Perforin-like proteins are expressed by many bacterial and protozoan pathogens, yet little is known about their function or mode of action. Here we describe TgPLP1, a secreted perforin-like protein of the intracellular protozoan pathogen Toxoplasma gondii that displays structural features necessary for pore-formation. Following intracellular growth, TgPLP1-deficient parasites failed to exit normally, resulting in entrapment within host cells. We show that this defect is due to an inability to permeabilize rapidly the parasitophorous vacuole membrane and host plasma membrane during exit. TgPLP1 ablation had little effect on growth in culture, but resulted in a >5-order of magnitude reduction of acute virulence in mice. Perforin-like proteins from other intracellular pathogens may play a similar role in microbial egress and virulence.Perforin (PF) and members of the membrane attack complex (MAC; complement proteins C6-9) are pore-forming proteins of the innate and adaptive immune response that constitute the founding members of the MACPF domain family (1). Recent studies (2,3) have suggested a shared mechanism of pore formation between the MACPF domain and cholesterol-dependent cytolysins, important virulence factors of many pathogenic bacteria (4).Perforin-like proteins (PLPs) are found in the genomes of bacterial (5,6) and protozoan pathogens (7, fig. S1), including the intracellular parasite Toxoplasma gondii. Toxoplasma causes congenital birth defects, ocular disease, and life-threatening encephalitis in immunocompromised individuals (8). It also serves as a model of other parasites in the phylum Apicomplexa (9) that cause important human diseases such as malaria. Despite their expression by many pathogens, no mode of action or pore-forming activity has been demonstrated for any microbial PLP. Here we show that a Toxoplasma perforin-like protein (TgPLP1) aids parasite egress by rapidly compromising the integrity of membranes encasing the parasite.MACPF domain proteins of the mammalian immune system induce cell death by oligomerizing on the surface of target cells and inserting to form large (~100Å diameter) pores (10). The
Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes severe central nervous system disorders of immunocompromised (AIDS/transplant/lymphoma) individuals and birth defects in congenitally infected neonates worldwide (16). Toxoplasma infects a wide range of mammalian hosts and is capable of infecting virtually any nucleated cell type from these organisms. The parasite actively invades its host cell, establishing a specialized parasitophorous vacuole (PV) within the host cytoplasm (22). This vacuole fails to fuse with the host endocytic or exocytic pathways, thus avoiding lysosomal destruction, and provides a residence in which parasites can replicate within the host cell (29, 37). The processes of invasion and vacuole formation therefore establish an intimate yet separate association between the parasite and its host cell.Host cell invasion and PV formation are mediated in part by the action of the rhoptries, specialized secretory organelles that release their contents at the onset of invasion (32). The club-shaped rhoptries are composed of two suborganellar domains, the bulbous rhoptry bodies and the duct-like rhoptry necks. These domains appear to carry out very different roles in host cell invasion and establishment of the intracellular niche for survival. Proteins secreted from the rhoptry necks have recently been shown to be released into the moving junction, a ring-shaped structure that forms the intersection between the invading parasite and the host plasma membrane (1, 6). Rhoptry neck proteins in the moving junction likely serve to filter host transmembrane proteins from the nascent PV during invasion, a process that contributes to the nonfusogenic nature of the vacuole within the host cell. Rhoptry proteins from the other subcompartment, the rhoptry bodies, are secreted into the nascent PV, where they are destined to remain within the vacuole or a...
The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.The single-celled eukaryote Toxoplasma gondii infects warmblooded animals throughout the world. Although the complex life cycle of this obligate intracellular parasite includes sexual and asexual stages, Toxoplasma can propagate asexually indefinitely because sexual reproduction is not required for transmission. The parasite has the capacity to invade any nucleated cell of its host and employs an array of proteins to facilitate invasion and to alter host cell physiology. These proteins are secreted from micronemes, rhoptries, and dense granules, which are specialized secretory organelles unique to organisms of the phylum Apicomplexa.
The phylum Apicomplexa consists of a diverse group of obligate, intracellular parasites. The distinct evolutionary pressures on these protozoans as they have adapted to their respective niches have resulted in a variety of methods that they use to interact with and modify their hosts. One of these is the secretion and trafficking of parasite proteins into the host cell. We review this process for Theileria, Toxoplasma and Plasmodium. We also present what is known about the mechanisms by which parasite proteins are exported into the host cell, as well as information on their known and putative functions once they have reached their final destination.
Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa that is able to infect a wide variety of host cells. During its active invasion process it secretes proteins from discrete secretory organelles: the micronemes, rhoptries and dense granules. Although a number of rhoptry proteins have been shown to be involved in important interactions with the host cell, very little is known about the mechanism of secretion of any Toxoplasma protein into the host cell. We used a chemical inhibitor of phospholipase A2s, 4-bromophenacyl bromide (4-BPB), to look at the role of such lipases in the secretion of Toxoplasma proteins. We found that 4-BPB was a potent inhibitor of rhoptry secretion in Toxoplasma invasion. This drug specifically blocked rhoptry secretion but not microneme secretion, thus effectively showing that the two processes can be de-coupled. It affected parasite motility and invasion, but not attachment or egress. Using propargyl- or azido-derivatives of the drug (so-called click chemistry derivatives) and a series of 4-BPB-resistant mutants, we found that the drug has a very large number of target proteins in the parasite that are involved in at least two key steps: invasion and intracellular growth. This potent compound, the modified “click-chemistry” forms of it, and the resistant mutants should serve as useful tools to further study the processes of Toxoplasma early invasion, in general, and rhoptry secretion, in particular.
Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.Pseudomonas syringae is a phytopathogenic proteobacterium whose host-specific pathovars collectively attack a wide variety of crop plants (21). P. syringae has a type III secretion system (T3SS), which is encoded by hrp and hrc genes (3). The former are so named because they are required (or in operons required) for P. syringae to elicit the defense-associated hypersensitive response (HR) in nonhost plants or to be pathogenic in host plants; the latter encode a subset of nine proteins that are required for the HR and are highly conserved components of the T3SS of both plant and animal pathogens. The Hrp T3SS is required to inject effectors, known as Hop (Hrp outer protein) or Avr (avirulence) proteins, into plant cells, which is an essential process in P. syringae pathogenesis (4).P. syringae pv. syringae strain 61 is a weak pathogen of bean whose Hrp system has been extensively characterized because cosmid pHIR11, which expresses the system, enables nonpathogens such as Pseudomonas fluorescens to secrete harpin proteins in culture and inject test effectors in planta, which facilitates study of the T3SS and the action of individual effectors in activating or suppressing HR and basal defenses (5,25,30,42). Although the tomato and Arabidopsis pathogen P. syringae pv. tomato DC3000 now has emerged as the primary model for studying P. syringae T3SS-related virulence mechanisms (7, 46), much early work on the Hrp system was done with P. syringae pv. syringae 61 (5,8,10,11,20,22,24,26,35,55,58). (As part of this study we have collected the strain 61 hrp-hrc sequences carried on pHIR11 into a single file with GenBank accession number EF514224, and we have also made available the complete sequence, along with a list of corrections [http://pseudomonas-syringae.org].) The Hrp systems of these two strains are functionally similar, and the P. syringae pv. syringae 61 hrp-hrc gene cluster can restore pathogenicity on tomato (but not Arabidopsis) to a DC3000 ⌬hrp-hrc mutant (14). The P. syringae pv. syringae 61 Hrp system is also representative of Hrp1 T3SSs, which are carried by phytopathogens in the Pseudomonadaceae and Enterobacteriaceae and differ in many ways from the Hrp2 T3SSs of phytopathogenic Ralstonia and Xanthomonas spp. (3, 9).
Macrolide antibacterial agents inhibit parasite proliferation by targeting the apicoplast ribosome. Motivated by the long-term goal of identifying antiparasitic macrolides that lack antibacterial activity, we have systematically analyzed the structure-activity relationships among erythromycin analogues and have also investigated the mechanism of action of selected compounds. Two lead compounds, N-benzyl-azithromycin (11) and N-phenylpropyl-azithromycin (30), were identified with significantly higher antiparasitic activity and lower antibacterial activity than erythromycin or azithromycin. Molecular modeling based on the co-crystal structure of azithromycin bound to the bacterial ribosome suggested that a substituent at the N-9 position of desmethyl-azithromycin could improve selectivity due to species-specific interactions with the ribosomal L22 protein. Like other macrolides, these lead compounds display a strong “delayed death phenotype”; however, their early effects on T. gondii replication are more pronounced.
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