Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections.
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.
Department of Surgery, University of Chicago, Chicago, IL 60637, USA.
Bo-Shiun
BS
Lai
William H
WH
Witola
Kamal
K
El Bissati
Ying
Y
Zhou
Ernest
E
Mui
Alina
A
Fomovska
Rima
R
McLeod
D000818:Animals; D016782:Apicomplexa / Q000201:enzymology / Q000254:growth & development; D001426:Bacterial Proteins / Q000235:genetics; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000469:parasitology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods* / Q000592:standards; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D060172:Morpholinos / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014122:Toxoplasma / Q000201:enzymology / Q000254:growth & development*; D014123:Toxoplasmosis / Q000235:genetics / Q000628:therapy*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D060172:Morpholinos; D012333:RNA, Messenger; C054562:yellow fluorescent protein, Bacteria; D008156:Luciferases; D013762:Tetrahydrofolate Dehydrogenase