Although waves of bloodstream-stage parasite (merozoite) replication typify the pathology of Plasmodium falciparum malaria, surprisingly little is known about how parasites exit from infected erythrocytes. A paper published in Cell reveals that the PfSUB1 subtilisin-like serine protease is required for the exit of merozoites from infected erythrocytes. This enzyme is sequestered in exonemes, which are newly discovered parasite organelles.

During the bloodstream stage of the malaria life cycle, merozoites that are derived either from infected eryth-rocytes or infected hepatocytes infect erythrocytes by a well-characterized process that involves three organelles that are present in the apical part of the cell: rhoptries, micronemes and dense granules. These organelles mediate merozoite attachment, invasion and formation of the parasi-tophorous vacuole (PV), in which the parasite grows. Eventually, numerous daughter merozoites erupt out of the infected erythrocyte, but to escape they must breach the membranes of the PV and the cell.

PfSUB1 was identified by Blackman and colleagues in 1998 as one of three subtilisin-like serine proteases that are present in P. falciparum. Here, Yeoh et al. sought to probe the role of PfSUB1 in the biology of malaria. Unsuccessful attempts to disrupt the pfsub1 gene proved that it is essential. By tagging the carboxyl terminus of PfSUB1 with a triple haemagglutinin (HA) tag to yield PfSUB1HA3, they were

able to scrutinize the subcel-lular localization of PfSUB1. In accordance with their previous observations, PfSUB1 was localized to the api-cal part of the cell, but was absent from rhop-tries and micronemes. Using antibodies to ring-infected erythrocyte surface antigen (RESA), a marker protein for dense granules, and antibodies to either the HA tag or the PfSUB1 protein, they showed that PfSUB1 is present in a subset of dense granules with an altered morphology that they named exonemes. Further studies showed that although RESA was released in newly infected erythro-cytes, PfSUB1 was only found in the supernatants of ruptured maturing merozoites (schizonts). This led the authors to propose that PfSUB1 was involved in a pre-invasion event, such as merozoite egress.

A large-scale screen of more than 170,000 low-molecular-weight compounds led Yeoh et al. to iden-tify an inhibitor that is specific for PfSUB1. By adding this inhibitor to synchronized P. falciparum cultures, they showed that PfSUB1 activity is required for schizont rupture and erythrocyte re-invasion. An analysis of parasite proteins from cultures treated with excess inhibitor identi-fied the P. falciparum protease serine repeat antigen 5 (SERA5; a member of the nine-gene fam-ily of serine repeat antigens of P. falciparum) as a potential target of PfSUB1. The SERA proteases are

associated with destruction of the PV and the erythrocyte membrane.

Using purified proteins, Yeoh et al. confirmed that PfSUB1 proc-esses inactive SERA5 to an active form and also processes SERA4 and SERA6; all three of these SERA pro-teins are expressed in the PV. These intriguing results led the authors to propose that PfSUB1 is released from the exoneme into the PV when the schizonts are mature, where it activates the SERA4, 5 and 6 pro-teases. The SERA proteases might then disrupt the PV, and perhaps even the erythrocyte membrane, thereby enabling the merozoites to escape.

Clarifying the roles of the dif-ferent proteases in egress will be a major focus of future work and might ultimately provide a new impetus in the search for improved therapeutics to treat the millions of people who are infected with P. falciparum.

Susan Jones

M a l a r i a

Malaria eats out

Original research PaPer Yeoh, S. et al. Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell 131, 1072–1083 (2007)

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nATURE REVIEwS | microbiology VolUmE 6 | FEBRUARY 2008

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