http://orcid.org/0000-0002-7828-8118
ABSTRACT
The malaria causative parasite, Plasmodium, has received intense focus due to its complex life cycle and threat to human health. When infecting human hepatocytes, the parasite manages to escape clearance by macroautophagy/autophagy. In a recent paper by Real et al., the authors discovered that the parasitophorous vacuole (PV) membrane protein UIS3 encoded by Plasmodium interacts with MAP1LC3/LC3, an important component of the autophagy machinery. This interaction interferes with the association between LC3 and its receptors, which helps the parasite avoid sequestration by a phagophore, and subsequent elimination by the host. This study expands our knowledge about the Plasmodium-host interaction, as well as provides information for a potential anti-Plasmodium drug target.
Malaria has been a major threat to global health for many years, putting more than 3 million people at risk in approximately 100 countries [Citation1]. The causative parasite, Plasmodium, is drawing increasing interest in the field of microbiology and cell biology. Plasmodium has a complex life cycle alternating between mosquitos and human. After the parasites are transferred to a human through a blood feeding, the sporozoites first infect hepatocytes, termed the liver stage, where they reproduce asexually into merozoites. Release from liver cells is followed by a blood stage where Plasmodium infect red blood cells. Exit from the hepatocytes appears to involve a process whereby the parasite becomes enwrapped within host cell plasma membrane in the form of a merosome [Citation2]; using host membrane likely serves as part of the mechanism for avoiding the host defense against invasive pathogens.
Autophagy is an important degradation system in cells; its functions vary from removing damaged proteins or organelles to serving as a part of innate immunity. However, parasites such as Plasmodium manage to escape autophagy-dependent clearance. The details of the ‘hidden’ mechanism behind this evasion, as well as the interaction between the parasite and host, are not completely known. The replication of Plasmodium takes place within a membrane-bound compartment referred to as a parasitophorous vacuole (PV), which protects the parasites from host attack. In the recent study by Real et al., the authors reported that the PV membrane protein UIS3 interacts with host LC3 to prevent autophagy-mediated clearance by the host cells [Citation3].
UIS3 is important for Plasmodium replication [Citation4]. Accordingly, the authors first investigated the role of UIS3 in parasite survival during the liver stage using HepG2 cells. Plasmodium lacking UIS3 (uis3−) show impaired replication as expected when infecting hepatocytes. Interestingly, knocking down host cell ATG5, which is important for LC3 conjugation to phosphatidylethanolamine, and RAB7, which is important for fusion between autophagosomes and lysosomes, restores replication of the UIS3-lacking parasites. This phenotype indicated that UIS3 functions to protect parasites from autophagy clearance.
Because successful replication in the liver stage will release blood infective merozoites within merosomes, the authors next looked into whether uis3− parasites can produce merosomes when autophagy is impaired; they collected cell culture supernatant from uis3+- or uis3--infected cells and use them to infect mice. uis3+ parasites from both wild-type and ATG5- or RAB7-deficient cells can produce merosomes and infect mice. Conversely, uis3− parasites can only infect mice when recovered from cells lacking ATG5 or RAB7, but not wild-type cells. The authors also observed a similar phenotype in mouse embryonic fibroblasts, where knocking out different Atg genes increases the survival rates of uis3− parasites. These results, taken together, indicate that UIS3 is important for Plasmodium to escape autophagic surveillance.
The authors next investigated how UIS3 cross-talks with the autophagy machinery. Immunofluorescence data indicate that UIS3 colocalizes with host LC3 at the PV membrane, an observation that is further supported by co-immunoprecipitation data. The authors also measured the binding affinity by between these proteins through surface plasmon resonance. The resulting dissociation constant (KD), 0.244 μM, indicates a strong interaction between LC3 and UIS3. Proteins that interact with LC3 usually contain an LC3-interacting region (LIR) [Citation5]. However, sequence analysis does not reveal the presence of a LIR within UIS3. Thus, the authors used a molecular docking algorithm to establish a potential model for the interaction. This model predicts that the interaction happens between helices α2 and α3 of UIS3 and the β2 strand of LC3. Mutations in several key residues in these regions results in a diminished interaction between the 2 proteins, without causing an obvious change to the structure of the proteins.
A logical direction for these studies was to examine whether the UIS3-LC3 interaction is the mechanism through which UIS3 interferes with autophagy. SQSTM1/p62 is a well-studied autophagy receptor and substrate, that interacts with Atg8-family members such as LC3; degradation of SQSTM1 serves as one method for detecting autophagic flux. Monitoring SQSTM1 degradation in the presence of different forms of UIS3, revealed that the UIS3 mutants that show diminished LC3-interaction display normal SQSTM1 degradation. Conversely, wild-type UIS3 results in an SQSTM1 degradation defect. This result indicates that UIS3 functions as an autophagy inhibitor via its interaction with LC3. Furthermore, this finding raises the possibility that UIS3 may compete with other autophagy substrates in binding LC3 to block autophagy. To test this hypothesis, the authors conducted a luminescence-based mammalian interactome mapping (LUMIER) competition assay. In this experiment, UIS3 and SQSTM1 were fused to luciferase and co-incubated with GST-LC3. Increasing concentrations of SQSTM1 LIR fragments were added as a competitor, and luciferase activity was measured for affinity-isolated proteins. The results showed that a higher concentration of the SQSTM1 LIR is needed to compete with UIS3 in binding LC3 compared to SQSTM1, indicating that UIS3 has a higher binding affinity for LC3.
This work sheds light on the potential mechanism of how some parasites escape autophagy clearance, and provides more information about the cross-talk between the parasite and the host autophagy machinery. The study also elucidated the autophagy-relevant function of the parasite protein UIS3, which provides useful information for drug design and potential vaccine discovery for the treatment of malaria patients.
Disclosure statement
No potential conflict of interest was reported by the authors.
Additional information
Funding
References
- Cowman AF, Healer J, Marapana D, et al. Malaria: biology and disease. Cell. 2016;167(3):610–624.
- Graewe S, Rankin KE, Lehmann C, et al. Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress. PLoS Pathog. 2011;7(9):e1002224.
- Real E, Rodrigues L, Cabal GG, et al. Plasmodium UIS3 sequesters host LC3 to avoid elimination by autophagy in hepatocytes. Nat Microbiol. 2018;3(1):17–25.
- Spielmann T, Montagna GN, Hecht L, et al. Molecular make-up of the Plasmodium parasitophorous vacuolar membrane. Int J Med Microbiol. 2012;302(4–5):179–186.
- Birgisdottir AB, Lamark T, Johansen T. The LIR motif - crucial for selective autophagy. J Cell Sci. 2013;126(Pt 15):3237–3247.