Many intracellular pathogens, including Toxoplasma gondii, survive within macrophages by residing in vacuoles that avoid fusion with lysosomes. It is important to determine whether cell-mediated immunity can trigger macrophage antimicrobial activity by rerouting these vacuoles to lysosomes. We report that CD40 stimulation of human and mouse macrophages infected with T. gondii resulted in fusion of parasitophorous vacuoles and late endosomes/lysosomes. Vacuole/lysosome fusion took place even when CD40 was ligated after the formation of parasitophorous vacuoles. Genetic and pharmacological approaches that impaired phosphoinositide-3-class 3 (PIK3C3), Rab7, vacuolar ATPase, and lysosomal enzymes revealed that vacuole/lysosome fusion mediated antimicrobial activity induced by CD40. Ligation of CD40 caused colocalization of parasitophorous vacuoles and LC3, a marker of autophagy, which is a process that controls lysosomal degradation. Vacuole/lysosome fusion and antimicrobial activity were shown to be dependent on autophagy. Thus, cell-mediated immunity through CD40 stimulation can reroute an intracellular pathogen to the lysosomal compartment, resulting in macrophage antimicrobial activity.

Apicomplexan parasites divide and replicate through a complex process of internal budding. Daughter cells are preformed within the mother on a cytoskeletal scaffold, endowed with a set of organelles whereby in the final stages the mother disintegrates and is recycled in the emerging daughters. How the cytoskeleton and the various endomembrane systems interact in this dynamic process remains poorly understood at the molecular level. Through a random YFP fusion screen we have identified two Toxoplasma gondii proteins carrying multiple membrane occupation and recognition nexus (MORN) motifs. MORN1 is highly conserved among apicomplexans. MORN1 specifically localizes to ring structures at the apical and posterior end of the inner membrane complex and to the centrocone, a specialized nuclear structure that organizes the mitotic spindle. Time-lapse imaging of tagged MORN1 revealed that these structures are highly dynamic and appear to play a role in nuclear division and daughter cell budding. Overexpression of MORN1 resulted in severe but specific defects in nuclear segregation and daughter cell formation. We hypothesize that MORN1 functions as a linker protein between certain membrane regions and the parasite's cytoskeleton. Our initial biochemical analysis is consistent with this model. Whereas recombinant MORN1 produced in bacteria is soluble, in the parasite MORN1 was associated with the cytoskeleton after detergent extraction.

Summary The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member sub-family of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatio-temporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.

Toxoplasmosis is the clinical and pathological consequence of acute infection with the obligate intracellular apicomplexan parasite Toxoplasma gondii. Symptoms result from tissue destruction that accompanies lytic parasite growth. This review updates current understanding of the host cell invasion, parasite replication and eventual egress that comprise the lytic cycle, as well as the ways T. gondii manipulates host cells to assure survival. Since the publication of a previous iteration of this review 15 years ago, important advances have been made in our molecular understanding of parasite growth and mechanisms of host cell egress, and knowledge of the parasite’s manipulation of the host has rapidly progressed. Here we cover molecular advances and current conceptual frameworks that include each of these topics, with an eye to what might be known 15 years from now.

Nucleotide metabolic pathways provide numerous successful targets for antiparasitic chemotherapy, but the human pathogen Cryptosporidium parvum thus far has proved extraordinarily refractory to classical treatments. Given the importance of this protist as an opportunistic pathogen afflicting immunosuppressed individuals, effective treatments are urgently needed. The genome sequence of C. parvum is approaching completion, and we have used this resource to critically assess nucleotide biosynthesis as a target in C. parvum. Genomic analysis indicates that this parasite is entirely dependent on salvage from the host for its purines and pyrimidines. Metabolic pathway reconstruction and experimental validation in the laboratory further suggest that the loss of pyrimidine de novo synthesis is compensated for by possession of three salvage enzymes. Two of these, uridine kinase-uracil phosphoribosyltransferase and thymidine kinase, are unique to C. parvum within the phylum Apicomplexa. Phylogenetic analysis suggests horizontal gene transfer of thymidine kinase from a proteobacterium. We further show that the purine metabolism in C. parvum follows a highly streamlined pathway. Salvage of adenosine provides C. parvum's sole source of purines. This renders the parasite susceptible to inhibition of inosine monophosphate dehydrogenase, the rate-limiting enzyme in the multistep conversion of AMP to GMP. The inosine 5 monophosphate dehydrogenase inhibitors ribavirin and mycophenolic acid, which are already in clinical use, show pronounced anticryptosporidial activity. Taken together, these data help to explain why widely used drugs fail in the treatment of cryptosporidiosis and suggest more promising targets.Cryptosporidium parvum ͉ horizontal gene transfer ͉ drug target ͉ thymidine kinase ͉ IMPDH