Researchers at Ludwig Maximilian University of Munich (LMU) and the University of Glasgow say they have developed a novel genetic screening technique that blocks the protein regulation of the parasite Toxoplasma gondii and causes it to die off inside the host cell.
The findings are published in the journal Nature Microbiology in a paper titled, “A splitCas9 phenotypic screen in Toxoplasma gondii identifies proteins involved in host cell egress and invasion.”
“Apicomplexan parasites, such as Toxoplasma gondii, have specific adaptations that enable invasion and exit from the host cell,” wrote the researchers. “Owing to the phylogenetic distance between apicomplexan parasites and model organisms, comparative genomics has limited capacity to infer gene functions. Further, although CRISPR/Cas9-based screens have assigned roles to some Toxoplasma genes, the functions of encoded proteins have proven difficult to assign. To overcome this problem, we devised a conditional Cas9-system in T. gondii that enables phenotypic screens. Using an indicator strain for F-actin dynamics and apicoplast segregation, we screened 320 genes to identify those required for defined steps in the asexual life cycle.”
Until now, it was not known which genes encode the proteins that control the exit from the host cell. To identify them, the team of researchers led by Markus Meißner, professor and chair of experimental parasitology at LMU, collaborated with colleagues from the University of Glasgow to develop a novel genetic screening technique and investigate a library of 320 parasite-specific genes. The researchers discovered two genes without which cell egress is impossible.
Targeting the destruction of these genes led to a blockade of the egress and thus to the death of the next generation of parasites within the host cell. “This paves the way potentially for the development of active substances that could block the function of the corresponding proteins and so put a halt to propagation,” observed Meißner.
“We assume that similar processes control the propagation of the malaria pathogen,” explained LMU parasitologist Elena Jimenez-Ruiz, PhD. “Next, we will investigate what functions these proteins have in the malaria pathogen and whether there are possible starting points for the development of new drugs.”