In Silico Identification of Specialized Secretory-Organelle Proteins in Apicomplexan Parasites and In Vivo Validation in Toxoplasma gondii

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions

Mechanistic Elucidation of the Stepwise Formation of a Tetranuclear Manganese Pinned Butterfly Cluster via N–N Bond Cleavage, Hydrogen Atom Transfer, and Cluster Rearrangement

A mechanistic pathway for the formation of the structurally characterized manganese-amide-hydrazide pinned butterfly complex, Mn₄(μ₃‑PhN-NPh-κ³<i>N</i>,<i>N</i>′)₂­(μ‑PhN-NPh-κ²-<i>N</i>,<i>N</i>′)­(μ‑NHPh)₂L₄ (L = THF, py), is proposed and supported by the use of labeling studies, kinetic measurements, kinetic competition experiments, kinetic isotope effects, and hydrogen atom transfer reagent substitution, and via the isolation and characterization of intermediates using X-ray diffraction and electron paramagnetic resonance spectroscopy. The data support a formation mechanism whereby bis­[bis­(trimethylsilyl)­amido]­manganese­(II) (Mn­(NR₂)₂, where R = SiMe₃) reacts with <i>N</i>,<i>N</i>′-diphenyl­hydrazine (PhNHNHPh) via initial proton transfer, followed by reductive N–N bond cleavage to form a long-lived Mn^IV imido multinuclear complex. Coordinating solvents activate this cluster for abstraction of hydrogen atoms from an additional equivalent of PhNHNHPh resulting in a Mn­(II)­phenylamido dimer, Mn₂­(μ‑NHPh)₂­(NR₂)₂L₂. This dimeric complex further assembles in fast steps with two additional equivalents of PhNHNHPh replacing the terminal silylamido ligands with η¹-hydrazine ligands to give a dimeric Mn₂­(μ‑NHPh)₂­(PhN-NHPh)₂L₄ intermediate, and finally, the addition of two additional equivalents of Mn­(NR₂)₂ and PhNHNHPh gives the pinned butterfly cluster