LAI-1, but not azido-LAI-1, co-localized less extensively with LCV-associated calnexin and PtdIns(4)P (Fig 2D), validating that UV specifically affects diazirine- but not azido-LAI-1. Moreover, the results are in agreement with the notion that at later stages of the infection, the LAI-1-binding LCV membranes dynamically reorganize. In summary, LAI-1 and clickable azido-derivatives inhibit D. discoideum migration and localize to LCVs in the amoebae.
LAI-1 localizes to LCV-ER membrane contact sites and the LCV membrane
Next, we sought to assess the subcellular localization of LAI-1 in more detail and at different early timepoints p.i. To this end, we used D. discoideum producing calnexin-GFP (ER), P4C-GFP (LCV membrane/PtdIns(4)P), GREMIT (mitochondria), or Plin-GFP (lipid droplets, LD). The amoebae were treated with 10 µM azido-LAI-1, infected with mCerulean-producing L. pneumophila JR32 and clicked with DIBO594 dye (Fig 3A). Fluorescence intensity profiles and the quantification of co-localization by Pearson’s correlation coefficient revealed that in L. pneumophila-infected D. discoideum, LAI-1 co-localizes with calnexin-GFP as well as with P4C-GFP on LCVs (Fig 3B) and in whole cells (Fig 3C). Throughout the infection (0.5-8 h p.i.), LAI-1 did not co-localize with either GREMIT or Plin-GFP, indicating that LAI-1 does not localize to mitochondria or LD. Interestingly, LAI-1 specifically localized to LCVs harboring wild-type L. pneumophila but not to the AmtA-positive vacuole harboring ΔicmT mutant bacteria lacking a functional T4SS (Fig 3B). In summary, these results reveal that LAI-1 co-localizes with the ER at LCV-ER MCS as well as with the PtdIns(4)P-positive LCV membrane.
(A) D. discoideum Ax2 producing calnexin (CnxA)-GFP (ER; pAW016), P4C-GFP (LCV membrane; pWS034), GREMIT (mitochondria), GFP-Plin (LD; pHK101), or AmtA (early endosomes; pDM1044-AmtA-mCherry) was treated with clickable azido-LAI-1 (10 µM, 1 h), infected (MOI 5, 4 h) with mCerulean-producing L. pneumophila JR32 or ΔicmT (pNP99), clicked with DIBO594 dye, fixed, and analyzed by confocal microscopy. Scale bars, 3 µm (fluorescence images; left panels). Fluorescence intensity profiles were generated for the GFP fusion proteins and DIBO594 dye using the RGB profile from ImageJ (right panels). The co-localization of azido-LAI-1 (DIBO594 dye) with different organelle markers was quantified by Pearson’s correlation coefficient for (B) LCVs or (C) entire cells (0.5-8 h p.i.). Data shown (B, C) are means and standard deviations of biological triplicates.
LAI-1-dependent inhibition of Dictyostelium migration involves GBP
D. discoideum produces different large GTPases [56,57], including the fusion GTPase Sey1 and GBP, both of which have been identified by MS in the proteome of purified LCVs [59]. Sey1 represents the single atlastin homolog in D. discoideum, localizes to the ER and is implicated in ER dynamics, LCV maturation and intracellular replication of L. pneumophila [63–65]. Building on these insights, we sought to characterize the single D. discoideum GBP in the context of an infection with L. pneumophila.
An alignment of DdGBP with human GBP1 (hGBP1) revealed that the P-loop and the GTPase domain are conserved (S2A Fig). However, while DdGBP harbors a putative N-terminal signal peptide and a C-terminal transmembrane segment, it lacks the C-terminal CaaX motif that confers membrane-association by prenylation of hGBP1 [66]. A structural model of DdGBP generated by AlphaFold revealed an extended α-helical portion following the GTPase domain (S2B Fig), which was substantially larger than in hGBP1 [54,55].
We constructed and tested a D. discoideum mutant strain lacking the single GBP family large GTPase. To this end, the gnbp gene was deleted from the D. discoideum Ax2 genome by double homologous recombination, yielding the Δgnbp mutant strain. The mutant strain lacking GBP grew like the parental amoebae under axenic conditions. Since mammalian GBPs are implicated in cell migration [67], we sought to assess the role of DdGBP for amoeba migration. Compared to the parental strain, Δgnbp amoebae showed increased random migration (Fig 4A) and moved with significantly increased velocity (Fig 4B). The Δgnbp migration phenotype was reverted by plasmid-borne production of GBP-GFP, validating the genetic setup of the mutant strain and indicating that the C-terminal fusion of GBP with GFP is functional. Taken together, DdGBP negatively regulates random amoebae motility.
(A) Single cell migration of D. discoideum Ax2, Δgnbp or Δgnbp producing GFP (pDM317) or GBP-GFP was recorded continuously for 2 h with 2 min time intervals (n ≈ 15 cells per sample). (B) Amoebae velocity was quantified using the ImageJ manual tracker and Ibidi chemotaxis software. Data shown are means and standard deviations of at least 3 biological replicates (Student’s t-test; ***, p ≤ 0.001; ****, p ≤ 0.0001; n = 45-60 cells per sample). (C) D. discoideum Ax2 or Δgnbp producing GFP (pDM317) was left untreated or treated (10 µM, 1 h) with LAI-1, azido-LAI-1, or DMSO (solvent control), and cell migration towards 1 mM folate was assessed by under-agarose assay (4 h p.i.). The white lines represent the edge of the sample wells. Data shown are representative of at least three biological replicates.
Next, we assessed the migration towards folate of D. discoideum Ax2 and Δgnbp amoebae left untreated or treated with LAI-1 or azido-LAI-1 (Fig 4C). Similarly to random migration, amoebae lacking GBP showed enhanced chemotactic migration towards folate. Interestingly, compared to the parental amoebae, Δgnbp cells showed a reduced response to treatment with LAI-1 or azido-LAI-1, revealing the involvement of GBP in LAI-1-dependent migration inhibition. While the chemotactic migration of the parental D. discoideum strain was dose-dependently impaired by 1-10 µM LAI-1, the migration of the Δgnbp strain was not inhibited (S3 Fig). In summary, these results indicate that the single D. discoideum GBP is implicated in random and chemotactic amoebae migration as well as in LAI-1-dependent inhibition of migration.
GBP restricts growth of L. pneumophila and co-localizes with LAI-1 and the ER at LCV-ER MCS
Given the role of GBP for D. discoideum migration and LAI-1-dependent migration inhibition, we next assessed whether GBP affects the intracellular growth of L. pneumophila (Fig 5A). Compared to the parental D. discoideum strain, L. pneumophila grew significantly more efficiently in the Δgnbp mutant strain, indicating that GBP restricts intracellular growth of the pathogen. In contrast, the avirulent L. pneumophila ΔicmT mutant strain did not grow in the mutant amoebae (S4A Fig). L. pneumophila was taken up with the same efficiency by the parental D. discoideum strain and Δgnbp mutant amoebae (S4B and S4C Fig), and therefore, the enhanced intracellular bacterial growth is not due to enhanced uptake. Treatment of D. discoideum Ax2 or Δgnbp with LAI-1 prior to an infection did neither substantially alter the course of the infection (Fig 5A) nor bacterial uptake (S4B and S4C Fig). Intriguingly, while DdGBP restricted the intracellular growth of L. pneumophila, it did not affect the intracellular growth of the amoebae-resistant pathogen Mycobacterium marinum (S5A Fig). In summary, L. pneumophila wild-type but not ΔicmT mutant bacteria grow more efficiently in the Δgnbp mutant strain, while bacterial uptake was not affected. These findings identify DdGBP as an antimicrobial factor.
(A) D. discoideum Ax2 or Δgnbp was treated with LAI-1 (10 µM, 1 h) or DMSO (solvent control), infected (MOI 1, 10 d) with GFP-producing L. pneumophila JR32 (pNT28), and intracellular replication was assessed by RFU. Data shown are means and standard deviations of biological triplicates (Student’s t-test; *, p ≤ 0.05; **, p ≤ 0.01) for comparing D. discoideum Ax2 +/-LAI-1 and Δgnbp +/-LAI-1, respectively. (B) Dually labeled D. discoideum Ax2 producing GBP-GFP and calnexin (CnxA)-mCherry (pAW012) was fixed and analyzed by confocal microscopy. Scale bar, 5 µm. Dually labeled D. discoideum Ax2 producing GBP-GFP and (C) CnxA-mCherry (pAW012) or (D) P4C-mCherry (pWS032) was left untreated or treated with LAI-1 (10 µM, 1 h) or DMSO (solvent control), infected (MOI 5, 2-12 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed, and analyzed by confocal microscopy (8 h p.i., left panels). Scale bars, 5 µm. The colocalization of GBP with the ER (CnxA; C) or with the LCV membrane (P4C; D) was quantified by Pearson’s correlation coefficient (right panels). Fluorescence intensity profiles were generated for the GFP fusion proteins and CnxA (C; lower panels) or P4C (D; lower panels) using the RGB profile from ImageJ. Data shown (right panels) are biological triplicates of means and standard deviations. (E) D. discoideum Ax2 or Δgnbp producing GBP-GFP or P4C-GFP, respectively, was treated with clickable azido-LAI-1 (10 µM, 1 h), infected (MOI 5, 4 h) with mCerulean-producing L. pneumophila JR32 (pNP99), clicked with DIBO594 dye, fixed, and analyzed by confocal microscopy. Scale bars, 3 µm. Fluorescence intensity profiles were generated for GBP-GFP or P4C-GFP and DIBO594 dye using the RGB profile from ImageJ.
The GBP-GFP fusion construct is biologically active upon ectopic production (Fig 4A and 4B), and thus, we sought to test where the fusion protein localizes in the cell. In uninfected D. discoideum GBP-GFP co-localized with calnexin-mCherry, indicating that the large GTPase localizes to the ER (Fig 5B). To further assess the localization of GBP, we used dually labeled D. discoideum producing GBP-GFP and either the ER marker calnexin-mCherry or the LCV/PtdIns(4)P marker P4C-mCherry. Upon infection of the dually labeled D. discoideum strains with mCerulean-producing L. pneumophila JR32, GBP-GFP co-localized with calnexin-mCherry but not with P4C-mCherry (Figs 5C, 5D and S6). Thus, GBP localizes to the ER at LCV-ER MCS but not to the PtdIns(4)P-positive LCV membrane. This localization pattern was observed in L. pneumophila-infected D. discoideum at 2-12 h p.i., regardless of whether the amoebae were treated with 10 µM LAI-1 or not (Figs 5C, 5D and S6). Taken together, GBP-GFP localizes to the ER but not to the LCV membrane throughout an infection with L. pneumophila, and the localization is not affected by LAI-1.
To assess the topology of DdGBP, we performed a proteinase K digestion assay using intact or Triton X-100-treated crude membranes of D. discoideum producing calnexin (CnxA)-GFP, DdGBP-GFP, or GFP-DdGBP (S2C Fig). This approach revealed that N- as well as C-terminally GFP-tagged GBP fusion proteins were proteolytically degraded, indicating that GBP is exposed to the cytoplasm and not sequestered to an organelle lumen. CnxA-GFP, used as a positive control, was susceptible to proteinase K treatment, as the GFP tag of the ER-residing protein is positioned towards the cytosol. In contrast, the canonical ER enzyme protein disulfide isomerase (PDI) was protected from proteinase K digestion, validating its localization to the ER lumen. Taken together, bioinformatic, microscopic and biochemical data indicate that D. discoideum GBP localizes to the ER with its N-terminal portion including the GTPase domain exposed to the cytoplasm (S2D Fig).
Next, we employed clickable azido-LAI-1 to further characterize the localization of DdGBP-GFP and to test whether the absence of GBP in the Δgnbp mutant strain affects LAI-1 localization (Fig 5E). In D. discoideum Ax2 infected with mCerulean-producing L. pneumophila JR32, GBP-GFP co-localized with azido-LAI-1 around LCVs. As expected, GBP-GFP did not localize around intracellular M. marinum, which does not reside in an ER-associated compartment (S5B Fig). Moreover, azido-LAI-1 co-localized with the LCV marker P4C-GFP in D. discoideum Ax2 (Fig 3A) as well as in Δgnbp mutant amoebae (Fig 5E). Hence, LAI-1 co-localizes with GBP-GFP and with P4C-GFP independently of GBP.
LAI-1 reduces the size of GBP-positive LCVs
Within the first two hours of L. pneumophila infection, LCVs expand in size and are remodeled, likely reflecting the formation of a replication-permissive compartment [68]. To assess the role of LAI-1 for LCV size expansion and remodeling, we used dually labeled D. discoideum producing GBP-GFP and AmtA-mCherry. Prior to an infection with mCerulean-producing L. pneumophila JR32 or ΔicmT, the amoebae were treated with 10 µM LAI-1 or not, and the LCV area was quantified by confocal microscopy (Figs 6A and S7). Interestingly, throughout the infection (2-8 h p.i.), treatment with LAI-1 resulted in a significantly smaller size of LCVs harboring wild-type L. pneumophila (Fig 6B), but did not affect phagosomes harboring ΔicmT mutant bacteria (Fig 6C). Analogously, treatment of D. discoideum producing P4C-mCherry with 10 µM LAI-1 or azido-LAI-1 significantly reduced the size of LCVs harboring mCerulean-producing wild-type L. pneumophila (S1C Fig). Taken together, treatment with LAI-1 or azido-LAI-1 resulted in significantly smaller LCVs harboring L. pneumophila wild-type but not ΔicmT mutant bacteria, suggesting that LAI-1 impairs LCV remodeling at early stages of infection.
(A) Dually labeled D. discoideum Ax2 producing GBP-GFP and AmtA-mCherry (pDM1044-AmtA-mCherry) was left untreated or treated with LAI-1 (10 µM, 1 h) or DMSO (solvent control), infected (MOI 5, 8 h) with mCerulean-producing L. pneumophila JR32 or ΔicmT (pNP99) and analyzed by confocal microscopy. Scale bars, 5 μm. The area of LCVs containing (B) strain JR32 or (C) strain ΔicmT was quantified using ImageJ software. Data shown are means and standard deviations of biological triplicates (Student’s t-test; *, p ≤ 0.05).
LAI-1-dependent LCV size remodeling involves GBP
Since GBP is implicated in LAI-1-dependent inhibition of D. discoideum migration (Fig 4C), we sought to assess whether GBP also plays a role in LAI-1-dependent LCV remodeling. To address this question, we used dually labeled D. discoideum or Δgnbp mutant amoebae producing calnexin-GFP and P4C-mCherry. Prior to an infection with mCerulean-producing wild-type L. pneumophila, the amoebae were treated with 10 µM LAI-1 or not, and the LCV area was quantified by confocal microscopy (Figs 7A and S8A). Throughout the infection (2-20 h p.i.), the treatment of the parental D. discoideum strain with LAI-1 resulted in a significantly smaller size of calnexin-GFP-positive LCVs (Fig 7B) and P4C-positive LCVs (Fig 7C). Intriguingly, however, the LCV size reduction by LAI-1 did no longer occur in D. discoideum Δgnbp. Accordingly, GBP is implicated in LAI-1-dependent LCV size remodeling, analogously to LAI-1-dependent cell migration inhibition.
(A-C) Dually labeled D. discoideum Ax2 or Δgnbp producing calnexin (CnxA)-GFP (pAW016) and P4C-mCherry (pWS032) was treated with LAI-1 (10 µM, 1 h), or DMSO (solvent control), infected (MOI 5, 4 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed and analyzed by confocal microscopy. Scale bars, 3 µm. (D-G) D. discoideum Ax2 or Δgnbp producing CnxA-mCherry (pAW012) or P4C-mCherry (pWS032) was infected (MOI 5) for (D, E) 4 h or (F, G) 2-20 h with GFP-producing L. pneumophila JR32 harboring pMF16 (P6SRNA-lqsA) or pMF17 (P6SRNA-lqsAK258A), fixed and analyzed by confocal microscopy. Scale bars, 3 µm. The area of LCVs positive for (E, F) CnxA or (E, G) P4C was quantified using ImageJ software. Data shown are means and standard deviations of biological triplicates (Student’s t-test; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).
We also tested whether the effects of synthetic LAI-1 on LCV size remodeling are observed with endogenously produced LAI-1. To this end, we used GFP-tagged L. pneumophila JR32 overproducing wild-type LqsA or, as a negative control, the catalytically inactive mutant LqsAK258A under control of the strong P6SRNA promoter [24]. The D. discoideum parental strain Ax2 or Δgnbp mutant amoebae producing calnexin-mCherry or P4C-mCherry were infected with these bacterial strains, and the LCV area was quantified by confocal microscopy (Figs 7D and S8B). In the parental D. discoideum Ax2 strain, calnexin-positive (Fig 7E and 7F) LCVs as well as PtdIns(4)P-positive (Fig 7E and 7G) LCVs harboring L. pneumophila overproducing LqsA were significantly smaller than LCVs harboring L. pneumophila overproducing LqsAK258A. These findings indicate that endogenously produced LAI-1 affects LCV remodeling. In Δgnbp mutant amoebae, LqsA-dependent size remodeling was not statistically significant for PtdIns(4)P-positive LCVs (Fig 7E) but still happened with calnexin-positive LCVs (Fig 7E). In summary, LAI-1- and LqsA affect LCV size remodeling, and a role for GBP in this process is more pronouncedly observed with synthetic LAI-1 than with endogenously produced LAI-1.
In mammalian cells, GBPs compromise the integrity of pathogen vacuoles, thereby contributing to the antimicrobial effect of this protein family [44,45]. To test whether LAI-1 and/or DdGBP affect LCV integrity, we used D. discoideum producing cytoplasmic mCherry, which is excluded from intact vacuoles but enters compromised pathogen vacuoles [69]. Dually labeled D. discoideum or Δgnbp producing cytoplasmic mCherry and P4C-GFP were treated with 10 µM LAI-1 or not, infected with mCerulean-producing L. pneumophila JR32 and analyzed by confocal microscopy (S9 Fig). This approach revealed that LCV integrity is not compromised in either the parental D. discoideum Ax2 strain or the Δgnbp mutant at 4 h p.i. Taken together, neither LAI-1 nor DdGBP affect LCV integrity, suggesting specific roles for LAI-1 and GBP in LCV remodeling and intracellular growth of L. pneumophila rather than the mere disruption of the LCV architecture.
Discussion
In this study, we investigated the role of the single member of the GBP family of large GTPases in D. discoideum for LAI-1-dependent inter-kingdom signaling and intracellular replication of L. pneumophila. We show that LAI-1 and clickable derivatives impair D. discoideum migration, modulate LCV size and localize to ER-LCV MCS (Figs 2, 3 and 6), and that DdGBP impedes D. discoideum migration (Fig 4), restricts intracellular growth of L. pneumophila (Fig 5) and localizes to ER-LCV MCS (Fig 5). Intriguingly, LAI-1-dependent inhibition of D. discoideum migration (Fig 4) and LCV expansion/remodeling involves DdGBP (Fig 7) without compromising the integrity of LCVs (S9 Fig). Current insights on LAI-1- and GBP-dependent inter-kingdom signaling between L. pneumophila and host cells are summarized in a working model (Fig 1A).
DdGBP was previously identified in the proteome of LCVs purified from L. pneumophila-infected amoebae [59]. We validated and corroborated this finding by demonstrating that GBP-GFP is functional (Fig 4A and 4B) and indeed localizes to the ER in uninfected D. discoideum and to LCV-ER MCS in L. pneumophila-infected amoebae (Fig 5B–5D). The LCV-ER MCS form within 1-2 h after the uptake of L. pneumophila and are maintained throughout the infection [68].
D. discoideum GBP restricts the intracellular replication of L. pneumophila (Fig 5A), without compromising LCV integrity (S9 Fig). LCV rupture takes place only late in the L. pneumophila infection cycle (> 48 h p.i.), followed by only a very short stay in the host cytoplasm before lysis of the amoeba within minutes [40]. Given the very late and short exposure of L. pneumophila to the host cytoplasm, it is unlikely that DdGBP restricts bacterial growth by directly targeting and killing the pathogen. In agreement with this notion, we did not observe GBP-GFP localizing to cytoplasmic L. pneumophila. Rather, GBP might affect the formation and/or maturation of the LCV. Hence, it appears that by affecting the expansion and remodeling of LCVs, GBP impairs the formation of a replication-permissive compartment and thus might restrict intracellular growth of L. pneumophila. In contrast, GBP does not affect intracellular growth of M. marinum (S5 Fig). This difference might be explicable by the fact that the LCV associates with ER, while the Mycobacterium-containing vacuole (MCV) does not. Hence, the mechanism of intracellular growth restriction by GBP might specifically involve the ER.
The complex process of LCV formation and maturation occurs within 1-2 h after uptake of L. pneumophila and involves a PI lipid conversion from PtdIns(3)P to PtdIns(4)P [70–72], ER acquisition [73,74] and pathogen vacuole size expansion/remodeling [68]. Synthetic LAI-1 localizes to the ER at LCV-ER MCS as well as to the PtdIns(4)P-positive LCV membrane (Figs 2 and 3). Intriguingly, LAI-1 specifically localizes to LCVs harboring wild-type L. pneumophila but not to the membrane of AmtA-positive vacuoles harboring avirulent L. pneumophila ΔicmT (Fig 6). Therefore, specific LCV membrane components might determine LAI-1 acquisition and accumulation. Such factors are currently unknown but might comprise lipids and/or proteins.
Synthetic LAI-1 inhibits the expansion/remodeling of LCVs (Fig 6) in a GBP-dependent manner during the initial 8 h of infection (Fig 7A–7C). Perhaps due to effects of LAI-1 on early LCV formation events, synthetic LAI-1 might be more effective than (continuously) released endogenous LAI-1 synthesized by overproduced autoinducer synthase LqsA (Fig 7D–7G). Alternatively, the relatively high concentration of 10 µM synthetic LAI-1 might not be reached upon production of the autoinducer by L. pneumophila, and/or the solubility and bioavailability of synthetic LAI-1 and endogenous LAI-1 might differ. While synthetic LAI-1 inhibits the expansion/remodeling of LCVs in D. discoideum in a GBP-dependent manner, L. pneumophila overexpressing lqsA grows more efficiently in macrophages [24]. This apparent discrepancy might be explained by different functions of DdGBP and mammalian GBPs. In addition, L. pneumophila does not produce detectable LAI-1, unless lqsA is overexpressed under control of the strong promoters PflaA or P6SRNA [24]. The exogenous addition of synthetic LAI-1 might have different effects on L. pneumophila infection compared to the (continuous) production of endogenous LAI-1. Endogenously produced LAI-1 is associated with and release by outer membrane vesicles [24], and therefore, the solubility and bioavailability of synthetic and “natural” LAI-1 might differ: while the former might be delivered in micelles, the latter appears to be solubilized in phospholipid bilayer vesicles.
The LAI-1-dependent size reduction of LCVs is relatively small – untreated LCVs were ca. 4-5 µm2, compared to LAI-1-treated LCVs which were ca. 3-4 µm2 at 8 h p.i. – and thus likely reflects a structural remodeling of the pathogen vacuole rather than a substantial LCV expansion. A massive expansion of the pathogen vacuole occurs at later infection time points (> 8 h p.i.), likely through the interception of (anterograde and retrograde) vesicular trafficking between the ER and the Golgi apparatus [75–77], and through the fusion of Golgi-derived vesicles [72].
Previously, LAI-1 has been shown to inhibit the migration of D. discoideum, mouse macrophages and human epithelial cells [42]. In epithelial cells, LAI-1-dependent cell migration inhibition requires the scaffold protein IQGAP1, the small GTPase Cdc42 and the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or the small GTPases RhoA, Rac1 or Ran [42]. In the eukaryotic signal transduction pathway triggered by LAI, GBP likely participates upstream of IQGAP1, Cdc42 and ARHGEF9. It remains to be elucidated, which of the 7 human GBP paralogue(s) is implicated in LAI-1-dependent migration inhibition.
Given that the effect of LAI-1 on cell migration involves Cdc42 and the actin cytoskeleton, the effect of LAI-1 on LCV expansion (Fig 6) might also involve this small GTPase and the cytoskeleton. This is even more plausible, since in D. discoideum GBP is implicated in cell migration (Fig 4) as well as in LCV expansion (Fig 7). Early steps in LCV formation also involve the formation of ER-LCV MCS [68]. In agreement with a role of the cytoskeleton in these processes, the formation of endosome-ER MCS involves the actin cytoskeleton [78]. However, the hypothesis that Cdc42 promotes LCV expansion/remodeling is challenging to test in mammalian cells, since appropriate LCV markers are scarce, and it is difficult to assess in D. discoideum, since the small GTPases and their modulators are different.
Mammalian GBPs function as PRRs [47], which bind conserved bacterial structures such as LPS of Gram-negative bacteria, and thus, contribute to pathogen detection and elimination [48,49]. Analogously, D. discoideum GBP might recognize conserved bacterial patterns such as LPS or certain classes of bacterial low molecular weight molecules. Accordingly, DdGBP might represent an evolutionarily ancient PRRs, which has evolved to bind and detect small lipophilic molecules such as the lipid A anchor of LPS or hydrophobic signaling molecules such as the aliphatic α-hydroxyketones LAI-1 (3-hydroxypentadecane-4-one) and V. cholerae CAI-1 (3-hydroxytridecane-4-one). It is currently unknown whether DdGBP directly binds LAI-1 and what effects this binding might have on a molecular and cellular level.
Overall, our findings agree with the notion that similarly to mammalian cells, the D. discoideum GBP is implicated in the recognition of and/or defense against intracellular pathogens, and therefore, functions as an antimicrobial compound. However, since the protozoan amoebae do not produce cytokines, caspases, or their activation platforms like the inflammasome, the output of GBP-dependent pathogen detection by amoebae does not involve pyroptotic or apoptotic cell death, and thus, is clearly different from mammalian cells. Given that DdGBP restricts the intracellular replication of L. pneumophila but not M. marinum (S5A Fig) and GBP-GFP localizes to LCVs but not MCVs (S5B Fig), the ER-association of GBP might underly the specificity of bacterial killing.
In summary, LAI-1-dependent inter-kingdom signaling of L. pneumophila comprises cell migration inhibition and cytoskeleton remodeling, as well as LCV expansion and dynamics. LAI-1 is detected and taken up by eukaryotic host cells by unknown mechanisms. The single D. discoideum GBP family large GTPase restricts intracellular growth of L. pneumophila, localizes to LCV-ER contact sites and is implicated in LAI-1-dependent LCV remodeling. Thus, LAI-1 links small molecule inter-kingdom signaling and GBP-dependent cell autonomous immunity, as outlined in the working model (Fig 1A). These results collectively suggest a novel mechanism of inter-kingdom signaling mediated by LAI-1 and GBP, shedding light on the intricate pathogen-host interactions between L. pneumophila and host cells. Further studies will elucidate the pathways underlying the inter-kingdom detection of and response to LAI-1 by eukaryotic host cells.
Materials and methods
Bacteria, D. discoideum, and Δgnbp mutant strain
The bacterial strains and cell lines used in this study are listed in Table 1. L. pneumophila was grown for 3 days on charcoal yeast extract (CYE) agar plates [79], with or without chloramphenicol (Cam; 10 µg/ml) at 37°C. Bacterial colonies were used to inoculate liquid cultures on a wheel (starting concentration OD600 of 0.1, 80 rpm) in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) medium [80] and grown for approximately 21 h at 37°C to an early stationary phase (2 × 109 bacteria/ml), with Cam (5 µg/ml) added to maintain plasmids if required.
V. cholerae strain MM920 was cultured overnight at 30°C in LB broth supplemented with tetracycline (Tet; 5 μg/ml) prior to an experiment. V. cholerae MM920 lacks the sensor kinase gene luxQ and the autoinducer synthase gene cqsA, and therefore, does not respond to AI-2, and does not produce but responds to the α-hydroxyketone compounds CAI-1 and LAI-1. Strain MM920 harbors plasmid pBB1, which contains the luxCDABE luciferase operon of V. harveyi and produces light upon detection of CAI-1 and LAI-1.
D. discoideum strains were grown at 23°C in HL5 medium without glucose (ForMedium) supplemented with D-maltose (Roth). The amoebae were grown in T75 flasks and split every second day. D. discoideum was transformed and selected with geneticin (G418; 20 μg/ml), hygromycin (Hyg; 50 μg/ml), or blasticidin S (Bls; 5 µg/ml) as described previously [69,71,97,98].
The D. discoideum gnbp gene (DDB_G0281639) was deleted in D. discoideum Ax2(Ka) by double homologous recombination using plasmid pKOSG-IBA-dicty1, yielding strain Δgnbp (Table 1). To this end, strain Ax2(Ka) was grown in axenic conditions at 22°C in HL5c medium (Formedium) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen). The Δgnbp deletion mutant strain was generated by homologous recombination following a one-step cloning in pKOSG-IBA-dicty1 as previously described [92]. The GFP-GBP or GBP-GFP constructs were generated by cDNA library PCR-amplification of the gnbp open reading frame using the oligonucleotides indicated (S1 Table), flanked by BglII/ XbaI restriction sites and inserted into the pDM317 or pDM323 backbones digested with BglII/ SpeI (XbaI complementary). The GBP-GFP construct was used throughout this study, while the GFP-GBP construct appeared to produce unspecific aggregates in D. discoideum.
V. cholerae LAI-1 reporter assay
The V. cholerae strain MM920 was inoculated in LB liquid medium supplemented with Tet (5 μg/ml) and incubated for 18 h at 37 °C. The overnight culture was diluted to an OD600 of 0.25 with fresh medium supplemented with synthetic LAI-1 or derivatives (1-50 µM), or DMSO as solvent control. The mixtures were then transferred to a 96-well plate (Chemie Brunschwig AG), and luminescence intensity (bottom read) was measured every 0.5 h for 8 to 10 h at 30°C using a Biotek Cytation 5 microplate reader with continuous orbital shaking. Images were captured after 4 to 5 h incubation (when bioluminescence intensity usually reached maximum levels) using the FluorChem SP imaging system (Alpha-InnoTec) with an exposure time of 15 min.
Synthesis of LAI-1, azido-LAI-1, and diazirine-axido-LAI-1
(S)-LAI-1 was synthesized as described [42] and is referred to as “LAI-1” throughout the manuscript. Azido-(S)-LAI-1 and diazirine-azido-(S)-LAI-1 are referred to as “azido-LAI-1” and “diazirine-LAI-1”, respectively, and were synthesized as follows (Figs S10 and S11 and S1 Text):
The chemical synthesis of clickable azido-LAI-1 (9) and bifunctional photoreactive diazirine-LAI-1 (22) started with commercially available (S)-2-hydroxybutyric acid 1. In contrast to the previously described synthesis of (S)-LAI-1 [42], the carboxylic acid was first converted to the corresponding Weinreb amide 2 [60], and then the alcohol was protected as a silyl ether 3. This modification was needed as the original synthesis route yielded only poor results during scaling up. For the synthesis of azido-LAI-1, the Weinreb amide 3 was reacted with the acetal-protected bromide 4 via Grignard reaction to form the ketone 5. The hydroxyl group was released via acid-catalyzed cleavage in a protic medium and subsequently converted into bromide 7 using Appel reaction conditions. The bromide was converted to the azide [99], and deprotection of the silyl ether with TBAF yielded azido-LAI-1 (9).
The synthesis of diazirine-LAI-1 was also initiated via a Grignard reaction with the Weinreb amide 3, but an acetal-protected bromide 10 with a shorter chain length was used to allow subsequent functionalization of the side chain. The terminal acetal 11 was acid-catalyzed and cleaved to obtain the free alcohol 12. To facilitate the upcoming Grignard reaction and diazirine synthesis, the carbonyl group had to be protected as a 1,3-dioxolane 13 to ensure selective conversion. Camphorsulfonic acid was required for this step, as stronger acids such as para-toluenesulfonic acid [100] led to complete decomposition of the reactant, while weaker acids such as pyridinium para-toluenesulfonate resulted in no conversion at all. The primary alcohol 13 was oxidized to carboxylic acid 14 by pyridinium dichromate [101], which was directly converted into the Weinreb amide 15. The ketone 17, containing a carbonyl group in the middle of the side chain, was formed through a Grignard reaction of the Weinreb amide 15 with the acetal-protected bromide 16. The carbonyl group was transformed in methanolic ammonia solution with hydroxylamine-O-sulfonic acid (HOSA) into the relatively unstable diaziridine, which was then directly oxidized with iodine to form diazirine 18 [102]. The diaziridine synthesis was also attempted in liquid ammonia [103], but it was unsuccessful due to insolubility. Subsequently, the carbonyl group was very gently deprotected via a Lewis acid-catalyzed transacetalization with acetone and a catalytic amount of iodine to avoid cleaving the silyl ether [104]. The acetal deprotection to the primary alcohol 19 and the following reaction steps to diazirine-LAI-1 (22) were carried out analogously to the synthesis of azido-LAI-1 (9).
Click chemistry of azido-LAI-1
D. discoideum strains producing GFP-labeled organelle markers were seeded in a 12-well plate (5 × 105 cells/well) in HL5 medium and cultured overnight at 23°C. On the day of infection, the amoebae were treated with 10 µM azido-LAI-1 or DMSO (solvent control) for 1 h. Subsequently, the amoebae were infected (MOI 5) with mCerulean-producing L. pneumophila JR32 (pNP99), centrifuged (450 × g, 10 min; room temperature, RT) and incubated at 25°C for 1 h. The infected amoebae were washed three times with HL5 medium and further incubated for the indicated times. 30 min before stopping the infection at the specific time point, 10 µM of clickable DIBO594 dye (ThermoScientific, C10407) was added and further incubated at 25°C. Cells were collected from the 12-well plates and centrifuged (500 × g, 5 min, RT). The supernatant was discarded, the cell pellet resuspended in HL5 medium and centrifuged again (500 × g, 5 min, RT). The samples were fixed with 4% PFA (Electron Microscopy Sciences; 30 min, RT), washed twice with PBS, transferred to an 18‐well μ‐slide dish (Ibidi) and immobilized by adding 0.5% agarose in PBS.
For imaging of the samples, a confocal laser scanning microscope Leica TCS SP8 X CLSM (HC PL APO CS2, objective 63×/1.4-0.60 oil; Leica Microsystems) was used, with a scanning speed of 100 Hz and bi‐directional laser scan. Acquisition was performed with a pixel/voxel size close to the instrument’s Nyquist criterion of 43 × 43 × 130 nm (xyz). Deconvolution of the images was performed with Huygens professional version 19.10 software (Scientific Volume Imaging, http://svi.nl) using the CMLE algorithm, set to 10-20 iterations and 0.05 quality thresholds. The colocalization of clickable LAI-1 with different cellular markers was quantified by using ImageJ plugin “Coloc 2” obtaining Pearson’s correlation coefficients.
Photo-crosslinking of diazirine-LAI-1
One day before the experiment, D. discoideum Ax2 was seeded (5 × 105 cells per well) in 12-well plates and cultured overnight at 23°C. 1 h before infection, the amoebae were treated with 10 µM diazirine-LAI-1 or azido-LAI-1. The amoebae were infected (MOI 5) with mCerulean‐producing L. pneumophila JR32 (pNP99), centrifuged (450 × g, 10 min, RT) and incubated at 25°C for 1 h. Subsequently, infected cells were washed three times with HL5 medium to remove extracellular bacteria, the medium was supplemented again with the respective LAI-1 derivative and further incubated at 25°C for the time indicated. At 8 h p.i., given samples were UV-irradiated for 5 min on ice using a 40 W Hg lamp (8 W, 5 bulbs), operated at 1000 W at a distance of ca. 35 cm from the light source. After 24 h p.i., cells (including supernatant) were collected by vigorous pipetting, centrifuged (500 × g, 5 min, RT) and fixed with 4% PFA (30 min, RT). After fixation, the amoebae were washed twice with PBS, transferred to an 18‐well μ‐slide dish (Ibidi) and immobilized by adding 0.5% agarose in PBS to the wells. For imaging of the samples, a confocal laser scanning microscope Leica TCS SP8 X CLSM was used as described above.
Proteinase K protection assay
D. discoideum producing calnexin-GFP (CnxA-GFP), DdGBP-GFP, or GFP-DdGBP was used to isolate crude membranes, which include the ER and other membrane-bound organelles. To this end, the strains were washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended in buffer A (20 mM HEPES, pH 7, 250 mM sucrose, 100 mM KCl, 2 mM MgCl₂, 1 mM KH₂PO₄, and protease inhibitors) at a density of 6 × 10⁶ cells/ml. The cells were disrupted using a ball homogenizer (8.02-mm bore, 8.002-mm ball, 10 strokes). Intact cells were removed by low-speed centrifugation (1,500 × g, 5 min). The resulting supernatant was centrifuged at 100,000 × g for 60 min, and the pellet was resuspended in buffer A.
For the proteinase K digestion, 100 µg of crude membranes were incubated with 0.2 μg/ml proteinase K (30 min, 4°C) with or without 1% Triton X-100. After digestion, proteins were precipitated by adding trichloroacetic acid (TCA). The samples were then analyzed by SDS-PAGE using 4-16% gradient gels, and the proteins were transferred to nitrocellulose membranes. The blots were incubated with anti-GFP (rabbit polyclonal, TP401; Torrey Pines Biolabs Inc., NJ, USA), followed by anti-PDI antibodies (mouse monoclonal, 221-64-1; [105]). Protein detection was performed using enhanced chemiluminescence (ECL, Bio-Rad, #1705062, Clarity Max ECL substrate), and images were captured with a ChemiDoc imaging system (Bio-Rad).
Single amoeba tracking
D. discoideum strains Ax3, Ax2, Δgnbp, Δgnbp/GFP or Δgnbp/GBP-GFP were seeded at a density of 2×104 cells/well into an 8-well μ‐slide dish (Ibidi) and incubated for 3-4 h in HL5 medium to allow attachment. The medium was then replaced by MB medium, and the amoebae were incubated at 23°C for 1 h before imaging. During microscopy, three fields of interest were randomly selected for each sample and recorded continuously for 2 h with 2 min time interval. Image analyses were performed using ImageJ and the Chemotaxis and Migration Tool version 2.0 (Ibidi).
Chemotaxis migration assay
Under-agarose migration assays with D. discoideum were performed as described [106,107]. The day before the assay, GFP-producing D. discoideum Ax2 or Δgnbp (pDM317) were seeded into 6-well plates in HL5 medium (1 × 106 cells/well), and microscopy dishes (μ-dish, 35 mm, Ibidi) were filled with a mixture of melted agarose in SM medium [10 g bacteriological peptone (Oxoid), 1 g Bacto yeast extract (BD Biosciences), 1.9 g KH2PO4, 0.6 g K2HPO4, 0.43 g MgSO4, 10 g glucose per liter, pH 6.5]. After solidification, 3 parallel slots of 2 × 4 mm (for cells and chemo-attractant solution) were manually cut 5 mm apart into the agarose. On the day of the experiment, the amoebae were washed once with MB medium [14 g bacteriological peptone (Oxoid), 7 g Bacto yeast extract (BD Biosciences), 4.26 g MES (Sigma-Aldrich) per liter, pH 6.9] and kept for 1 h in MB medium. During this period, LAI-1 or DMSO were added at the concentrations indicated. In parallel, the dishes were prepared by adding the chemo-attractant solution, 1 mM folic acid (Sigma-Aldrich) in SM medium, into the central slot 30 min before the cell suspensions were filled into the neighboring slots. After 2 washing steps with MB medium (450 × g, 10 min), the amoebae were detached by scratching into 500 μl MB, and 30 μl of the cell suspension was filled into the slots. The dishes were incubated for 4 h at 23°C to let the amoebae migrate. The migration was tracked using a Leica TCS SP8 X CLSM microscope as described above.
Uptake and intracellular replication of L. pneumophila
Uptake of GFP-producing L. pneumophila JR32 or ΔicmT by D. discoideum Ax2 or Δgnbp was assessed by flow cytometry. To this end, exponentially growing D. discoideum was seeded onto a 24-well plate (1 × 105 cells/well) in HL5 medium and cultured overnight at 25°C. On the day of the infection, the amoebae were treated with 10 µM LAI-1 or DMSO (solvent control) for 1 h and infected (MOI 50) with the L. pneumophila strains harboring plasmid pNT28. L. pneumophila strains were grown for 21 h in AYE/Cam, diluted in HL5 medium, centrifuged onto the cells (450 × g, 10 min, RT) and further incubated for 30 min at 25°C, followed by washing three times with HL5 medium to remove extracellular bacteria. Infected D. discoideum were detached by vigorously pipetting, and 2 × 104 amoebae per sample were analyzed using a LSR II Fortessa cell analyzer (Becton Dickinson, Palo Alto, United States). Scatter plot gating was based on uninfected amoebae, and GFP fluorescence intensity was quantified using FlowJo software.
Intracellular growth of GFP-producing L. pneumophila JR32 or ΔicmT in D. discoideum Ax2 or Δgnbp was assessed by fluorescence increase (relative fluorescence units, RFU). To this end, D. discoideum amoebae were seeded (2 × 104 cells per well) in 96‐well culture‐treated plates (ThermoFisher) and cultured in HL5 medium overnight at 23°C. Amoebae were treated with 10 µM LAI-1 or DMSO for 1 h and infected (MOI 1) with the L. pneumophila strains harboring plasmid pNT28. L. pneumophila strains were grown for 21 h in AYE medium, diluted in MB medium, centrifuged onto the cells (450 × g, 10 min, RT) and incubated for 1 h at 25°C. Subsequently, the medium was exchanged with fresh MB medium (supplemented with LAI-1 or DMSO) and further incubated for the time indicated at 25°C. GFP fluorescence was measured every two days using a BioTek Cytation 5 microplate reader (Agilent Technologies).
Quantification of LCV area sizes in D. discoideum
Dually fluorescence‐labeled D. discoideum strains were grown in exponential phase in HL5 medium containing G418 (20 μg/ml) and/or Hyg (50 μg/ml). One day before the experiment, the amoebae were seeded (5 × 105 cells per well) in 12-well plates and cultured overnight at 23°C. 1 h before infection, the amoebae were treated with 10 µM LAI-1, LAI-1 derivative or DMSO (solvent control). The amoebae were infected (MOI 5) with mCerulean‐producing L. pneumophila JR32 (pNP99), centrifuged (450 × g, 10 min, RT) and incubated at 25°C for 1 h. Subsequently, infected cells were washed three times with HL5 medium to remove extracellular bacteria, the medium was supplemented again with the respective LAI-1 derivative or DMSO and further incubated at 25°C for the time indicated. At given infection time points, cells (including supernatant) were collected by vigorous pipetting, centrifuged (500 × g, 5 min, RT) and fixed with 4% PFA (30 min, RT). After fixation, the amoebae were washed twice with PBS, transferred to an 18‐well μ‐slide dish (Ibidi) and immobilized by adding 0.5% agarose in PBS to the wells. For imaging of the samples, a confocal microscope Leica TCS SP8 X CLSM was used as described above.
Statistical methods
Each experiment was independently performed at least three times and representative images are shown. All statistical analyses were performed using GraphPad Prism (www.graphpad.com). The two‐tailed Student’s t‐test (Mann-Whitney test, no assumption of Gaussian distributions) was used to show significant differences between samples and control. Significances are indicated in the figures as follows: *, **, *** or **** to indicate probability values of less than 0.05, 0.01, 0.001 and 0.0001, respectively. The value of “n” represents the number of biological replicates performed or the number of analyzed cells/LCVs per condition.
Supporting information
S1 Fig. LAI-1 and clickable derivatives promote luminescence of a Vibrio reporter strain and LCV size modulation.
(A) The Vibrio cholerae α-hydroxyketone reporter strain MM920 was left untreated or treated with the indicated concentrations of synthetic (S)-LAI-1, azido-LAI-1, diazirine-LAI-1, or DMSO (solvent control), and luminescence intensity was measured by a plate reader (30°C, 10 h). RLU, relative light units. Data shown are biological triplicates of means and standard deviations. (B) D. discoideum Ax2 was treated with LAI-1 or clickable azido-LAI-1 (10 µM, 1 h), infected (MOI 5, 4 h) with mCerulean-producing L. pneumophila JR32 (pNP99), clicked with DIBO594 dye, and analyzed by confocal laser scanning microscopy. Scale bars: 20 μm. (C) D. discoideum Ax2 producing P4C-mCherry (pWS032) was treated (10 µM, 1 h) with LAI-1, azido-LAI-1, or DMSO (solvent control), infected (MOI 5, 4 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed and analyzed by confocal microscopy. LCV areas were quantified using ImageJ software. Data shown are means and standard deviations of biological triplicates (Student’s t-test; *, p ≤ 0.05; **, p ≤ 0.01).
https://doi.org/10.1371/journal.ppat.1013026.s001
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S2 Fig. Analysis of DdGBP topology in the ER membrane.
(A) Sequence alignment of DdGBP and human GBP1 (hGBP1) was made using T-Coffee (www.ebi.ac.uk/ jdispatcher/msa/tcoffee) and analyzed with ESPript 3.0 (espript.ibcp.fr/ESPript/ESPript/) with published (hGBP1) or predicted (DdGBP) structural data (hGBP1 – UniProtKB: P32455, PDB: 1DG3; DdGBP – UniProtKB: Q54TN9). P-Loop, GTPase, and helical domains are displayed, as well as the predicted N-terminal signal peptide and C-terminal transmembrane domains of DdGBP, and the C-terminal CaaX prenylation motif of hGBP1. The α-helical domain is more extended in DdGBP than in hGBP1. (B) Structural model of DdGBP as predicted by AlphaFold3 (AlphaFoldDB: AF-Q54TN9-F1-v4): N-terminus (blue), C-terminus (red). (C) Proteinase K digestion (30 min) using intact or Triton X-100-treated crude membranes of D. discoideum producing the indicated GFP-tagged proteins. Immunoblot using anti-GFP and anti-protein disulfide isomerase (PDI) antibodies is shown (left: molecular weight markers (kDa), below: digestion (%) of GFP-tagged proteins). PDI was protected from proteinase K digestion due to its localization to the lumen of the ER, while GFP-tagged calnexin A (CnxA-GFP) was susceptible to proteinase K treatment, as the GFP tag is positioned towards the cytosol. Both N- and C-terminally GFP-tagged DdGBP was degraded by proteinase K, indicating that DdGBP is anchored in the ER membrane with its GTPase domain facing the cytosol. The data shown is representative of two independent biological replicates. (D) Presumed topology of GFP-tagged DdGBP and CnxA, taking into account the predicted C-terminal transmembrane domain of DdGBP.
https://doi.org/10.1371/journal.ppat.1013026.s002
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S3 Fig. LAI-1-dependent migration inhibition of D. discoideum involves GBP.
D. discoideum Ax2 or Δgnbp producing GFP (pDM317) was left untreated or treated with LAI-1 (1 µM, 5 µM or 10 µM; 1 h) or DMSO (solvent control), and cell migration towards 1 mM folate (4 h) was assessed by under-agarose assay. The white lines represent the edge of the sample wells.
https://doi.org/10.1371/journal.ppat.1013026.s003
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S4 Fig. D. discoideum Δgnbp does not permit replication of L. pneumophila ΔicmT and does not affect L. pneumophila uptake.
(A) D. discoideum Ax2 or Δgnbp was infected (MOI 1, 10 d) with GFP-producing L. pneumophila JR32 or ΔicmT (pNT28), and intracellular replication was assessed by RFU. Data shown are means and standard deviations of biological triplicates (Student’s t-test; *, p ≤ 0.05; **, p ≤ 0.01). (B, C) D. discoideum Ax2 or Δgnbp was treated with LAI-1 (10 µM, 1 h) or DMSO (solvent control), infected (MOI 50, 30 min) with GFP-producing L. pneumophila JR32 (pNT28) and analyzed by flow cytometry. Untreated, uninfected amoebae were used for gating. Data shown are (B) counts vs. GFP fluorescence intensity, and (C) percentage of GFP-positive amoebae (means and standard deviations of biological triplicates).
https://doi.org/10.1371/journal.ppat.1013026.s004
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S5 Fig. GBP does not affect growth of intracellular M. marinum.
(A) D. discoideum Ax2 or Δgnbp was infected (MOI 10) for the time indicated with luciferase-producing M. marinum, and intracellular growth was assessed by bioluminescence. Means and SEM of biological triplicates are shown for three independent Δgnbp clones (c3, c17, c19); r.l.u., relative light units. (B) D. discoideum Ax2 producing GBP-GFP was infected (MOI 10, 1.5 h or 24 h) with mCherry-producing M. marinum, fixed and analyzed by confocal microscopy. Representative maximum projections of live time-lapse spinning disk confocal images are shown. Scale bars, 10 µm. Images are representative of at least 3 independent experiments.
https://doi.org/10.1371/journal.ppat.1013026.s005
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S6 Fig. GBP localizes to ER at LCV-ER contact sites.
Dually labeled D. discoideum Ax2 producing GBP-GFP and (A) calnexin (CnxA)-mCherry (pAW012) or (B) P4C-mCherry (pWS032) was left untreated or treated with LAI-1 (10 µM, 1 h), or DMSO (solvent control), infected (MOI 5, 4 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed, and analyzed by confocal microscopy. Scale bars, 3 µm. Single channels and merge are shown (related to Fig 5).
https://doi.org/10.1371/journal.ppat.1013026.s006
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S7 Fig. LAI-1 prevents expansion of GBP-positive LCVs.
Dually labeled D. discoideum Ax2 producing GBP-GFP and AmtA-mCherry (pDM1044-AmtA-mCherry) was left untreated or treated with LAI-1 (10 µM, 1 h), or DMSO (solvent control), infected (MOI 5, 8 h) with mCerulean-producing L. pneumophila JR32 or ΔicmT (pNP99) and analyzed by confocal microscopy. Scale bars, 3 μm. Single channels and merge are shown (related to Fig 6).
https://doi.org/10.1371/journal.ppat.1013026.s007
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S8 Fig. LAI-1-dependent LCV remodeling involves GBP.
(A) Dually labeled D. discoideum Ax2 or Δgnbp producing calnexin (CnxA)-GFP (pAW016) and P4C-mCherry (pWS032) was treated with LAI-1 (10 µM, 1 h), or DMSO (solvent control), infected (MOI 5, 4 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed and analyzed by confocal laser microscopy. Scale bars, 3 µm. (B) D. discoideum Ax2 or Δgnbp producing CnxA-mCherry (pAW012) or P4C-mCherry (pWS032) was infected (MOI 5, 4 h) with GFP-producing L. pneumophila JR32 harboring pMF16 (P6SRNA-lqsA) or pMF17 (P6SRNA-lqsAK258A), fixed and analyzed by confocal microscopy. Scale bars, 3 µm. Single channels and merge are shown (related to Fig 7).
https://doi.org/10.1371/journal.ppat.1013026.s008
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S9 Fig. LAI-1 and GBP do not affect LCV integrity.
Dually labeled D. discoideum Ax2 or Δgnbp producing cytoplasmic mCherry (pDM1042) and P4C-GFP (pWS034) was left untreated or treated with LAI-1 (10 µM, 1 h), or DMSO (solvent control), infected (MOI 5, 4 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and analyzed by confocal microscopy. Scale bars, 3 μm. Single channels and merge are shown.
https://doi.org/10.1371/journal.ppat.1013026.s009
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S10 Fig. Synthesis and application of azido-(S)-LAI-1.
(A) Reagents and conditions: a) HNMe(OMe)•HCl (1.15 eq.), NMM (1.15 eq.), EDC•HCl (1.15 eq.), CH2Cl2 –15 °C→rt, 65 h, quant.; b) TBDPSCl (1.15 eq.), imidazole (4.60 eq.), DMF, 0 °C→rt→55 °C, 85%; c) DHP (1.50 eq.), PpTs (0.10 eq.), CH2Cl2, 0 °C→rt, 19 h, 99%; d), Mg (8.00 eq.), 4 (2.10 eq.), THF, 0 °C→rt, 16 h, 56%; e) PpTs (0.30 eq.), THF:MeOH (3:1), 60 °C, 20 h, 92%; f) CBr4 (1.50 eq.), PPh3 (1.50 eq.), CH2Cl2, 0 °C→rt, 17 h, 99%; NaN3 (3.00 eq.), DMF, 60 °C, 16 h, quant.; h) TBAF (1 m in THF, 1.20 eq.), THF, 0 °C→rt, 1.5 h, 90%. DHP = 3,4-Dihydro-2H-pyran, DMF = N,N-Dimethylformamide, EDC = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, eq. = equivalents, NMM = 4-Methylmorpholine, PpTs = pyridinium p-toluenesulfonate, quant. = quantitative; TBAF = tetra-n-butylammonium fluoride; TBDPSCl = tert-butyldiphenylsilyl chloride, THF = tetrahydrofuran. (B) Azido-LAI-1 can be attached to various conjugation partners (e.g., dyes) using SPAAC.
https://doi.org/10.1371/journal.ppat.1013026.s010
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S11 Fig. Synthesis and application of diazirine-(S)-LAI-1.
(A) Reagents and conditions: a) DHP (1.50 eq.), PpTs (0.10 eq.), CH2Cl2, 0 °C→rt, 18 h, 95%; b) Mg (8.00 eq.), 10 (2.10 eq.), THF, 0 °C→rt, 16 h, 91%; c) PpTs (0.25 eq.), THF:MeOH (3:1), 60 °C, 20 h, 91%; d) CSA (1.00 eq.), 1,2-ethanediol, (24.6 eq.), ethyl orthoformate (8.30 eq.), 50 °C, 16 h, 74%; e) PDC (3.50 eq.), DMF, rt, 16 h, quant.; f) HNMe(OMe)•HCl (1.15 eq.), NMM (1.15 eq.), EDC•HCl (1.15 eq.), CH2Cl2 0 °C→rt, 49 h, 72%; g) DHP (1.50 eq.), PpTs (0.10 eq.), CH2Cl2, 0 °C→rt, 18 h, 87%; h) Mg (8.00 eq.), 16 (2.35 eq.), THF, 0 °C→rt, 17 h, 91%; i) NH3 (7 m in MeOH, 75 eq.), HOSA (1.15 eq), MeOH, 0 °C→rt, 51 h; j) I2 (1.25 eq.), NEt3 (2.00 eq.), MeOH, 0 °C→rt, 16 h, 42% over two steps; k) I2 (0.10 eq.), acetone (58.4 eq.), 60 °C, 10 min; l)) PpTs (0.25 eq.), THF:MeOH (3:1), 60 °C, 55 h, 89% over two steps; m) CBr4 (1.50 eq.), PPh3 (1.50 eq.), CH2Cl2, 0 °C→rt, 17 h, 73%; n) NaN3 (3.00 eq.), DMF, 60 °C, 16 h, 99%; o) TBAF (1 m in THF, 1.20 eq.), THF, 0 °C→rt, 1.5 h, 85%. CSA = camphorsulfonic acid, PDC = pyridinium dichromate, HOSA = hydroxylamine-O-sulfonic acid. (B) The diazirine function of azido-diazirine-LAI-1 is stimulated by UV light and forms a carbene by releasing nitrogen. The highly reactive carbene can interact with various chemical moieties and thus covalently binds to its biological environment. The covalently fixed azido-LAI-1-derivative can then be attached to various conjugation partners (e.g., dyes, biotin) using SPAAC.
https://doi.org/10.1371/journal.ppat.1013026.s011
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S1 Table. Oligonucleotides used in this study.
https://doi.org/10.1371/journal.ppat.1013026.s012
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References
- 1. Ng W-L, Bassler BL. Bacterial quorum-sensing network architectures. Annu Rev Genet. 2009;43:197–222. pmid:19686078
- 2. Shank EA, Kolter R. New developments in microbial interspecies signaling. Curr Opin Microbiol. 2009;12(2):205–14. pmid:19251475
- 3. Pacheco AR, Sperandio V. Inter-kingdom signaling: chemical language between bacteria and host. Curr Opin Microbiol. 2009;12(2):192–8. pmid:19318290
- 4. Kendall MM, Sperandio V. What a dinner party! Mechanisms and functions of interkingdom signaling in host-pathogen associations. mBio. 2016;7(2):e01748. pmid:26933054
- 5. Hochstrasser R, Hilbi H. Intra-species and inter-kingdom signaling of Legionella pneumophila. Front Microbiol. 2017;8:79. pmid:28217110
- 6. Boamah DK, Zhou G, Ensminger AW, O’Connor TJ. From many hosts, one accidental pathogen: the diverse protozoan hosts of Legionella. Front Cell Infect Microbiol. 2017;7:477. pmid:29250488
- 7. Hoffmann C, Harrison CF, Hilbi H. The natural alternative: protozoa as cellular models for Legionella infection. Cell Microbiol. 2014;16(1):15–26. pmid:24168696
- 8. Swart AL, Harrison CF, Eichinger L, Steinert M, Hilbi H. Acanthamoeba and Dictyostelium as cellular models for Legionella infection. Front Cell Infect Microbiol. 2018;8:61. pmid:29552544
- 9. Newton HJ, Ang DKY, van Driel IR, Hartland EL. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 2010;23(2):274–98. pmid:20375353
- 10. Hilbi H, Hoffmann C, Harrison CF. Legionella spp. outdoors: colonization, communication and persistence. Environ Microbiol Rep. 2011;3(3):286–96. pmid:23761274
- 11. Mondino S, Schmidt S, Rolando M, Escoll P, Gomez-Valero L, Buchrieser C. Legionnaires’ disease: state of the art knowledge of pathogenesis mechanisms of Legionella. Annu Rev Pathol. 2020;15:439–66. pmid:31657966
- 12. Personnic N, Bärlocher K, Finsel I, Hilbi H. Subversion of retrograde trafficking by translocated pathogen effectors. Trends Microbiol. 2016;24(6):450–62. pmid:26924068
- 13. Qiu J, Luo Z-Q. Legionella and Coxiella effectors: strength in diversity and activity. Nat Rev Microbiol. 2017;15(10):591–605. pmid:28713154
- 14. Swart AL, Gomez-Valero L, Buchrieser C, Hilbi H. Evolution and function of bacterial RCC1 repeat effectors. Cell Microbiol. 2020;22(10):e13246. pmid:32720355
- 15. Hilbi H, Buchrieser C. Microbe profile: Legionella pneumophila - a copycat eukaryote. Microbiology (Reading). 2022;168(3):10.1099/mic.0.001142. pmid:35230931
- 16. Lockwood DC, Amin H, Costa TRD, Schroeder GN. The Legionella pneumophila Dot/Icm type IV secretion system and its effectors. Microbiology (Reading). 2022;168(5):10.1099/mic.0.001187. pmid:35639581
- 17. Hubber A, Roy CR. Modulation of host cell function by Legionella pneumophila type IV effectors. Annu Rev Cell Dev Biol. 2010;26:261–83. pmid:20929312
- 18. Asrat S, de Jesús DA, Hempstead AD, Ramabhadran V, Isberg RR. Bacterial pathogen manipulation of host membrane trafficking. Annu Rev Cell Dev Biol. 2014;30:79–109. pmid:25103867
- 19. Steiner B, Weber S, Hilbi H. Formation of the Legionella-containing vacuole: phosphoinositide conversion, GTPase modulation and ER dynamics. Int J Med Microbiol. 2018;308(1):49–57. pmid:28865995
- 20. Swart AL, Hilbi H. Phosphoinositides and the fate of Legionella in phagocytes. Front Immunol. 2020;11:25. pmid:32117224
- 21. Tiaden A, Spirig T, Hilbi H. Bacterial gene regulation by alpha-hydroxyketone signaling. Trends Microbiol. 2010;18(7):288–97. pmid:20382022
- 22. Tiaden A, Hilbi H. α-Hydroxyketone synthesis and sensing by Legionella and Vibrio. Sensors (Basel). 2012;12(3):2899–919. pmid:22736983
- 23. Michaelis S, Gomez-Valero L, Chen T, Schmid C, Buchrieser C, Hilbi H. Small molecule communication of Legionella: the ins and outs of autoinducer and nitric oxide signaling. Microbiol Mol Biol Rev. 2024;88(3):e0009723. pmid:39162424
- 24. Fan M, Kiefer P, Charki P, Hedberg C, Seibel J, Vorholt JA, et al. The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote interbacterial and interkingdom signaling. J Biol Chem. 2023;299(12):105376. pmid:37866633
- 25. Spirig T, Tiaden A, Kiefer P, Buchrieser C, Vorholt JA, Hilbi H. The Legionella autoinducer synthase LqsA produces an alpha-hydroxyketone signaling molecule. J Biol Chem. 2008;283(26):18113–23. pmid:18411263
- 26. Tiaden A, Spirig T, Sahr T, Wälti MA, Boucke K, Buchrieser C, et al. The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila. Environ Microbiol. 2010;12(5):1243–59. pmid:20148929
- 27. Kessler A, Schell U, Sahr T, Tiaden A, Harrison C, Buchrieser C, et al. The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit. Environ Microbiol. 2013;15(2):646–62. pmid:23033905
- 28. Tiaden A, Spirig T, Weber SS, Brüggemann H, Bosshard R, Buchrieser C, et al. The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA. Cell Microbiol. 2007;9(12):2903–20. pmid:17614967
- 29. Tiaden A, Spirig T, Carranza P, Brüggemann H, Riedel K, Eberl L, et al. Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host interactions. J Bacteriol. 2008;190(22):7532–47. pmid:18805977
- 30. Schell U, Kessler A, Hilbi H. Phosphorylation signalling through the Legionella quorum sensing histidine kinases LqsS and LqsT converges on the response regulator LqsR. Mol Microbiol. 2014;92(5):1039–55. pmid:24720786
- 31. Hochstrasser R, Hutter CAJ, Arnold FM, Bärlocher K, Seeger MA, Hilbi H. The structure of the Legionella response regulator LqsR reveals amino acids critical for phosphorylation and dimerization. Mol Microbiol. 2020;113(6):1070–84. pmid:31997467
- 32. Hochstrasser R, Kessler A, Sahr T, Simon S, Schell U, Gomez-Valero L, et al. The pleiotropic Legionella transcription factor LvbR links the Lqs and c-di-GMP regulatory networks to control biofilm architecture and virulence. Environ Microbiol. 2019;21(3):1035–53. pmid:30623561
- 33. Michaelis S, Chen T, Schmid C, Hilbi H. Nitric oxide signaling through three receptors regulates virulence, biofilm formation, and phenotypic heterogeneity of Legionella pneumophila. mBio. 2024;15(6):e0071024. pmid:38682908
- 34. Hochstrasser R, Hilbi H. Legionella quorum sensing meets cyclic-di-GMP signaling. Curr Opin Microbiol. 2020;55:9–16. pmid:32045871
- 35. Striednig B, Hilbi H. Bacterial quorum sensing and phenotypic heterogeneity: how the collective shapes the individual. Trends Microbiol. 2022;30(4):379–89. pmid:34598862
- 36. Personnic N, Striednig B, Hilbi H. Legionella quorum sensing and its role in pathogen-host interactions. Curr Opin Microbiol. 2018;41:29–35. pmid:29190490
- 37. Schell U, Simon S, Sahr T, Hager D, Albers MF, Kessler A, et al. The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophila. Mol Microbiol. 2016;99(4):778–93. pmid:26538361
- 38. Hochstrasser R, Hilbi H. The Legionella Lqs-LvbR regulatory network controls temperature-dependent growth onset and bacterial cell density. Appl Environ Microbiol. 2022;88(5):e0237021. pmid:34985976
- 39. Personnic N, Striednig B, Lezan E, Manske C, Welin A, Schmidt A, et al. Quorum sensing modulates the formation of virulent Legionella persisters within infected cells. Nat Commun. 2019;10(1):5216. pmid:31740681
- 40. Striednig B, Lanner U, Niggli S, Katic A, Vormittag S, Brülisauer S, et al. Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery. EMBO Rep. 2021;22(9):e52972. pmid:34314090
- 41. Personnic N, Striednig B, Hilbi H. Quorum sensing controls persistence, resuscitation, and virulence of Legionella subpopulations in biofilms. ISME J. 2021;15(1):196–210. pmid:32951019
- 42. Simon S, Schell U, Heuer N, Hager D, Albers MF, Matthias J, et al. Inter-kingdom signaling by the Legionella quorum sensing molecule LAI-1 modulates cell migration through an IQGAP1-Cdc42-ARHGEF9-dependent pathway. PLoS Pathog. 2015;11(12):e1005307. pmid:26633832
- 43. Hochstrasser R, Michaelis S, Brülisauer S, Sura T, Fan M, Maaß S, et al. Migration of Acanthamoeba through Legionella biofilms is regulated by the bacterial Lqs-LvbR network, effector proteins and the flagellum. Environ Microbiol. 2022;24(8):3672–92. pmid:35415862
- 44. Tretina K, Park E-S, Maminska A, MacMicking JD. Interferon-induced guanylate-binding proteins: guardians of host defense in health and disease. J Exp Med. 2019;216(3):482–500. pmid:30755454
- 45. Kutsch M, Coers J. Human guanylate binding proteins: nanomachines orchestrating host defense. FEBS J. 2021;288(20):5826–49. pmid:33314740
- 46. Rafeld HL, Kolanus W, van Driel IR, Hartland EL. Interferon-induced GTPases orchestrate host cell-autonomous defence against bacterial pathogens. Biochem Soc Trans. 2021;49(3):1287–97. pmid:34003245
- 47. Kirkby M, Enosi Tuipulotu D, Feng S, Lo Pilato J, Man SM. Guanylate-binding proteins: mechanisms of pattern recognition and antimicrobial functions. Trends Biochem Sci. 2023;48(10):883–93. pmid:37567806
- 48. Kutsch M, Sistemich L, Lesser CF, Goldberg MB, Herrmann C, Coers J. Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions. EMBO J. 2020;39(13):e104926. pmid:32510692
- 49. Santos JC, Boucher D, Schneider LK, Demarco B, Dilucca M, Shkarina K, et al. Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria. Nat Commun. 2020;11(1):3276. pmid:32581219
- 50. Bass AR, Egan MS, Alexander-Floyd J, Lopes Fischer N, Doerner J, Shin S. Human GBP1 facilitates the rupture of the Legionella-containing vacuole and inflammasome activation. mBio. 2023;14(5):e0170723. pmid:37737612
- 51. Feeley EM, Pilla-Moffett DM, Zwack EE, Piro AS, Finethy R, Kolb JP, et al. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems. Proc Natl Acad Sci U S A. 2017;114(9):E1698–706. pmid:28193861
- 52. Liu BC, Sarhan J, Panda A, Muendlein HI, Ilyukha V, Coers J, et al. Constitutive interferon maintains GBP expression required for release of bacterial components upstream of pyroptosis and anti-DNA responses. Cell Rep. 2018;24(1):155-168.e5. pmid:29972777
- 53. Pilla DM, Hagar JA, Haldar AK, Mason AK, Degrandi D, Pfeffer K, et al. Guanylate binding proteins promote caspase-11-dependent pyroptosis in response to cytoplasmic LPS. Proc Natl Acad Sci U S A. 2014;111(16):6046–51. pmid:24715728
- 54. Prakash B, Praefcke GJ, Renault L, Wittinghofer A, Herrmann C. Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins. Nature. 2000;403(6769):567–71. pmid:10676968
- 55. Ji C, Du S, Li P, Zhu Q, Yang X, Long C, et al. Structural mechanism for guanylate-binding proteins (GBPs) targeting by the Shigella E3 ligase IpaH9.8. PLoS Pathog. 2019;15(6):e1007876. pmid:31216343
- 56. Dunn JD, Bosmani C, Barisch C, Raykov L, Lefrançois LH, Cardenal-Muñoz E, et al. Eat prey, live: Dictyostelium discoideum as a model for cell-autonomous defenses. Front Immunol. 2018;8:1906. pmid:29354124
- 57. Katic A, Hüsler D, Letourneur F, Hilbi H. Dictyostelium dynamin superfamily GTPases implicated in vesicle trafficking and host-pathogen interactions. Front Cell Dev Biol. 2021;9:731964. pmid:34746129
- 58. Journet A, Klein G, Brugière S, Vandenbrouck Y, Chapel A, Kieffer S, et al. Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry. Proteomics. 2012;12(2):241–5. pmid:22120990
- 59. Hoffmann C, Finsel I, Otto A, Pfaffinger G, Rothmeier E, Hecker M, et al. Functional analysis of novel Rab GTPases identified in the proteome of purified Legionella-containing vacuoles from macrophages. Cell Microbiol. 2014;16(7):1034–52. pmid:24373249
- 60. Fink J, Schumacher F, Schlegel J, Stenzel P, Wigger D, Sauer M, et al. Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis. Org Biomol Chem. 2021;19(10):2203–12. pmid:33496698
- 61. Sternstein C, Böhm T-M, Fink J, Meyr J, Lüdemann M, Krug M, et al. Development of an effective Functional Lipid Anchor for Membranes (FLAME) for the bioorthogonal modification of the lipid bilayer of mesenchymal stromal cells. Bioconjug Chem. 2023;34(7):1221–33. pmid:37328799
- 62. Rühling M, Kersting L, Wagner F, Schumacher F, Wigger D, Helmerich DA, et al. Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy. Nat Commun. 2024;15(1):7456. pmid:39198435
- 63. Steiner B, Swart AL, Welin A, Weber S, Personnic N, Kaech A, et al. ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila. EMBO Rep. 2017;18(10):1817–36. pmid:28835546
- 64. Hüsler D, Steiner B, Welin A, Striednig B, Swart AL, Molle V, et al. Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole. Cell Microbiol. 2021;23(5):e13318. pmid:33583106
- 65. Hüsler D, Stauffer P, Keller B, Böck D, Steiner T, Ostrzinski A, et al. The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila. Elife. 2023;12:e85142. pmid:37158597
- 66. Nantais DE, Schwemmle M, Stickney JT, Vestal DJ, Buss JE. Prenylation of an interferon-gamma-induced GTP-binding protein: the human guanylate binding protein, huGBP1. J Leukoc Biol. 1996;60(3):423–31. pmid:8830800
- 67. Yamakita I, Mimae T, Tsutani Y, Miyata Y, Ito A, Okada M. Guanylate binding protein 1 (GBP-1) promotes cell motility and invasiveness of lung adenocarcinoma. Biochem Biophys Res Commun. 2019;518(2):266–72. pmid:31421831
- 68. Vormittag S, Hüsler D, Haneburger I, Kroniger T, Anand A, Prantl M, et al. Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling. EMBO Rep. 2023;24(3):e56007. pmid:36588479
- 69. Koliwer-Brandl H, Knobloch P, Barisch C, Welin A, Hanna N, Soldati T, et al. Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol. Cell Microbiol. 2019;21(6):e13008. pmid:30656819
- 70. Weber SS, Ragaz C, Reus K, Nyfeler Y, Hilbi H. Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog. 2006;2(5):e46. pmid:16710455
- 71. Weber S, Wagner M, Hilbi H. Live-cell imaging of phosphoinositide dynamics and membrane architecture during Legionella infection. mBio. 2014;5(1):e00839-13. pmid:24473127
- 72. Weber S, Steiner B, Welin A, Hilbi H. Legionella-containing vacuoles capture PtdIns(4)P-Rich vesicles derived from the Golgi apparatus. mBio. 2018;9(6):e02420-18. pmid:30538188
- 73. Lu H, Clarke M. Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. Cell Microbiol. 2005;7(7):995–1007. pmid:15953031
- 74. Ragaz C, Pietsch H, Urwyler S, Tiaden A, Weber SS, Hilbi H. The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole. Cell Microbiol. 2008;10(12):2416–33. pmid:18673369
- 75. Kagan JC, Roy CR. Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat Cell Biol. 2002;4(12):945–54. pmid:12447391
- 76. Nagai H, Kagan JC, Zhu X, Kahn RA, Roy CR. A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science. 2002;295(5555):679–82. pmid:11809974
- 77. Robinson CG, Roy CR. Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila. Cell Microbiol. 2006;8(5):793–805. pmid:16611228
- 78. Striepen JF, Voeltz GK. Endosome biogenesis is controlled by ER and the cytoskeleton at tripartite junctions. Curr Opin Cell Biol. 2023;80:102155. pmid:36848759
- 79. Feeley JC, Gibson RJ, Gorman GW, Langford NC, Rasheed JK, Mackel DC, et al. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol. 1979;10(4):437–41. pmid:393713
- 80. Horwitz MA. Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med. 1983;158(4):1319–31. pmid:6619736
- 81. Bloomfield G, Tanaka Y, Skelton J, Ivens A, Kay RR. Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum. Genome Biol. 2008;9(4):R75. pmid:18430225
- 82. Loovers HM, Kortholt A, de Groote H, Whitty L, Nussbaum RL, van Haastert PJM. Regulation of phagocytosis in Dictyostelium by the inositol 5-phosphatase OCRL homolog Dd5P4. Traffic. 2007;8(5):618–28. pmid:17343681
- 83. Sadosky AB, Wiater LA, Shuman HA. Identification of Legionella pneumophila genes required for growth within and killing of human macrophages. Infect Immun. 1993;61(12):5361–73. pmid:8225610
- 84. Segal G, Shuman HA. Intracellular multiplication and human macrophage killing by Legionella pneumophila are inhibited by conjugal components of IncQ plasmid RSF1010. Mol Microbiol. 1998;30(1):197–208. pmid:9786196
- 85. Miller MB, Skorupski K, Lenz DH, Taylor RK, Bassler BL. Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. Cell. 2002;110(3):303–14. pmid:12176318
- 86. Perry CJ, Warren EC, Damstra-Oddy JL, Storey C, Francione LM, Annesley SJ, et al. A Dictyostelium discoideum mitochondrial fluorescent tagging vector that does not affect respiratory function. Biochem Biophys Rep. 2020;22:100751. pmid:32258439
- 87. Bärlocher K, Hutter CAJ, Swart AL, Steiner B, Welin A, Hohl M, et al. Structural insights into Legionella RidL-Vps29 retromer subunit interaction reveal displacement of the regulator TBC1D5. Nat Commun. 2017;8(1):1543. pmid:29146912
- 88. Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, Wiles S, Robertson BD, Ripoll J, et al. Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters. PLoS One. 2010;5(3):e9823. pmid:20352111
- 89. Veltman DM, Akar G, Bosgraaf L, Van Haastert PJM. A new set of small, extrachromosomal expression vectors for Dictyostelium discoideum. Plasmid. 2009;61(2):110–8. pmid:19063918
- 90. Barisch C, Paschke P, Hagedorn M, Maniak M, Soldati T. Lipid droplet dynamics at early stages of Mycobacterium marinum infection in Dictyostelium. Cell Microbiol. 2015;17(9):1332–49. pmid:25772333
- 91. Manstein DJ, Schuster HP, Morandini P, Hunt DM. Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene. 1995;162(1):129–34. pmid:7557400
- 92. Wiegand S, Kruse J, Gronemann S, Hammann C. Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning. Genomics. 2011;97(5):321–5. pmid:21316445
- 93. Cosma CL, Humbert O, Ramakrishnan L. Superinfecting mycobacteria home to established tuberculous granulomas. Nat Immunol. 2004;5(8):828–35. pmid:15220915
- 94. Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, et al. Optimisation of bioluminescent reporters for use with mycobacteria. PLoS One. 2010;5(5):e10777. pmid:20520722
- 95. Weber SS, Ragaz C, Hilbi H. The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE. Cell Microbiol. 2009;11(3):442–60. pmid:19021631
- 96. Welin A, Weber S, Hilbi H. Quantitative imaging flow cytometry of Legionella-infected Dictyostelium amoebae reveals the impact of retrograde trafficking on pathogen vacuole composition. Appl Environ Microbiol. 2018;84(11):e00158-18. pmid:29602783
- 97. Weber S, Hilbi H. Live cell imaging of phosphoinositide dynamics during Legionella infection. Methods Mol Biol. 2014;1197:153–67. pmid:25172280
- 98. Knobloch P, Koliwer-Brandl H, Arnold FM, Hanna N, Gonda I, Adenau S, et al. Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes. Cell Microbiol. 2020;22(5):e13163. pmid:31945239
- 99. Lang J, Bohn P, Bhat H, Jastrow H, Walkenfort B, Cansiz F, et al. Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease. Nat Commun. 2020;11(1):1338. pmid:32165633
- 100. Yang Z, Attygalle AB, Meinwald J. Reptilian chemistry: enantioselective syntheses of novel components from a crocodile exocrine secretion. Synthesis. 2000;2000(13):1936–43.
- 101. Corey EJ, Schmidt G. Useful procedures for the oxidation of alcohols involving pyridinium dichromate in aprotic media. Tetrahedron Lett. 1979;20(5):399–402.
- 102. Procacci B, Roy SS, Norcott P, Turner N, Duckett SB. Unlocking a diazirine long-lived nuclear singlet state via photochemistry: NMR detection and lifetime of an unstabilized diazo-compound. J Am Chem Soc. 2018;140(48):16855–64. pmid:30407809
- 103. Wang L, Ishida A, Hashidoko Y, Hashimoto M. Dehydrogenation of the NH-NH bond triggered by potassium tert-butoxide in liquid ammonia. Angew Chem Int Ed Engl. 2017;56(3):870–3. pmid:27936299
- 104. Sun J, Dong Y, Cao L, Wang X, Wang S, Hu Y. Highly efficient chemoselective deprotection of O,O-acetals and O,O-ketals catalyzed by molecular iodine in acetone. J Org Chem. 2004;69(25):8932–4. pmid:15575776
- 105. Monnat J, Hacker U, Geissler H, Rauchenberger R, Neuhaus EM, Maniak M, et al. Dictyostelium discoideum protein disulfide isomerase, an endoplasmic reticulum resident enzyme lacking a KDEL-type retrieval signal. FEBS Lett. 1997;418(3):357–62. pmid:9428745
- 106. Laevsky G, Knecht DA. Under-agarose folate chemotaxis of Dictyostelium discoideum amoebae in permissive and mechanically inhibited conditions. Biotechniques. 2001;31(5):1140–2, 1144, 1146–9. pmid:11730020
- 107. Simon S, Wagner MA, Rothmeier E, Müller-Taubenberger A, Hilbi H. Icm/Dot-dependent inhibition of phagocyte migration by Legionella is antagonized by a translocated Ran GTPase activator. Cell Microbiol. 2014;16(7):977–92. pmid:24397557