Sweet and CRISP(R)y parasite engineering

Abstract

Toxoplasma gondii is an intracellular parasite that is highly prevalent within human populations. Its genome encodes a range of enzymes involved in glycan biosynthesis and metabolism. A new study presents a library of CRISPR/Cas9-based glyco-relevant gene knockouts and their examination in glycomic and functional assays. This new resource can pave the way for a better understanding of the role of carbohydrates in infection and immunomodulation by this significant protozoan parasite.

Introduction

The “sugary” coat of any cell is the first interaction surface when two cells meet, regardless of the biological kingdom from which the cells originate. Thus, it should be no surprise that binding or entry of pathogens into host cells depends on carbohydrate chains (i.e. glycans) and carbohydrate-binding proteins. Parasite-derived carbohydrates also typically differ from those found in their host and so can trigger the host's innate immune system and induce adaptive immune responses. Some antiglycan responses can block invasion or indicate that glycans are potential targets for vaccine development; certainly, they can prove diagnostic for a parasite infection as is the case with nematodes such as Toxocara canis. Tackling the details of these processes is difficult when one does not know the identity of the genes involved, whether these encode enzymes required for glycan synthesis and degradation (glycosyltransferases and glycosidases), carbohydrate-binding proteins (lectins, agglutinins, and adhesins), or the proteins to which glycans are attached. However, as exemplified by Gas-Pascual et al. in this issue (1), CRISPR/Cas9-mediated genetic engineering opens up new possibilities.

Toxoplasma gondii is one of the most common parasites that infect humans and can cause blindness or encephalitis, particularly in immunocompromised patients. Although acute infections can be treated, there is no cure for a chronic infection. There are three stages (infective oocytes, replicative tachyzoites, and latent bradyzoites); tachyzoites are primarily responsible for the acute infection, infect migratory cells, such as dendritic cells and macrophages (but can invade all nucleated cells in warm-blooded hosts), replicate until the host cell lyses, and rapidly disseminate throughout the host. Similar to other parasites, T. gondii has developed highly sophisticated mechanisms to counteract or regulate the host immune responses in order to establish a life-long chronic infection; especially the down-regulation of components of the immune system leads to some sort of tolerance. This effect, associated with reduced responses to both allergens and vaccination, has been summarized under the “hygiene hypothesis” or “old friend's hypothesis,” which seeks to explain the mutual evolution of host and parasite over millennia. In the case of T. gondii, epidemiological studies show lower allergic sensitization and asthma in T. gondii–positive patients (2), while in experimental studies in mice, a T. gondii infection or tachyzoite extract prevents allergy in mice, whereby carbohydrates could play a role (3, 4). The relevance of sugars for T. gondii is reflected in the critical nature of different forms of O-glycosylation for its cell wall structure and its growth and by the immunomodulatory potential of its glycosylphosphatidylinositols, as well as the ability of some of the MIC proteins, associated with a secretory organelle known as the microneme, to recognize host carbohydrates. Thus, glycobiological research on T. gondii should result in identifying those glycan-biosynthesizing enzymes crucial for its fitness, survival, infectivity, and immunomodulation; these may then serve as potential drug targets in the search for new therapeutics against T. gondii-induced morbidity and mortality.

Gas-Pascual et al. (1) have bioinformatically analyzed and systematically knocked out a large set of glycosylation-related genes in Toxoplasma. In total, some 60 genes were identified by the homology of the encoded proteins to glycosyltransferases, transporters, or enzymes involved in generating activated sugars from other organisms. Some of these homologues are not present in other apicomplexan protist parasites such as Plasmodium or Cryptosporidium, which raises the possibility of horizontal gene transfer from the host during T. gondii evolution. The parasite can only endogenously produce truncated N-glycans, but its genome also encodes the capacity to make four different sorts of O-glycans as well as a type of C-linked glycan. Some of the homologues identified are members of known glycosyltransferase gene families, but their specific function in T. gondii is yet to be characterized.

Gas-Pascual et al. (1) then used a double CRISPR/Cas9 approach (with two guide RNAs rather than one as in most studies) to prepare 17 knockouts and systematically examined the glycomic repercussions using MS, lectin and antibody blots, and a monolayer cellular infection model (see Fig. 1 for a schematic summary). By and large, the results of this systematic analysis are compatible with the literature, although some details are different. For example, they found only limited processing of N-glycans in T. gondii with partial removal of glucose residues from the N-glycans actually attached to protein. Furthermore, three O-glycan types associated with the secretory pathway could be detected, and the most complex of which are disaccharides. Despite this simplicity, there is even current controversy whether O-fucosylation of secreted T. gondii proteins (e.g. MIC2) is or is not important for invasion (5, 6), but a relevant small plaque pofut2 phenotype was observed by Gas-Pascual et al. (1). The authors also developed a new method to analyze glycophosphatidylinositol-type glycolipid anchors, but could not isolate any mutants affecting their core structure. The most unusual glycan is a pentasaccharide modification attached via hydroxyproline residues to the cytoplasmic ubiquitin ligase subunit Skp1 and is similar (but not identical) to one found in the slime mold Dictyostelium discoideum (7); in contrast, the cytoplasmic O-fucosylation found in T. gondii is due to an orthologue of the Spindly protein from Arabidopsis thaliana. Such examples highlight the value of interspecies comparisons and also remind us of the value to parasitology of research on nonpathogens such as the cellular slime mold or model plants.

Figure 1.

CRISPR/Cas9-based genomic tools in combination with glycomics and fitness assays to study Toxoplasma glycobiology. CRISPR/Cas9-based gene editing strategy enables alteration of the genomes of parasites, such as T. gondii. A double-CRISPR/Cas9 approach was designed to promote the replacement of candidate glycogenes and a resistance gene by two guide RNAs (gRNAs; for simplicity only one is shown). T. gondii tachyzoite–derived fractions were subject to glycomic analysis by MS. The effects of glycogene disruption on plaque formation on fibroblast monolayer and other markers of fitness can be assessed, leading to the identification of novel N-glycans, O-glycans, and Tn-antigens that are essential for T. gondii biology. PAM, protospacer adjacent motif.

With some exceptions, parasites have proven refractory to targeted genetic modification; CRISPR/Cas9 technology, though, increases the access to knockouts in a range of organisms, and the “rate determining steps” are the design of the guide RNAs, how to drive their expression and how to transform the organism. In this and many systems, the CRISPR/Cas9 approach will help unravel the many mysteries about glycans and their functions, especially how and why the adaptation to specific ecological or host niches is accompanied by wide variations in the glycomic capacity. As a result, genetically engineered Toxoplasma can be used for comparisons with other pathogenic apicomplexans, including Cryptosporidium parvum or Plasmodium spp., and the public availability of the new resource will undoubtedly catalyze this sort of work.

Beyond the specific glycobiological aspects, CRISPR/Cas9 technology should facilitate interdisciplinary studies aimed at understanding the biology and pathogenicity of T. gondii. A major challenge is to determine whether, how, and which glycans aid infection and survival or mediate cross-talk between the host and parasite. For instance, the relevance of glycan interactions for the pro- and anti-inflammatory responses during acute and chronic phases of infection could be examined. Moreover, this resource may aid the identification of glycoconjugates for vaccination or diagnosis. Ultimately, the definition of glyco-related drug targets to prevent or ameliorate infections would be a particularly “sweet” outcome of this work.