Epigenetic gene regulation has emerged as a major mechanism for gene regulation in all eukaryotes. Histones are small, basic proteins that constitute the major protein component of chromatin, and posttranslational modifications (PTM) of histones are essential for epigenetic gene regulation. The different combinations of histone PTM form the histone code for an organism, marking functional units of chromatin that recruit macromolecular complexes that govern chromatin structure and regulate gene expression. To characterize the repertoire of Toxoplasma gondii histone PTM, we enriched histones using standard acid extraction protocols and analyzed them with several complementary middle-down and bottom-up proteomic approaches with the high-resolution Orbitrap mass spectrometer using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and/or electron transfer dissociation (ETD) fragmentation. We identified 249 peptides with unique combinations of PTM that comprise the T. gondii histone code. T. gondii histones share a high degree of sequence conservation with human histones, and many modifications are conserved between these species. In addition, T. gondii histones have unique modifications not previously identified in other species. Finally, T. gondii histones are modified by succinylation, propionylation, and formylation, recently described histone PTM that have not previously been identified in parasitic protozoa. The characterization of the T. gondii histone code will facilitate in-depth analysis of how epigenetic regulation affects gene expression in pathogenic apicomplexan parasites and identify a new model system for elucidating the biological functions of novel histone PTM.

SummarySuccessful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin-like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore-forming toxin, we conclude that rupture of the erythrocyte membrane is blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforinlike protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.

Protein ubiquitination plays key roles in protein turnover, cellular signalling, and intracellular transport. The genome of Toxoplasma gondii encodes ubiquitination machinery but the roles of this posttranslational modification (PTM) are unknown. To examine the prevalence and function of ubiquitination in T. gondii, we mapped the ubiquitin proteome of tachyzoites. Over 500 ubiquitin-modified proteins, with almost 1000 sites, were identified on proteins with diverse localisations and functions. Enrichment analysis demonstrated that 35% of ubiquitinated proteins are cell cycle-regulated. Unexpectedly, most classic cell cycle regulators conserved in T. gondii were not detected in the ubiquitinome. Furthermore, many ubiquitinated proteins localise to the cytoskeleton and inner membrane complex, a structure beneath the plasma membrane facilitating division and host invasion. Comparing the ubiquitinome with other PTM proteomes reveals waves of PTM enrichment during the cell cycle. Thus, T. gondii PTMs are implicated as critical regulators of cell division and cell cycle transitions.

The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important merozoite surface and PV proteins. These include the essential merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptryassociated protein 1) and the merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target.Malaria is a huge global health threat, causing immense suffering and up to 2.7 million fatalities per annum worldwide, largely in children below the age of five. There is no licensed malaria vaccine, and while some effective drugs are available at present, the parasite often rapidly acquires resistance in the face of drug pressure. Recent reports of resistance to some of the latest artemisinin-based antimalarial drugs are particularly alarming (22,76), stressing the need to expand our understanding of the basic biology of the malaria parasite and to seek parasite-specific processes that can be exploited as new drug targets. Malaria is caused by obligate intracellular protozoan parasites of the genus Plasmodium, transmitted by female Anopheline mosquitoes. Of the five Plasmodium species that cause human malaria, Plasmodium falciparum is responsible for the most acute disease.Like other apicomplexan pathogens, P. falciparum is an obligate intracellular parasite, and all the clinical manifestations of malaria result from replication of the parasite in circulating erythrocytes. Following erythrocyte invasion, the parasite occupies a membrane-bound parasitophorous vacuole (PV) where it divides asexually to form a mature schizont containing 16 or more daughter merozoites. In a poorly understood process called egress, the enclosing PV and residual host erythrocyte membranes eventually rupture, releasing the merozoites, which at once invade fresh erythrocytes to perpetuate the cycle. Successive cycles of replication lead to increasing parasitemia and pathology. Both egress and subsequent invasion can be blocked by b...

Pathogens have evolved strategies to promote their survival by dramatically modifying the transcriptional profile and protein content of the host cells they infect. Modifications of the host transcriptome and proteome are mediated by pathogen-encoded effector molecules that modulate host cells through a variety of different mechanisms. Recent studies highlight the importance of the host chromatin and other epigenetic regulators as targets of pathogens. Host gene regulatory mechanisms may be targeted through cytoplasmic signaling, directly by pathogen effector proteins, and possibly by pathogen RNA. Although many of these changes are short-lived and persist only during the course of infection, several studies indicate that pathogens are able to induce long-term, heritable changes that are essential to pathogenesis of infectious diseases and persistence of pathogens within their hosts. In this review, we discuss how pathogens modulate the epigenome of host cells, a new and flourishing avenue of host-pathogen interaction studies.