A Plasmodium cysteine protease required for efficient transition from the liver infection stage

Abstract

The transitions between developmental stages are critical points in the Plasmodium life cycle. The development of Plasmodium in the livers of their mammalian hosts bridges malaria transmission and the onset of clinical symptoms elicited by red blood cell infection. The egress of Plasmodium parasites from the liver must be a carefully orchestrated process to ensure a successful switch to the blood stage of infection. Cysteine protease activity is known to be required for liver-stage Plasmodium egress, but the crucial cysteine protease(s) remained unidentified. Here, we characterize a member of the papain-like cysteine protease family, Plasmodium berghei serine repeat antigen 4 (PbSERA4), that is required for efficient initiation of blood-stage infection. Through the generation PbSERA4-specific antisera and the creation of transgenic parasites expressing fluorescently tagged protein, we show that PbSERA4 is expressed and proteolytically processed in the liver and blood stages of infection. Targeted disruption of PbSERA4 results in viable and virulent blood-stage parasites. However, upon transmission from mosquitoes to mice, Pbsera4(-) parasites displayed a reduced capacity to initiate a new round of asexual blood-stage replication. Our results from cultured cells indicate that this defect results from an inability of the PbSERA4-deficient parasites to egress efficiently from infected cells at the culmination of liver-stage development. Protection against infection with wildtype P. berghei could be generated in animals in which Pbsera4(-) parasites failed to establish infection. Our findings confirm that liver-stage merozoite release is an active process and demonstrate that this parasite-encoded cysteine protease contributes to parasite escape from the liver. Plasmodium parasites cause over 200 million cases of malaria every year. When parasites are transmitted by mosquito bite, they initially colonize the liver before they move into the blood and cause disease. During successful transition from the liver into the blood, Plasmodium cloak themselves in host plasma membrane as they egress from the liver cells. Although some aspects of how Plasmodium exit their host hepatocytes appear unique, certain attributes are shared across diverse pathogens. For example, protease activity is required not only for multiple stages of Plasmodium exit, but is also involved in the egress of some bacteria and other protozoan. Here we characterize a protease in Plasmodium berghei that is expressed in the liver and conserved across Plasmodium species. Through gene targeting, we found PbSERA4 is required for efficient egress of Plasmodium from the liver. In the absence of this protease the transition between the liver and blood stages of growth is prolonged due to inefficient parasite release from liver cells. These findings provide new insights into the function of a conserved Plasmodium protease and into the process of Plasmodium escape from the liver.