Host Cell Egress and Invasion Induce Marked Relocations of Glycolytic Enzymes in Toxoplasma gondii Tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K+] experienced during egress and invasion, a signal that requires changes of [Ca2+]c in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites

Proteomic Analysis of Fractionated Toxoplasma Oocysts Reveals Clues to Their Environmental Resistance

Toxoplasma gondii is an obligate intracellular parasite that is unique in its ability to infect a broad range of birds and mammals, including humans, leading to an extremely high worldwide prevalence and distribution. This work focuses on the environmentally resistant oocyst, which is the product of sexual replication in felids and an important source of human infection. Due to the difficulty in producing and working with oocysts, relatively little is known about how this stage is able to resist extreme environmental stresses and how they initiate a new infection, once ingested. To fill this gap, the proteome of the wall and sporocyst/sporozoite fractions of mature, sporulated oocysts were characterized using one-dimensional gel electrophoresis followed by LC-MS/MS on trypsin-digested peptides. A combined total of 1021 non-redundant T. gondii proteins were identified in the sporocyst/sporozoite fraction and 226 were identified in the oocyst wall fraction. Significantly, 172 of the identified proteins have not previously been identified in Toxoplasma proteomic studies. Among these are several of interest for their likely role in conferring environmental resistance including a family of small, tyrosine-rich proteins present in the oocyst wall fractions and late embryogenesis abundant domain-containing (LEA) proteins in the cytosolic fractions. The latter are known from other systems to be key to enabling survival against desiccation

Transcriptomic Analysis of Toxoplasma Development Reveals Many Novel Functions and Structures Specific to Sporozoites and Oocysts

Sexual reproduction of Toxoplasma gondii occurs exclusively within enterocytes of the definitive felid host. The resulting immature oocysts are excreted into the environment during defecation, where in the days following, they undergo a complex developmental process. Within each oocyst, this culminates in the generation of two sporocysts, each containing 4 sporozoites. A single felid host is capable of shedding millions of oocysts, which can survive for years in the environment, are resistant to most methods of microbial inactivation during water-treatment and are capable of producing infection in warm-blooded hosts at doses as low as 1–10 ingested oocysts. Despite its extremely interesting developmental biology and crucial role in initiating an infection, almost nothing is known about the oocyst stage beyond morphological descriptions. Here, we present a complete transcriptomic analysis of the oocyst from beginning to end of its development. In addition, and to identify genes whose expression is unique to this developmental form, we compared the transcriptomes of developing oocysts with those of in vitro-derived tachyzoites and in vivo-derived bradyzoites. Our results reveal many genes whose expression is specifically up- or down-regulated in different developmental stages, including many genes that are likely critical to oocyst development, wall formation, resistance to environmental destruction and sporozoite infectivity. Of special note is the up-regulation of genes that appear “off” in tachyzoites and bradyzoites but that encode homologues of proteins known to serve key functions in those asexual stages, including a novel pairing of sporozoite-specific paralogues of AMA1 and RON2, two proteins that have recently been shown to form a crucial bridge during tachyzoite invasion of host cells. This work provides the first in-depth insight into the development and functioning of one of the most important but least studied stages in the Toxoplasma life cycle

A SNARE involved in protein transport through the Golgi apparatus

In eukaryotic cells, the Golgi apparatus receives newly synthesized proteins from the endoplasmic reticulum (ER) and delivers them after covalent modification to their destination in the cell. These proteins move from the inside (cis) face to the plasma-membrane side (trans) of the Golgi, through a stack of cisternae, towards the trans-Golgi network (TGN), but very little is known about how proteins are moved through the Golgi compartments. In a model known as the maturation model, no special transport process was considered necessary, with protein movement along the Golgi being achieved by maturation of the cisternae. Alternatively, proteins could be transported by vesicles or membrane tubules. Although little is known about membrane-tubule-mediated transport, the molecular mechanism for vesicle-mediated transport is quite well understood, occurring through docking of SNAREs on the vesicle with those on the target membrane. We have now identified a protein of relative molecular mass 27K which is associated with the Golgi apparatus. The cytoplasmic domain of this protein or antibodies raised against it quantitatively inhibit transport in vitro from the ER to the trans-Golgi/TGN, acting at a stage between the cis/medial- and the trans-Golgi/TGN. This protein, which behaves like a SNARE and has been named GS27 (for Golgi SNARE of 27K), is identical to membrin, a protein implicated earlier in ER-to-Golgi transport. Our results suggest that protein movement from medial- to the trans-Golgi/TGN depends on SNARE-mediated vesicular transport