RNG1 is a late marker of the apical polar ring in Toxoplasma gondii.

The asexually proliferating stages of apicomplexan parasites cause acute symptoms of diseases such as malaria, cryptosporidiosis and toxoplasmosis. These stages are characterized by the presence of two independent microtubule organizing centers (MTOCs). Centrioles are found at the poles of the intranuclear spindle. The apical polar ring (APR), a MTOC unique to apicomplexans, organizes subpellicular microtubules which impose cell shape and apical polarity on these protozoa. Here we describe the characteristics of a novel protein that localizes to the APR of Toxoplasma gondii which we have named ring-1 (RNG1). There are related RNG1 proteins in Neospora caninum and Sarcocystis neurona but no obvious homologs in Plasmodium spp., Cryptosporidium spp. or Babesia spp. RNG1 is a small, low-complexity, detergent-insoluble protein that assembles at the APR very late in the process of daughter parasite replication. We were unable to knock-out the RNG1 gene, suggesting that its gene product is essential. Tagged RNG1 lines have also allowed us to visualize the APR during growth of Toxoplasma in the microtubule-disrupting drug oryzalin. Oryzalin inhibits nuclear division and cytokinesis although Toxoplasma growth continues, and similar to earlier observations of unchecked centriole duplication in oryzalin-treated parasites, the APR continues to duplicate during aberrant parasite growth.

The cell-penetrating peptide, Pep-1, has activity against intracellular chlamydial growth but not extracellular forms of Chlamydia trachomatis.

OBJECTIVES: In the course of studies to identify novel treatment strategies against the pathogenic bacterium, Chlamydia, we tested the carrier peptide, Pep-1, for activity against an intracellular infection. METHODS: Using a cell culture model of Chlamydia trachomatis infection, the effect of Pep-1 was measured by incubating the peptide with extracellular chlamydiae prior to infection, or by adding Pep-1 to the medium at varying times after infection, and assaying for inhibition of inclusion formation. RESULTS: Pep-1 had a concentration-dependent effect on chlamydial growth with 100% inhibition of inclusion formation at 8 mg/L peptide. There was a window of susceptibility during the chlamydial developmental cycle with a maximal effect when treatment was begun within 12 h of infection. Pep-1 treatment caused a severe reduction in the production of infectious progeny even when started later, when the effect on inclusion formation was minimal. Furthermore, electron micrographs showed a paucity of progeny elementary bodies (EBs) in the inclusion. In contrast, pre-incubation of EBs with Pep-1 prior to infection did not affect inclusion formation. Taken together, these findings indicate that the antichlamydial effect was specific for the intracellular stage of chlamydial infection. By comparison, Pep-1 had no antimicrobial activity against Escherichia coli and Staphylococcus aureus or the obligate intracellular parasite, Toxoplasma gondii. CONCLUSIONS: Pep-1 has antichlamydial activity by preventing intracellular chlamydial growth and replication but has no effect on extracellular chlamydiae.