Too Much Sugar Puts a Parasite in Jeopardy
Authors: Stanislas Tomavo
Journal: Cell Host & Microbe (2015)

Toxoplasma gondii and other coccidian parasites accumulate starch-like amylopectin stores whose functional significance is unclear. In this issue of Cell Host & Microbe, Uboldi et al., 2015Uboldi A.D. McCoy J.M. Blume M. Gerlic M. Ferguson D.J.P. Dagley L.F. Beahan C.T. Stapleton D.I. Gooley P.R. Bacic A. et al.Cell Host Microbe. 2015; 18 (this issue): 670-681Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar present a pioneering investigation into a signaling cascade with a pivotal role in amylopectin metabolism and transmission of encysted parasites during chronic infection. Toxoplasma gondii and other coccidian parasites accumulate starch-like amylopectin stores whose functional significance is unclear. In this issue of Cell Host & Microbe, Uboldi et al., 2015Uboldi A.D. McCoy J.M. Blume M. Gerlic M. Ferguson D.J.P. Dagley L.F. Beahan C.T. Stapleton D.I. Gooley P.R. Bacic A. et al.Cell Host Microbe. 2015; 18 (this issue): 670-681Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar present a pioneering investigation into a signaling cascade with a pivotal role in amylopectin metabolism and transmission of encysted parasites during chronic infection. The Apicomplexa is an ancient phylum of some 5,000 diverse eukaryotic species. These intracellular parasites have gained notoriety because they cause acute disease and death in humans, as well as impacting the livestock industry worldwide. The phylum Apicomplexa includes Plasmodium species, which are responsible for malaria, and also Cryptosporidium, causing severe diarrhea in immunocompromised individuals; Eimeria, the agent of coccidiosis in poultry; and Toxoplasma gondii, one of the most ubiquitous intracellular parasites. While extremely common, infections by T. gondii generally remain subclinical because of a combination of a robust immune control and parasite-mediated subversion of the host enabling long-term survival. The infectious stages consist of the acute (tachyzoite), latent (bradyzoite), and spore-forming oocyst (sporozoite) stages. Infections initiated either by the ingestion of environmental oocysts or by tissue cysts are associated with the rapid replication and spread of tachyzoite forms. The control of this acute phase by the immune system eliminates the tachyzoites, while the few remaining parasites differentiate into the relatively quiescent and slowly growing bradyzoites, forming latent cysts that can persist mainly in the muscle and the central nervous system. The ensuing chronic infection is essentially asymptomatic, although cyst formation in the retina is a significant cause of blindness in some regions of South America (Glasner et al., 1992Glasner P.D. Silveira C. Kruszon-Moran D. Martins M.C. Burnier Junior M. Silveira S. Camargo M.E. Nussenblatt R.B. Kaslow R.A. Belfort Junior R. Am. J. Ophthalmol. 1992; 114: 136-144Abstract Full Text PDF PubMed Scopus (318) Google Scholar). Furthermore, in the absence of immune function, most notably observed in active HIV-AIDS, tissue cyst reactivation occurs, leading to life-threatening neurological disease and death if not treated. The chronic infection of T. gondii can also increase the risk of schizophrenia, bipolar disorder, and obsessive disorder (Arias et al., 2012Arias I. Sorlozano A. Villegas E. de Dios Luna J. McKenney K. Cervilla J. Gutierrez B. Gutierrez J. Schizophr. Res. 2012; 136: 128-136Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, Hurley and Taber, 2012Hurley R.A. Taber K.H. J. Neuropsychiatry Clin. Neurosci. 2012; 24: 376-383Crossref PubMed Scopus (23) Google Scholar). The complex lifestyle of T. gondii relies on stage conversion and differentiation of tachyzoite to bradyzoite and the oocyst-forming sporozoites. One of the evolutionary hallmarks of the coccidian parasites is the presence of starch-like amylopectin stores (Harris et al., 2004Harris J.R. Adrian M. Petry F. Parasitology. 2004; 128: 269-282Crossref PubMed Scopus (23) Google Scholar; Figure 1). In contrast to plants, which contain starch in the form of branched amylopectin and amylose in their chloroplasts, the amylopectin of T. gondii is exclusively composed of a linear polymer of α(1-4)-linked glucose backbone modified with α(1-6)-branched glucose residues (Guérardel et al., 2005Guérardel Y. Leleu D. Coppin A. Liénard L. Slomianny C. Strecker G. Ball S. Tomavo S. Microbes Infect. 2005; 7: 41-48Crossref PubMed Scopus (48) Google Scholar). It has been proposed that the loss of amylopectin from Eimeria sporozoites compromises their ability to infect mice (Nakai and Ogimoto, 1983Nakai Y. Ogimoto K. Jnp. J. Vet. Sci. 1983; 45: 127-129Crossref Scopus (11) Google Scholar), but almost nothing is known about the regulation of amylopectin accumulation and utilization during stage conversion of T. gondii. In this issue of Cell Host & Microbe, Uboldi et al., 2015Uboldi A.D. McCoy J.M. Blume M. Gerlic M. Ferguson D.J.P. Dagley L.F. Beahan C.T. Stapleton D.I. Gooley P.R. Bacic A. et al.Cell Host Microbe. 2015; 18 (this issue): 670-681Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar show that Ca2+-dependent protein kinase 2 (CDPK2) plays a central role in the regulation of T. gondii amylopectin formation and degradation. The authors present evidence that the ability of CDPK2 to bind amylopectin granules depends on the presence of the N-terminal carbohydrate binding module 20 (CBM20) domain. Furthermore, the authors elegantly demonstrate that the loss of CDPK2 in the two distinct parasite strains, RH type I and Pru type II, results in abnormal accumulation of amylopectin in the basal end of parasites and in the vacuolar space of the residual bodies. This suggests that the CDPK2 mutants have a defect in the synthesis or turnover of these polysaccharide granules. In addition, the CDPK2 protein lacking the Ca2+-binding EF-hand motif, known to activate the kinase in response to Ca2+, was unable to functionally complement the CDPK2 mutant, which points to a role of Ca2+ signaling in the control of Toxoplasma amylopectin levels. The authors also provide evidence for direct phosphorylation of pyruvate phosphate dikinase (PPDK), a key enzyme in gluconeogenesis, by CDPK2. This validates the involvement of the CDPK2 kinase in posttranslational regulation of at least some amylopectin synthetic enzymes. It is, however, surprising that neither the α-amylase, involved in amylopectin degradation, nor the cytosolic amylopectin synthase (Coppin et al., 2005Coppin A. Varré J.S. Lienard L. Dauvillée D. Guérardel Y. Soyer-Gobillard M.O. Buléon A. Ball S. Tomavo S. J. Mol. Evol. 2005; 60: 257-267Crossref PubMed Scopus (98) Google Scholar) was detected as CDPK2 substrates. In contrast to the encysted bradyzoites, RH type I strain tachyzoites can only accumulate amylopectin under stress conditions (Guérardel et al., 2005Guérardel Y. Leleu D. Coppin A. Liénard L. Slomianny C. Strecker G. Ball S. Tomavo S. Microbes Infect. 2005; 7: 41-48Crossref PubMed Scopus (48) Google Scholar), while tachyzoites of Pru or other type II cyst-forming parasites but also those of the RH strain only do so modestly under normal conditions. It is often hypothesized that amylopectin is necessary to fulfil the energy requirement of the oocyst-forming sporozoites and the encysted bradyzoites. However, this hypothesis has never been tested prior to the present study, which reports that CDPK2 loss, while leading to a massive amylopectin accumulation, also provokes the death of these mutants. Furthermore, a virulence study in mice revealed that the CDPK2 mutants were unable to form tissue cysts and thus establish a chronic infection (Uboldi et al., 2015Uboldi A.D. McCoy J.M. Blume M. Gerlic M. Ferguson D.J.P. Dagley L.F. Beahan C.T. Stapleton D.I. Gooley P.R. Bacic A. et al.Cell Host Microbe. 2015; 18 (this issue): 670-681Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Although the in vivo results are striking, the involvement of CDPK2-dependent Ca2+ signaling in the organ tropism of the parasite during infection was not tested and could bias the interpretation of the results. No human-use-approved vaccine exists against Toxoplasma, and no currently available drugs cure chronic T. gondii infections. The CDPK2 mutation prevented the establishment of the as-yet-incurable chronic toxoplasmosis in the mouse model, which suggests its prospective clinical utility as a metabolically attenuated vaccine. A recent paper demonstrates that bradyzoites, classically considered dormant, are replicating within the tissue cysts more actively than previously thought (Watts et al., 2015Watts E. Zhao Y. Dhara A. Eller B. Patwardhan A. Sinai A.P. MBio. 2015; 6: e01155Crossref Scopus (123) Google Scholar). In light of this, the following questions need to be investigated before considering CDPK2 mutants as attenuated vaccine candidates. (1) Do the mutants generated with an RH type I strain protect the infected mice against a second challenge using the same strain? (2) Can the RH type I CDPK2 mutants stimulate a crossprotective and sterile immunity against the cyst-forming type II strain, and vice versa? (3) Do the loss of CDPK2 and accumulation of amylopectin in these mutants lead to susceptibility or resistance to any known drugs that interfere with amylopectin metabolism? Our field now faces the challenge of understanding why the hugely increased cytoplasmic amylopectin stores are not toxic to the parasite but support the normal growth of tachyzoites, both in vitro and in vivo. Carbohydrate metabolism is essential not only for gluconeogenesis, replication, and virulence of T. gondii (Blume et al., 2015Blume M. Nitzsche R. Sternberg U. Gerlic M. Masters S.L. Gupta N. McConville M.J. Cell Host Microbe. 2015; 18: 210-220Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) but also for glycolysis and mitochondrial respiration in tachyzoites (Figure 1), and one could envisage a severe disturbance of all these connected metabolic pathways depending on the fluxes of the available glucose. Interestingly, the disruption of amylopectin synthesis and degradation appears to directly impact only the glycolytic fluxes, but not mitochondrial oxidative phosphorylation or gluconeogenesis. The present study raises several fascinating and important questions. For example, can we generate a knockout mutant lacking the amylopectin synthase in tachyzoites to explore the parasite’s ability to differentiate into encysted bradyzoites? Can we then follow their subsequent survival and sustained infection in mice? Uboldi et al., 2015Uboldi A.D. McCoy J.M. Blume M. Gerlic M. Ferguson D.J.P. Dagley L.F. Beahan C.T. Stapleton D.I. Gooley P.R. Bacic A. et al.Cell Host Microbe. 2015; 18 (this issue): 670-681Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar reveal that CDPK2-dependent regulation of amylopectin metabolism is not only essential for tachyzoite pathogenesis and the bradyzoite stages, but also completely abrogates chronic Toxoplasma infection. Thus, unchecked amylopectin synthesis is catastrophic for the parasite, leading to death and absence of cyst formation. It is becoming clear that regulation of the amylopectin stores is crucial, and that too much sugar puts the parasite in jeopardy. S.T. acknowledges financial support from the National Agency for Research (ANR), Laboratoire d’Excellence (LabEx) ParaFrap ANR-11-LABX-0024 and ANR-14-CE14-0002-01.