Genetic complementation in apicomplexan parasites

Abstract

A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from -proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.purine salvage ͉ lateral gene transfer ͉ Cryptosporidium parvum ͉ Toxoplasma gondii T he phylum Apicomplexa includes a large number of obligate intracellular parasites, among which are the human pathogens Plasmodium (malaria), Toxoplasma (AIDS-related encephalitis), Cryptosporidium, and Cyclospora (severe enteritis) as well as many parasites of substantial veterinary importance (Eimeria, Theileria, Sarcocystis, and Babesia). Toxoplasma gondii has emerged as a versatile genetic model organism to study the basic biology of this group of parasites (1). T. gondii is safe and easy to culture in vitro, and excellent animal models are available; it is also amendable to molecular genetic experimentation. The high transfection efficiencies, which can be achieved in this organism, have spurred the development of a powerful set of reverse genetic tools (2-4).Apicomplexa, as with most protists, diverged relatively early in the eukaryotic lineage and have many biological features not shared by major eukaryotic model systems (e.g., intracellular parasitism or the possession of secondary plastids). Several large scale sequencing efforts have increased the number of known apicomplexan genes drastically (5). Assigning biological functions to many of these genes, however, remains a major challenge. A forward genetic approach could bridge this gap and permit functional analysis of genes unique to Apicomplexa. The parasites maintain a haploid genome over most of their life cycle, which should greatly facilitate the generation of loss of function mutants. Indeed chemical as well as insertional mutagenesis has been applied successfully in several screens targeting nonessential genes involved in nucleotide biosynthesis (6-11). Parasites harboring conditional temperature-sens...

Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant

“…(Radke et al ., 2000). Inserts were recovered from the primary temperature-resistant population by recombination cloning (BP-recombination) (Hartley et al ., 2000;Striepen et al ., 2002) and following a second recombination (LR-recombination) (Hartley et al ., 2000) to shuttle the inserts back into a Toxoplasma expression context, the recovered cDNA inserts were transformed again into ts 11C9 mutant parasites. The number of temperatureresistant parasites increased > 200-fold after three rounds of enrichment (Fig.…”

“…Genomic DNA was purified from primary temperature-resistant parasites (polyclonal) and used directly to recover en mass all cDNA inserts in the pDONOR201 vector (Invitrogen) by BP in vitro recombination (designated 1∞-BP library) (Hartley et al, 2000;Striepen et al, 2002). Plasmids were transformed into bacteria and the resulting colonies scraped, collected by centrifugation, and plasmids purified using standard methods.…”

“…Recovery of cDNA inserts from the T. gondii genome by recombination followed published protocols (Hartley et al, 2000;Striepen et al, 2002). Genomic DNA was purified from primary temperature-resistant parasites (polyclonal) and used directly to recover en mass all cDNA inserts in the pDONOR201 vector (Invitrogen) by BP in vitro recombination (designated 1∞-BP library) (Hartley et al, 2000;Striepen et al, 2002).…”

What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

“…Although the generation of temperature sensitive mutants appears to be relatively straight forward, the isolation of the respective locus might be complicated in case several genes have been mutated or the respective mutation has a dominant effect. However, with the optimisation of complementation technologies (Striepen et al 2002, Gubbels et al 2008, this technique has been currently demonstrated to be very powerful for the identification and isolation of novel essential genes (Gubbels et al 2008). The obvious advantage of this strategy is that hypothetical genes that might otherwise be neglected in targeted approaches will be identified with equal probability.…”

Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer® peptides

“…Cryptosporidium does not contain guanine salvage enzymes, and consequently, this pathway appears to be the only route to source guanine nucleotides (Kirubakaran et al, 2012;Striepen et al, 2004). The inosine 5'-monophosphate dehydrogenase (IMPDH) gene appears to have been acquired through lateral gene transfer from a ε-proteobacterium (Striepen et al, 2002. Detailed kinetic analysis of this prokaryote-like enzyme demonstrated that the Cryptosporidium IMPDH is very different from its human homologs Umejiego et al, 2004).…”