A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites
Introduction
Intracellular pathogens have evolved to reside and proliferate within a multitude of host-cell types. Pore-forming proteins (PFPs) are emerging as a major part of the tool set used by a variety of prokaryotic and eukaryotic pathogens. PFPs play important roles in host-cell entry, nutrient acquisition, escape from the phagolysosomal system and final egress from the host cell [1]. Pathogenic bacteria employ PFPs to deliver activators of actin-mediated uptake mechanisms into the host cell leading to engulfment and internalization of the pathogen [2]. Protozoan pathogens, however, do not employ this strategy of induced passive uptake by non-phagocytic cells. While the protozoan parasite Leishmania is taken up passively by its phagocytic host cells [3] others like most apicomplexan parasites use a unique acto/myosin motor to actively enter their non-phagocytic host cells [4] and PFP involvement in this mode of entry has not been described. After host-cell entry the pathogen may grow and replicate in a vacuolar compartment [5], [6]. Life in a vacuole poses new challenges and PFPs may be used for the efficient uptake of nutrients across the vacuolar membrane [7] in order to sustain growth and replication or to avoid destruction by the host-cell phagolysosomal system [8]. Finally, after completion of the intracellular phase the pathogen must exit its host cell (egress) [9], [10] and PFPs are employed by some pathogens for egress [10].
The malaria parasite Plasmodium is a single-cell obligate intracellular parasite that cycles between a mosquito vector and a vertebrate host. Distinct stages of the parasite actively invade, replicate and egress from a number of different host cells. Considering their multiple interactions with target cells Plasmodia should be considered as prime suspect PFP producers. The merozoite stage invades red blood cells, replicates inside a vacuolar compartment, the parasitophorous vacuole (PV) and egresses to rapidly invade new red blood cells [11]. The motile ookinete stage penetrates the mosquito midgut epithelium and lyses the epithelial cell barrier to reach the basal lamina where it transforms into an oocyst [12]. Sporozoite stages that are released from a mature oocyst invade salivary gland cells, transmigrate through the cytoplasm and exit into the secretory cavity. When an infected mosquito bites, motile salivary gland sporozoite stages are then deposited into the skin of the host were they must traverse cellular barriers before entering the bloodstream that allows their transport to the liver. Here, they exit from the blood vessel, invade hepatocytes and transform into hepatic stages that reside in a PV where they grow and replicate, producing thousands of first generation merozoites [13]. Upon maturity merozoites must egress from the hepatocyte to invade red blood cells. It is conceivable that each parasite stage may utilize PFPs for its distinct interactions with host cells. Pores that may serve nutrient acquisition have been described in the PV of blood stages [14] yet the constituents of this pore remain to be characterized. Thus, we speculated that the Plasmodium genome [15], [16] should encode proteins with predicted pore-forming activity and the identification of such PFPs is an important step towards understanding the parasites interactions with target membranes.
Here, we describe five related genes, highly conserved among Plasmodium species, each encoding a putative secreted protein bearing a membrane-attack complex/perforin (MACPF)-like domain. This domain is well characterized in pore-forming proteins such as components of the mammalian complement system [17] and perforin [18]. We show that one Plasmodium Perforin-Like Protein (PPLP) is expressed in the secretory micronemes of the sporozoite stage and may thus function in its interaction with the mosquito and/or during the initial infection of the mammalian host.
Section snippets
Parasites
Anopheles stephensi mosquitoes were reared at 28 °C, 75% humidity under a 14-h light/10-h dark cycle and adults were fed on a 10% sucrose solution. 4–5-day-old female mosquitoes were blood-fed on anesthetized Balb/c mice that had been infected with the Plasmodium yoelii strain 17X. Rodents were assayed for parasitemia by blood smear and the abundance of gametocyte-stage parasites capable of exflagellation by wet-mount. After the infective blood meal, mosquitoes were maintained at 24 °C, 80%
Identification of a conserved Plasmodium gene family encoding perforin-like proteins
We initially observed expression of a multi-exon gene by identification of a cluster of 13 expressed sequences tags (ESTs) that were present in a salivary gland sporozoite-specific cDNA library of P. yoelii [19]. The complete open reading frame (ORF) and the exon/intron structure of the gene was determined by comparison of cDNA sequence generated from sporozoite RNA and the genomic DNA sequence available through the P. yoelii genome sequence database [16] (Fig. 1, Fig. 2). The spliced ORF
Discussion
Although a variety of pore-forming protein families have been identified in a number of prokaryotic and eukaryotic pathogens [1] there is little known about pore-forming proteins of the malaria parasite Plasmodium. We have herein described PPLPs, a new family of putative secreted Plasmodium proteins with each member bearing a single MACPF domain. This domain is present in pore-forming proteins of the mammalian complement system and perforin. The membrane-attack complex (MAC) of the complement
Acknowledgements
We thank the Wellcome Trust Sanger Institute (UK) for making genome sequence data publicly available before publication. This work was supported in part by a grant from the National Institute of Health (AI 053709) to S.H.I.K. The microarray work was supported by an NIH grant (R01 AI41879) to A.B.V.
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2022, Journal of Molecular BiologyThe key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell
2021, International Journal for ParasitologyCitation Excerpt :In T. gondii, PLP1 allows the tachyzoites to egress from an exhausted host cell to re-invade a new fresh one (Kafsack et al., 2009; Roiko and Carruthers, 2013). In Plasmodium spp. some PLPs are involved in egress while others facilitate movement of the parasite through different epithelia (Kadota et al., 2004; Kaiser et al., 2004; Ishino et al., 2005; Ecker et al., 2007; Amino et al., 2008; Deligianni et al., 2013, 2018; Garg et al., 2013, 2015, 2020; Wirth et al., 2014, 2015; Risco-Castillo et al., 2015; Yang et al., 2017). To date, all characterized PLPs are located in micronemes, an apicomplexan type of secretory organelles that play an important role in invasion and egress from the host cell, with the exception of Plasmodium berghei PLP4, which is located in apical vesicles, different from micronemes (Deligianni et al., 2018).
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2020, Molecular and Cellular ProteomicsImportant Extracellular Interactions between Plasmodium Sporozoites and Host Cells Required for Infection
2019, Trends in ParasitologyMembrane attack complex-associated molecules from redlip mullet (Liza haematocheila): Molecular characterization and transcriptional evidence of C6, C7, C8β and C9 in innate immunity
2018, Fish and Shellfish ImmunologyCitation Excerpt :There have also been no reports about TCC genes in the genomes of sharks and lampreys. Additionally, there are proteins that contain the MACPF domain but lack the other TCC-specific domains, that have been found in many organisms, including invertebrates [41,42], protozoans [43], plants [44], and bacteria [45]. Other TCC-specific domains such as TSP1, LDLa, CCP, and FIMAC regulate the interactions of the complement components.
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Present address: Seattle Biomedical Research Institute, Seattle, WA 98109-1651, USA.