Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum

Abstract

Graphical abstract

Morpholino-mediated in vivo silencing of Cryptosporidium parvum lactate dehydrogenase decreases oocyst shedding and infectivity

“…Morpholinos specific for inhibiting the translation of Cp LDH and C. parvum sporozoite 60K protein ( Cp 15/60) mRNA were designed as previously described ( Witola et al, 2017 ). Briefly, each morpholino was designed as a 25 base sequence including the start codon and downstream bases that would specifically bind to its complementary target mRNA site via Watson–Crick pairing, and sterically block the translation initiation complex.…”

“…Equal amounts of the samples were fractionated by SDS–PAGE and transferred onto nitrocellulose membranes. Immunoblotting was performed using mono-specific purified rat anti- Cp LDH antibodies ( Witola et al, 2017 ) or Cp 15/60 sporozoite 60K protein monoclonal antibody (LifeSpan Biosciences, Inc., USA) at 1:200 dilution as primary antibodies. Horseradish peroxidase (HRP)-conjugated chicken anti-rat (ThermoFisher Scientific, USA) and HRP-goat anti-mouse (ThermoFisher Scientific, USA) were used as secondary antibodies at 1:2000 dilution.…”

“…In efforts aimed at developing genetic tools for studying Cryptosporidium , a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene knockout system for Cryptosporidium was reported ( Vinayak et al, 2015 ). We have also recently reported an antisense approach for an in vitro targeted gene knockdown system in C. parvum using phosphorodiamidate morpholino oligomers (morpholinos) ( Witola et al, 2017 ). However, Cryptosporidium can only be cultured transiently in vitro ( Upton et al, 1994 ), and this does not allow for development of all the life cycle stages of the parasite.…”

Recent Breakthroughs and Ongoing Limitations in Cryptosporidium Research

“…Morpholinos are synthetic DNA analogs that inhibit protein translation initiation by base pairing to complementary mRNA. In a recent study, excysted sporozoites and HCT-8 cells were treated with morpholinos targeting C. parvum lactate dehydrogenase (CpLDH) and putative arginine n-methyltransferase (CpAMT) prior to in vitro infection, resulting in decreased protein expression of CpLDH and CpAMT 37 . In a follow-up study, morpholinos optimized for in vivo delivery against CpLDH and Cp15/60 were injected intraperitoneally into interferon-gamma knockout mice, which then were infected with C. parvum oocysts.…”

State of the Art and Future Directions ofCryptosporidiumspp.

“…No fully effective drug therapy or vaccine is available for Cryptosporidium, and the diagnosis of cryptosporidiosis has been based on the demonstration of oocysts in faeces, which present low sensibility [25]. However, the ability to culture relevant Cryptosporidium isolates in vitro, the development of novel gene-editing tools (knockout genes, CRISPR/ Cas9, and RNAi) [26][27][28][29][30], and 'omic' research (genomics, transcriptomics, and proteomics) represent essential paths towards significant advancements in the control of cryptosporidiosis [30][31][32][33][34][35][36][37][38]. In the future, those approaches will show a holistic view of the biology of Cryptosporidium.…”