The human malaria parasite Plasmodium falciparum relies on exogenous sources of fatty acids as it undergoes asexual replication within the host erythrocyte. A copious supply of fatty acids is required to support vigorous phospholipid and neutral lipid synthesis during the 48-hour replication cycle, which produces up to 32 daughter parasites (Vial and Ancelin, 1998). While the parasite genome encodes a fatty acid synthase, it can be knocked out in blood-stage parasites and is not considered to be a significant source of fatty acids during asexual replication (Vaughan et al., 2009, Yu et al., 2008). There are two major exogenous sources of fatty acids in the host circulation: unesterified or free fatty acids, which are present in serum at concentrations of ∼200 µM, and esterified fatty acids in the form of lysophosphatidylcholine (LPC), which are present at similar concentrations (Psychogios et al., 2011). Metabolic labeling experiments have revealed that both free fatty acids and those derived from LPC can be incorporated into P. falciparum lipids (Brancucci et al., 2017, Vial et al., 1989, Vial et al., 1982), demonstrating a flexibility with respect to fatty acid acquisition. Parasites can complete a replication cycle in media containing as few as two free fatty acids or LPC species, provided that one each of saturated and unsaturated fatty acid/acyl groups is present (Liu et al., 2024, Mi-Ichi et al., 2007). In addition to supplying fatty acids, LPC provides a critical source of choline for parasite phosphatidylcholine synthesis via the Kennedy pathway (Brancucci et al., 2017, Ramaprasad et al., 2022, Wein et al., 2018). Interestingly, depletion of exogenous LPC serves as an environmental cue for gametocytogenesis, a phenomenon that appears to be related to its provision of choline (Brancucci et al., 2017).

The enzymology of LPC catabolism in P. falciparum-infected erythrocytes has recently come into focus. We have identified two enzymes, termed exported lipase 2 (XL2) and exported lipase homolog 4 (XL4) that are responsible for over 90% of P. falciparum-encoded lysophospholipase activity (Liu et al., 2024). XL2 is exported to the host erythrocyte, whereas XLH4 is retained within the parasite. Both are α/β hydrolase-class serine hydrolases that exhibit A-type lysophospholipase activity, yielding a fatty acid and glycerophosphocholine (GPC) as the LPC hydrolysis products. Disruption of both XL2 and XLH4 reduced the parasite’s ability to scavenge fatty acids from LPC and sensitized parasites to LPC toxicity (Liu et al., 2024). Hydrolysis of GPC to choline and glycerol-3-phosphate is catalyzed by a parasite-encoded glycerophosphodiester phosphodiesterase (GDPD), which is located in the parasitophorous vacuole (PV) and cytosol and is therefore poised to act on GPC generated by both XL2 and XLH4 (Denloye et al., 2012, Ramaprasad et al., 2022). Conditional knockout of GDPD revealed that the enzyme is solely responsible for the release of choline from LPC-derived GPC and that this is an essential function under standard culture conditions (Ramaprasad et al., 2022). Thus, the parasite’s ability to efficiently metabolize LPC may be driven by its requirement for choline.

While XL2 and XLH4 constitute the dominant lysophospholipase activities in parasitized erythrocytes, disruption of both reduced, but did not abolish, the ability of parasites to replicate in a defined culture medium consisting of 16:0 and 18:1 LPC as the sole sources of exogenous fatty acids (Liu et al., 2024). One possible explanation for this finding is the presence of additional parasite-encoded lysophospholipase activities that, while not sufficient to fully compensate for the loss of XL2 and XLH4, are able to provide enough fatty acids to support a lower replication efficiency. Several other parasite serine hydrolases, termed LPL1, LPL3 and LPL20, have been shown to catalyze LPC hydrolysis in vitro (Asad et al., 2021, Sheokand et al., 2021, Sheokand et al., 2023) and are possible candidates; however, their contributions to LPC hydrolysis in vivo have not been defined. Alternately, LPC hydrolysis could be catalyzed by a host-derived lysophospholipase activity that has been detected in human erythrocytes (Podolski et al., 1983, Selle et al., 1993, Tamura et al., 1985).

In this study, we sought to identify the source of the residual lysophospholipase activity in erythrocytes infected with XL2/XLH4-deficient parasites and thereby to more completely define the cohort of enzymes responsible for scavenging fatty acids from LPC. We considered the parasite-encoded, α/β hydrolase-family enzyme “prodrug activation and resistance esterase” (PARE) to be a leading candidate, as prior activity-based profiling studies have revealed that PARE is an abundant serine hydrolase that is inhibited by the pan-lysophospholipase inhibitor AKU-010 (Liu et al., 2024). PARE was first characterized as a catalyst of pepstatin ester hydrolysis and its inactivation resulted in parasite resistance toward this compound (Istvan et al., 2017). A similar resistance-conferring activity has since been demonstrated for two unrelated ester-containing compounds (Butler et al., 2020, Sindhe et al., 2020), which suggests that PARE exhibits a promiscuous esterase activity. It is potently inhibited by monoacylglycerol lipase inhibitors (Elahi et al., 2019) and the lipid-like natural product Salinipostin A (Yoo et al., 2020), which suggests that it may have a lipid substrate, although its physiological substrate and role in the asexual parasite are currently unknown.

To test whether PARE contributes to LPC catabolism in XL/XLH-deficient parasites, we modified the PARE allele in a previously-described quadruple knockout line (QKO: ΔXL1/XL2/XLH3/XLH4; (Liu et al., 2024)) in order to enable a conditional knockdown approach. The effect of PARE knockdown on in situ LPC metabolism in P. falciparum-infected erythrocytes and on parasite growth on LPC as a sole source of exogenous fatty acids was assessed. In parallel, the effect of a functional PARE knockout on a wild-type background was examined to determine the effect of a PARE deletion in the presence of XL/XLH enzymes. The subcellular distribution of PARE was determined to provide a spatial context for its contribution to LPC hydrolysis.