Raykov, L. | bioRxiv Science

bioRxiv2024

Inter-kingdom signaling by the Legionella autoinducer LAI-1 involves the antimicrobial guanylate binding protein GBP

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 -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 ({Delta}gnbp) allows more efficient intracellular replication of L. pneumophila, without apparently compromising LCV remodeling 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 SummarySmall molecule inter-kingdom signaling between pathogens and host cells represents a crucial but only partly understood aspect of microbial virulence. The amoeba-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 GTPases 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.

microbiology↗

bioRxiv2021

The Dictyostelium discoideum E3 ubiquitin ligase TrafE coordinates endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum.

Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage, recognised as severe stress and controlled downstream by the endosomal sorting complex required for transport (ESCRT) and the autophagy machineries that are recruited to damaged membranes to either repair or to remove membrane remnants. Yet little is known about the upstream endolysosomal damage response (ELDR) factors that sense damage and lead to extensive tagging of the damaged organelles with signals, such as K63-polyubiquitin, required for the recruitment of ELDR components. To explore ELDR key factors responsible for detection and marking of damaged compartments we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase, TrafE, that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical compounds. TrafE acts at the intersection of ESCRT and autophagy pathways and plays a key role in functional recruitment of the ESCRT subunits ALIX, Vps32 and Vps4 to damage sites or maturing autophagosomes. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of bacteria as well as ESCRT-mediated and autophagy-mediated ELDR, resulting in early cell death.

microbiology↗