Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii.

A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms. Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution. Significant progress in studying the cell biology of this parasite has been accomplished over the last few years. Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T. gondii. In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.

Toxoplasma gondii ADP-ribosylation factor 1 mediates enhanced release of constitutively secreted dense granule proteins.

Toxoplasma gondii dense granules are morphologically similar to dense matrix granules in specialized secretory cells, yet are secreted in a constitutive, calcium-independent fashion. We previously demonstrated that secretion of dense granule proteins in permeabilized parasites was augmented by the non-hydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (Chaturvedi, S., Qi, H., Coleman, D. L., Hanson, P., Rodriguez, A., and Joiner, K. A. (1998) J. Biol. Chem. 274, 2424-2431). As now demonstrated by pharmacological and electron microscopic approaches, GTPgammaS enhanced release of dense granule proteins in the permeabilized cell system. To investigate the role of ADP-ribosylation factor 1 (ARF1) in this process, a cDNA encoding T. gondii ARF1 (TgARF1) was isolated. Endogenous and transgenic TgARF1 localized to the Golgi of T. gondii, but not to dense granules. An epitope-tagged mutant of TgARF1 predicted to be impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal, with localization to scattered vesicles, whereas a mutant impaired in nucleotide binding (T31N) was cytosolic in location. Both mutants caused partial dispersion of a Golgi/trans-Golgi network marker. TgARF1 mutants inhibited delivery of the secretory reporter, Escherichia coli alkaline phosphatase, to dense granules, precluding an in vivo assessment of the role of TgARF1 in release of intact dense granules. To circumvent this limitation, recombinant TgARF1 was purified using two separate approaches, and used in the permeabilized cell assay. TgARF1 protein purified on a Cibacron G3 column and able to bind GTP stimulated dense granule secretion in the permeabilized cell secretion assay. These results are the first to show that ARF1 can augment release of constitutively secreted vesicles at the target membrane.

The HSP70 gene family in Pneumocystis carinii: molecular and phylogenetic characterization of cytoplasmic members.

Pneumocystis carinii, a major opportunistic lung pathogen of AIDS patients, is found in a number of mammals and is proposed to be a member of the fungi. In this work, several members of the highly conserved HSP70 multigene family were characterized from rat-derived P. carinii. Previously, we reported characterization of the ER resident HSP70 homolog known as BiP from prototype (P.c. carinii) and variant (P. c. rattus) strains of the organism. We report here, from P. c. carinii, characterization of Pcsa1, an HSP70 homolog that encodes a cognate/stress-induced HSP70 homolog of the SSA subfamily in Saccharomyces cerevisiae. We also identify, from both rat strains and from a human isolate of P. carinii (P.c. hominis), a third set of HSP70 homologs that apparently encode a ribosome-associated cytoplasmic HSP70 homologous to the S. cerevisiae SSB subfamily. Our data indicate that Pcsal mRNA, like Pcbip mRNA, bears an intron in the 5' untranslated region, is induced by heat shock, and suggest that this gene undergoes alternative transcription and splicing. The SSB homologs display significant sequence heterogeneity between P. carinii source strains, supporting the genetic divergence and likely speciation of P. carinii isolates within and between host species. Phylogenetic analysis with the PcSA1 protein supports inclusion of P. carinii among the higher fungi.

Identification, characterization, and expression of the BiP endoplasmic reticulum resident chaperonins in Pneumocystis carinii.

We have isolated, characterized, and examined the expression of the genes encoding BiP endoplasmic reticulum (ER) resident chaperonins responsible for transport, maturation, and proper folding of membrane and secreted proteins from two divergent strains of Pneumocystis carinii. The BiP genes, Pcbip and Prbip, from the P. c. carinii (prototype) strain and the P. c. rattus (variant) strain, respectively, are single-copy genes that reside on chromosomes of approximately 330 and approximately 350 kbp. Both genes encode approximately 72.5-kDa proteins that are most homologous to BiP genes from other organisms and exhibit the amino-terminal signal peptides and carboxyl-terminal ER retention sequences that are hallmarks of BiP proteins. We established short-term P. carinii cultures to examine expression and induction of Pcbip in response to heat shock, glucose starvation, inhibition of protein transport or N-linked glycosylation, and other conditions known to affect proper transport, glycosylation, and maturation of membrane and secreted proteins. These studies indicated that Pcbip mRNA is constitutively expressed but induced under conditions known to induce BiP expression in other organisms. In contrast to mammalian BiP genes but like other fungal BiP genes, P. carinii BiP mRNA levels are induced by heat shock. Finally, the Prbip and Pcbip coding sequences surprisingly exhibit only approximately 83% DNA and approximately 90% amino acid sequence identity and show only limited conservation in noncoding flanking and intron sequences. Analyses of the P. carinii BiP gene sequences support inclusion of P. carinii among the fungi but suggest a large divergence and possible speciation among P. carinii strains infecting a given host.

An arrayed bacteriophage P1 genomic library of Pneumocystis carinii.

We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of approximately 4800 independent clones with an average insert size of approximately 55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4-5 positive clones for each, consistent with a several-fold coverage of the approximately 10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library of P. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen.