A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells

Abstract

Malaria parasites replicate within a parasitophorous vacuole in red blood cells (RBCs). Progeny merozoites egress upon rupture of first the parasitophorous vacuole membrane (PVM), then poration and rupture of the RBC membrane (RBCM). Egress is protease-dependent , but none of the effector molecules that mediate membrane rupture have been identified and it is unknown how sequential rupture of the two membranes is controlled. Minutes before egress, the parasite serine protease SUB1 is discharged into the parasitophorous vacuole where it cleaves multiple substrates including SERA6, a putative cysteine protease. Here, we show that Plasmodium falciparum parasites lacking SUB1 undergo none of the morphological transformations that precede egress and fail to rupture the PVM. In contrast, PVM rupture and RBCM poration occur normally in SERA6-null parasites but RBCM rupture does not occur. Complementation studies show that SERA6 is an enzyme that requires processing by SUB1 to function. RBCM rupture is associated with SERA6-dependent proteolytic cleavage within the actin-binding domain of the major RBC cytoskeletal protein β-spectrin. We conclude that SUB1 and SERA6 play distinct, essential roles in a coordinated proteolytic cascade that enables sequential rupture of the two bounding membranes and culminates in RBCM disruption through rapid, precise, SERA6-mediated disassembly of the RBC cytoskeleton.

The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites

“…SERA5 and SERA6 have been shown to be required for egress from the RBC, which would be after the PfGRP170-GFP-DDD parasites die (Collins, Hackett, Atid, Tan, & Blackman, 2017;Ruecker et al, 2012;Thomas et al, 2018). Seven of the proteins (including PfBiP) have a peak expression pattern around the time PfGRP170-GFP-DDD parasites begin to die.…”

Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes

“…Cysteine proteases were implicated in parasite egress: The broad‐spectrum cysteine protease inhibitor E64 blocks parasite egress by acting on an as‐yet‐unknown target (Salmon et al, ) and preventing erythrocyte membrane rupture (Glushakova et al, ). One candidate for the E64‐sensitive enzyme and an agent of erythrocyte cytoskeletal digestion is the parasite cysteine protease SERA6, which must be processed by SUB1 prior to PVM rupture and then is released in active form into the erythrocyte compartment upon PVM rupture (Thomas et al, ). This possibility is consistent with our data, which showed a very tight temporal association of PVM rupture with the abrupt initiation of the erythrocyte membrane breakdown.…”

“…The high precision of estimated time between PVM rupture and parasite egress shows that leakage of vacuolar content into the erythrocyte compartment is tightly associated with an initiation of morphological changes in the infected erythrocyte, described before (Glushakova et al, , , ; Hale et al, ; Langreth, Jensen, Reese, & Trager, ; Thomas et al, ; Wickham, Culvenor, & Cowman, ) but not previously attributed to the PVM rupture. During this interval of time, lasting on average 5–8 min, and only after PVM rupture, one sees the degradation of the vacuolar membrane and simultaneous transformation of erythrocyte membrane, observed as a gradual collapse of EPM onto the schizont body, possibly reflecting erythrocyte cytoskeleton digestion (Bowyer, Simon, Cravatt, & Bogyo, ; Millholland et al, ).…”

“…Combined observations contradict the notion that human calpain‐1 protease alone is responsible for erythrocyte membrane rupture (Chandramohanadas et al, ; Millholland et al, ). The data presented here and in recent publications reinforce the need for work on the biochemical identity of all proteases and mechanisms involved in erythrocyte cytoskeleton disruption (Das et al, ; Glushakova et al, ; Thomas et al, ).…”

“…At the time of egress, two membranes—the PV membrane (PVM) and the erythrocyte plasma membrane (EPM)—have to be breached by the parasites to egress for the initiation of a new replicative cycle. There is a growing understanding that parasite egress and invasion are two multistep events in parasite life orchestrated by multiple players, both regulatory and catalytic, under the control of parasite signalling systems (Alam et al, ; Beeson et al, ; Blackman & Carruthers, ; Koch & Baum, ; Thomas et al, ). Egress of merozoites, an invasive asexual form of malaria parasites, from erythrocytes was originally described by Trager () as an explosive release of separated parasites from an opening in the erythrocyte membrane.…”