LC3-association with the parasitophorous vacuole membrane ofPlasmodium bergheiliver stages follows a noncanonical autophagy pathway

Abstract

SummaryEukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with P. berghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in P. berghei- infected ATG5−/− host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3-independent pathway. | INTRODUCTIONMalaria, with more than 200 million estimated cases and more than 400 thousand deaths per year, remains one of the most devastating diseases worldwide (World Health Organization, 2015). Once Plasmodium sporozoites are injected by an infected female Anopheles mosquito during blood feeding, they migrate to the liver and infect hepatocytes by the formation of a parasitophorous vacuole (PV).Although the PV membrane (PVM) originates from invagination of the host cell plasma membrane, it is remodelled by the parasite upon infection (Spielmann, Montagna, Hecht, & Matuschewski, 2012).Inside the vacuole, the parasite starts its exoerythrocytic development by transforming into a multinuclear schizont that undergoes massive replication. Finally, several thousand erythrocyte-infective merozoites are formed and safely released into the blood within merosomes (Sturm et al., 2006). Within the hepatocyte, the Plasmodium parasite shows one of the fastest replication rates among eukaryotes, necessitating that massive amounts of nutrients are obtained from the host cell (Bano, Romano, Jayabalasingham, & Coppens, 2007).At the same time, the parasite needs to escape defence mechanisms of the host cell. homeostasis, cells can use selective autophagy to remove damaged or deleterious elements from the cytoplasm. Selective autophagy can also serve as a cellular antimicrobial defence against intracellular pathogens, a process called xenophagy (Deretic & Levine, 2009;Knodler & Celli, 2011;Levine, Mizushima, & Virgin, 2011;Mostowy, 2013). Starvation-induced and selective autophagy are both characterised by the formation of double membrane autophagosomes that sequester the autophagic cargo (Levine et al., 2011;Mizushima & Komatsu, 2011). In mammalian cells, starvation initiates autophag...

The plant–pathogen haustorial interface at a glance

“…Another similarity between plant-pathogen and mammalianparasite interfaces is the induction of autophagy responses that are directed towards the pathogens, which are contained in modified phagosomal compartments, similar to recent observations with P. infestans (Dagdas et al, 2016(Dagdas et al, , 2018. For instance, components of mammalian autophagy machinery such as ATG8 (the LC3/ GABRAP family in mammalian cells) as well as the autophagy cargo receptors p62 (also known as SQSTM) and NBR1 target the peri-microbial membrane interface engulfing the Plasmodium parasite (Schmuckli-Maurer et al, 2017;Wacker et al, 2017;Real et al, 2018) (see poster). Interestingly, similar to the P. infestans RXLR effector PexRD54, one of the PVM-embedded plasmodium effector proteins, called UIS3, binds to the mammalian ATG8 isoform LC3 to avoid being degraded by autophagy (Real et al, 2018).…”

“…Autophagy is a conserved eukaryotic trafficking process, in which cellular components and microbes are removed or relocated after engulfment in vesicular double-membrane-enclosed structures called autophagosomes (Lamb et al, 2013;Dagdas et al, 2018). Interestingly, selective forms of autophagy are induced at the perimicrobial interfaces in both plant and metazoan cells to counteract pathogen invasion (Thurston et al, 2012;Choi et al, 2014;Haldar et al, 2014;Schmuckli-Maurer et al, 2017;Wacker et al, 2017;Dagdas et al, 2018;Real et al, 2018). In plants, a defense-related autophagy machinery comprising the autophagy cargo receptor NBR1 (also known as Joka2) and the core autophagy adaptor ATG8 (ATG8CL isoform) target the EHM during P. infestans infection (see poster) (Dagdas et al, 2016).…”

Melanin processing by keratinocytes: A non‐microbial type of host‐pathogen interaction?

“…While most parasites can escape autophagy‐dependent clearance, the mechanisms involved are only now starting to be unravelled. For example, host LC3 is incorporated into the Plasmodium PVM early on during hepatocyte infection . LC3 incorporation and inhibition of autophagic degradation is mediated by the parasite protein, Up‐regulated in Infective Sporozoites gene 3 (UIS3).…”

Identification of Plasmodium falciparum proteoforms from liver stage models

“…After sporozoites enter the hepatocyte cytoplasm, they form a parasite vacuolar membrane that interfaces with the host autophagy system [35][36][37]. Parasites have designed a system to escape this endogenous cytoplasmic immunity that involves disrupting autophagy and lysosome interactions with the parasitophorous vacuole membrane (PVM) [38].…”