Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections

Abstract

Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii's fluorescence and luminescence. PPMO directed against T. gondii's dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO's specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.protozoan | Apicomplexan | toxoplasmosis | protein translation

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

“…PPMOs are produced following conjugation of a specific MO to a cell-penetrating, arginine-rich peptide, whereas VMOs are synthesized as conjugates between a MO molecule and an octa-guanidinium head group (13,17,24). To date, VMOs have been used to downregulate gene expression in human fibroblasts, mice, zebrafish, Xenopus oocytes, Toxoplasma gondii, and other model organisms (20,(25)(26)(27)(28)(29). However, these conjugates have not yet been assessed for their use in functional analysis of P. falciparum genes.…”

Toxoplasmosis and Vision Impairment

“…The parasite endures throughout a person's life and although research for a cure is ongoing, there is currently no treatment, which can eliminate it from the human system, nor an available vaccine. Dr Rima McLeod and her team of researchers at the University of Chicago, however, have been seeking to develop a successful approach using strands of DNA with a small peptide, inserted into cells which contain the parasite, in an attempt to disrupt genetic signals (Lai et al, 2012). The new treatment was tested in tissue culture containing infected cells and in live mice, artifi cially infected with the parasite.…”

Spiroindolone That Inhibits PfATPase4 Is a Potent, Cidal Inhibitor of Toxoplasma gondii Tachyzoites In Vitro and In Vivo

“…(5) The knockdown of T. gondii ATPase4 (TgATP4) with exon skipping or a translation-inhibiting peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO; a molecular transporter-conjugated antisense morpholino) (7) but not offtarget PPMO (7) showed that TgATP4 was also essential for replication (Fig. 1F).…”

“…Once the most effective spiroindolone is identified, the mode of solubilization and administration can be optimized. A variety of models are available for such future studies (1,4,7,12,13).…”

“…1A) was synthesized as described previously (5) by Reagents 4 Research. In vitro and in vivo assays were conducted as recently reported (1,6,7), with every experiment repeated at least twice (see the supplemental material). The significance of differences was determined using Student's t test, with a P value of Ͻ0.05 considered significant.…”