cyclic-gmp and Toxoplasmosis

To elicit effective invasion and egress from infected cells, obligate intracellular parasites of the phylum Apicomplexa rely on the timely and spatially controlled exocytosis of specialized secretory organelles termed the micronemes. The effector molecules and signaling events underpinning this process are intricate; however, recent advances within the field of Toxoplasma gondii research have facilitated a broader understanding as well as a more integrated view of this complex cascade of events and have unraveled the importance of phosphatidic acid (PA) as a lipid mediator at multiple steps in this process.

Topics: Animals; Calcium; Cyclic GMP; Exocytosis; Host-Parasite Interactions; Humans; Organelles; Phosphatidic Acids; Protein Transport; Protozoan Proteins; Signal Transduction; Toxoplasma; Toxoplasmosis

Apicomplexan parasites rely on cyclic nucleotide-dependent kinases for host cell infection, yet the mechanisms that control their activation remain unknown. Here we show that an apically localized guanylate cyclase (GC) controls microneme secretion and lytic growth in the model apicomplexan Toxoplasma gondii. Cell-permeable cGMP reversed the block in microneme secretion seen in a knockdown of TgGC, linking its function to production of cGMP. TgGC possesses an N-terminal P-type ATPase domain fused to a C-terminal heterodimeric guanylate cyclase domain, an architecture found only in Apicomplexa and related protists. Complementation with a panel of mutants revealed a critical requirement for the P-type ATPase domain for maximum GC function. We further demonstrate that knockdown of TgGC in vivo protects mice from lethal infection by blocking parasite expansion and dissemination. Collectively, this work demonstrates that cGMP-mediated signaling in Toxoplasma relies on a multi-domain architecture, which may serve a conserved role in related parasites.

Topics: Animals; Cyclic GMP; Female; Fibroblasts; Gene Knockdown Techniques; Guanylate Cyclase; Humans; Mice; Mice, Inbred C57BL; P-type ATPases; Protozoan Proteins; Toxoplasma; Toxoplasmosis; Virulence

Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKG

Topics: Acylation; Animals; Cell Membrane; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Editing; Humans; Indoleacetic Acids; Phosphorylation; Protein Isoforms; Protozoan Proteins; Signal Transduction; Toxoplasma; Toxoplasmosis

Host cell invasion, exit and parasite dissemination is critical to the pathogenesis of apicomplexan parasites such as Toxoplasma gondii and Plasmodium spp. These processes are regulated by intracellular Ca

Topics: Calcium Signaling; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Host-Parasite Interactions; Humans; Toxoplasma; Toxoplasmosis

The apical complex is the definitive cell structure of phylum Apicomplexa, and is the focus of the events of host cell penetration and the establishment of intracellular parasitism. Despite the importance of this structure, its molecular composition is relatively poorly known and few studies have experimentally tested its functions. We have characterized a novel Toxoplasma gondii protein, RNG2, that is located at the apical polar ring--the common structural element of apical complexes. During cell division, RNG2 is first recruited to centrosomes immediately after their duplication, confirming that assembly of the new apical complex commences as one of the earliest events of cell replication. RNG2 subsequently forms a ring, with the carboxy- and amino-termini anchored to the apical polar ring and mobile conoid, respectively, linking these two structures. Super-resolution microscopy resolves these two termini, and reveals that RNG2 orientation flips during invasion when the conoid is extruded. Inducible knockdown of RNG2 strongly inhibits host cell invasion. Consistent with this, secretion of micronemes is prevented in the absence of RNG2. This block, however, can be fully or partially overcome by exogenous stimulation of calcium or cGMP signaling pathways, respectively, implicating the apical complex directly in these signaling events. RNG2 demonstrates for the first time a role for the apical complex in controlling secretion of invasion factors in this important group of parasites.

Topics: Cell Line; Cyclic GMP; Gene Knockdown Techniques; Humans; Protozoan Proteins; Signal Transduction; Toxoplasma; Toxoplasmosis

The ability of Toxoplasma gondii to cycle between the tachyzoite and bradyzoite life stages in intermediate hosts is key to parasite survival and the pathogenesis of toxoplasmosis. Studies from a number of laboratories indicate that differentiation in T. gondii is a stress-induced phenomenon. The signalling pathways or molecular mechanisms that control formation of the latent bradyzoite stage are unknown and specific effectors of differentiation have not been identified. We engineered a reporter parasite to facilitate simultaneous comparison of differentiation and replication after various treatments. Chloramphenicol acetyltransferase (CAT), expressed constitutively from the alpha-tubulin promoter (TUB1), was used to quantitate parasite number. beta-galactosidase (beta-GAL), expressed from a bradyzoite specific promoter (BAG1), was used as a measure of bradyzoite gene expression. Sodium nitroprusside, a well-known inducer of bradyzoite differentiation, reduced reporter parasite replication and caused bradyzoite differentiation. Stress-induced differentiation in many other pathogens is regulated by cyclic nucleotide kinases. Specific inhibitors of the cAMP dependent protein kinase and apicomplexan cGMP dependent protein kinase inhibited replication and induced differentiation. The beta-GAL/CAT reporter parasite provides a method to quantify and compare agents that cause differentiation in T. gondii.

Topics: 1-Methyl-3-isobutylxanthine; Animals; beta-Galactosidase; Cell Differentiation; Chloramphenicol O-Acetyltransferase; Colforsin; Culture Media; Cyclic AMP; Cyclic GMP; Gene Expression Regulation, Developmental; Genes, Protozoan; Genes, Reporter; Heat-Shock Proteins; Life Cycle Stages; Nitroprusside; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Protozoan Proteins; Signal Transduction; Toxoplasma; Toxoplasmosis; Tubulin

Topics: Animals; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Lymphocytes; Lymphokines; Macrophages; Mice; Toxoplasma; Toxoplasmosis