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The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

ABSTRACT: The facultative intracellular bacterium Legionella pneumophila employs the Icm/Dot type IV secretion system (T4SS) to replicate in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). The ER-resident large fusion GTPase Sey1/atlastin-3 promotes remodeling and expansion of LCVs, and the GTPase is also implicated in the formation of ER-derived lipid droplets (LDs). Here we reveal that LCVs intimately interact with palmitate-induced LDs in Dictyostelium discoideum amoeba. Comparative proteomics of LDs isolated from the D. discoideum parental strain Ax3 or sey1 revealed 192 differentially produced proteins, of which 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or sey1, respectively. Using dually fluorescence-labeled amoeba producing the LCV marker P4C-GFP or AmtA-GFP and the LD marker mCherry-perilipin, we show that Sey1 and the L. pneumophila Icm/Dot T4SS as well as the effector LegG1 promote LCV-LD interactions. In vitro reconstitution of the LCV-LD interactions using purified LCVs and LDs from D. discoideum Ax3 or sey1 revealed that Sey1 and GTP promote this process. The LCV-LD interactions were impaired forsey1-derived LDs, suggesting that Sey1 regulates LD traits. Palmitate promoted the growth of L. pneumophila in D. discoideum Ax3 but not in sey1 and of wild-type but not mutant bacteria lacking a homologue of the E. coli fatty acid transporter FadL. Finally, isotopologue profiling indicated that intracellular L. pneumophila metabolizes 13C-palmitate, and its catabolism was reduced in D. discoideum sey1 and L. pneumophila fadL. Taken together, our results reveal that Sey1 mediates LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Dictyostelium Discoideum (slime Mold)

SUBMITTER: Sandra Maass

LAB HEAD: Dörte Becher

PROVIDER: PXD038200 | Pride | 2023-06-07

REPOSITORIES: Pride

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			Action	DRS
	220429_dictybase_primaryProteins.fasta	Fasta
	220712_VE1_PM4_AOs_2022-023_Dario_Ax3_A_01.raw	Raw
	220712_VE1_PM4_AOs_2022-023_Dario_Ax3_A_02.raw	Raw
	220712_VE1_PM4_AOs_2022-023_Dario_Ax3_A_03.raw	Raw
	220712_VE1_PM4_AOs_2022-023_Dario_Ax3_A_04.raw	Raw

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Project description:The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires’ Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs), leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR), requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as inhibition of host protein synthesis.

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The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

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