Protein pyrophosphorylation is an emerging, unusual posttranslational modification. This signaling mechanism can be driven by inositol pyrophosphate messengers, which can convert a prephosphorylated protein to the corresponding pyrophosphoprotein. Endogenous protein pyrophosphorylation influences various cellular processes and signaling pathways, including the regulation of rRNA synthesis and the modulation of vesicular trafficking. Herein, we will summarize the current detection and analysis methods that have established the occurrence of pyrophosphorylation. These methods have also been used to explore the effects of pyrophosphorylation on protein structure and function. Putative mechanisms for the regulation of this intriguing, understudied modification will be discussed. Finally, the future needs for this developing area of signal transduction research are highlighted.

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX-type hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange high-performance liquid chromatography (SAX-HPLC), polyacrylamide gel electrophoresis (PAGE), and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg2+ dependent. Specifically, Subclade I NUDTs preferentially hydrolyze 4-InsP7, while Subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher order mutants of Subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wild-type (WT) and Subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond Subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to Subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

Cells experience strong variations in the consumption and availability of inorganic phosphate (Pi). Since Pi is an essential macronutrient but excess Pi has negative impacts on nucleotide hydrolysis and metabolism, its concentration must be maintained in a suitable range. Conserved storage organelles, acidocalcisomes, provide this buffering function. We used acidocalcisome-like yeast vacuoles to study how such organelles are set up to perform this task. Our combined in vitro and in vivo analyses revealed that their ATP-driven polyphosphate polymerase VTC converts cytosolic Pi into inorganic polyphosphates (polyP), which it transfers into the vacuole lumen. Luminal polyphosphatases immediately hydrolyse this polyP to establish a growing reservoir of vacuolar Pi. Product inhibition by this Pi pool silences the polyphosphatases, caps Pi accumulation, and favours vacuolar polyP storage. Upon cytosolic Pi scarcity, the declining inositol pyrophosphate levels activate the vacuolar Pi exporter Pho91 to replenish cytosolic Pi. In this way, acidocalcisome-like vacuoles constitute a feedback-regulated buffering system for cytosolic Pi, which the cells can switch between Pi accumulation, Pi release, and high-capacity phosphate storage through polyP.

Abstract NUcleoside Diphosphate-linked to moiety X (NUDIX) hydrolases are ubiquitous enzymes that maintain metabolic homeostasis by hydrolyzing potentially toxic nucleoside diphosphates. In plants and other eukaryotes, inositol pyrophosphates (PP-InsPs) act as central signaling molecules, linking cellular phosphate status to gene expression via SPX-domain receptors. A recent study (McCombe et al., Science 387:955–962, 2025) showed that several plant pathogenic fungi secrete NUDIX effector proteins that hydrolyze PP-InsPs and manipulate host phosphate signaling. In the blast fungus Magnaporthe oryzae , a cytoplasmic NUDIX effector (MoNUDIX) hydrolyzes PP-InsPs, triggers a phosphate starvation response and suppresses immunity in rice, thereby facilitating disease progression. In contrast, the lentil anthracnose pathogen Colletotrichum lentis secretes CtNUDIX into the apoplast, where it disrupts PP-InsP-dependent endocytic machinery and elicits a hypersensitive cell death response. Collectively, these findings demonstrate how NUDIX effectors exemplify mechanistic diversification within a single effector family: manipulating phosphate signaling promotes biotrophic colonization, whereas disrupting host membrane integrity induces a switch to necrotrophy.

Capillary electrophoresis mass spectrometry (CE-MS) allows for the rapid and accurate quantitative analysis of inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). The recent discovery of new InsPs and PP-InsPs isomers in plants and mammals necessitates new heavy isotope references for quantitative analysis of complex cellular extracts. Here, we evaluate 18O-labeled InsPs and PP-InsPs as alternatives to 13C labeled internal standards for quantitation by CE-MS. In contrast to 13C labels, the 18O labels are introduced at the end of a synthetic campaign and not at the beginning, rendering 18O much more accessible and affordable as a label. A series of 18O-labeled InsPs and PP-InsPs with different numbers and positions of 18O atoms were synthesized, enabling systematic investigation of MS2 fragmentation pathways. We propose two major dissociation pathways to elucidate the 18O redistribution of the dominant product ion (the loss of H3PO4). Based on these insights, we identified the loss of HPO3 as a suitable transition for minimizing isotope redistribution in MS2 analysis. The ratios of this alternative product ion and dominant product ion were reproducible across replicates, concentration, and measurement days, supporting the use of this alternative product ion as a reliable product ion for quantitative analysis. Application to Saccharomyces cerevisiae, HCT116 cells, and Arabidopsis thaliana extracts confirmed accurate quantitation and precision comparable to 13C-based methods.

The homeostasis of intracellular inorganic phosphate is essential for eukaryotic metabolism and is regulated by the INPHORS signalling pathway, which employs inositol pyrophosphates (IPPs) as key intermediary messengers. This study investigates the metabolic pathways of inositol pyrophosphates (IPPs) in the yeast cell line PhoΔSPX and the human tumor cell line HCT116. Utilizing pulse-labelling experiments with 18O water and ordinary differential equation (ODE) models, we explore the synthesis and turnover of the highly phosphorylated IPP, 1,5-InsP8. Our findings challenge the notion that 1,5-InsP8 can be synthesized through distinct routes, revealing a linear reaction sequence in both systems. Employing model reduction via the profile likelihood method, we achieved statistically concise identifiability analysis that led to significant biological insights. In yeast, we determined that 1,5-InsP8 production primarily occurs through the phosphorylation of 5-InsP7, with the pathway involving 1-InsP7 deemed unnecessary as its removal did not compromise model accuracy. Crucially, this prediction of altered IPP concentrations was validated experimentally in vip1Δ and kcs1Δ knockout strains, providing orthogonal biological support for the reduced model. In HCT116 cells, 1,5-InsP8 synthesis is mainly driven by 1-InsP7, with variations observed across different experimental conditions. These results underscore the utility of model reduction in enhancing our understanding of metabolic pathways, coupling predictive modeling with experimental validation, and providing a framework for future investigations into the regulation and implications of linear IPP pathways in eukaryotic cells.

Inositol phosphates (InsPs) are intracellular signaling molecules that are essential for life. Inositol pyrophosphates, a subset of inositol phosphates, are the end products of inositol phosphate metabolism. In mammalian cells, up to90% of inositol pyrophosphates are 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5PP-InsP5), which is generated by inositol hexakisphosphate kinases (IP6Ks). 5PP-InsP5 can be further phosphorylated by diphosphoinositol pentakisphosphate kinases (PPIP5Ks) to generate 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). Unlike freely diffusible molecules, 5PP-InsP5 and InsP8 act locally at the sites where they are synthesized. Thus, individual IP6K and PPIP5K enzymes perform specific functions. Preclinical and clinical studies suggest that these molecules contribute to early life development, but mediate age-related diseases beyond adulthood. In this review, we summarize the functions and mechanisms of action of every individual IP6K and PPIP5K in both physiological processes and diseases and discuss the potential applications of these inositol pyrophosphate kinases as druggable targets for disease treatment.

The fission yeast phosphate acquisition (PHO) regulon is repressed under phosphate-replete conditions by upstream lncRNA-mediated transcriptional interference. Inositol-1-pyrophosphates control phosphate homeostasis via their action as agonists of precocious PHO lncRNA 3'-processing/termination. Inositol pyrophosphatase-inactivating mutations that increase inositol-1-pyrophosphates elicit derepression of the PHO genes and a severe growth defect in YES medium. Previous studies demonstrated suppression of inositol pyrophosphate toxicosis by targeted deletion or loss-of-function mutations in the nonessential Ssu72, Ppn1, Swd22, and Ctf1 subunits of the fission yeast cleavage and polyadenylation factor (CPF) complex. Here we conducted a selection for spontaneous mutations that suppress the precocious PHO lncRNA termination underlying the sickness of asp1-STF pyrophosphatase mutants. We thereby recovered and characterized novel hypomorphic missense mutations in five essential CPF subunits: Ysh1 (the cleavage endonuclease), Pta1 (an armadillo/HEAT-repeat protein), Pfs2 (a WD repeat protein), Cft1 (a WD repeat protein), and Msi2 (a tandem RRM RNA-binding protein). The suppressor screen also yielded an intron branchpoint mutation in the gene encoding essential CPF subunit Iss1. In addition, we found that asp1-STF toxicosis was suppressed by a missense mutation in the active site of Pla1, the essential poly(A) polymerase subunit of CPF. Genetic crosses revealed a hierarchy of mutational synergies between the essential CPF subunits, the inessential CPF subunits, termination factor Rhn1, the Thr4 "letter" of the RNA polymerase II CTD code, and the Asp1 kinase that synthesizes inositol-1-pyrophosphates. The synthetic lethality of msi2-G252E with ctf1Δ, swd22Δ, ppn1Δ, ssu72-C13S, rpb1-CTD-T4A, and asp1Δ establishes Msi2 as a central agent of 3'-processing/termination, functioning in parallel to inositol-1-pyrophosphates.

Inositol pyrophosphates (PP-InsPs) are soluble cellular messengers that integrate environmental cues to induce adaptive responses in eukaryotes. In plants, the biological functions of various PP-InsP species are poorly understood, largely due to the absence of canonical enzymes found in other eukaryotes. The recent identification of a new PP-InsP isomer with yet unknown enantiomeric identity, 4/6-InsP7 in the eudicot Arabidopsis thaliana, further highlights the intricate PP-InsP signalling network employed by plants. Yet, the abundance of 4/6-InsP7 in land plants, the enzyme(s) responsible for its synthesis, and the physiological functions of this species are all currently unknown. In this study, we show that 4/6-InsP7 is ubiquitous in the studied land plants. Our findings demonstrate that the Arabidopsis inositol polyphosphate multikinase (IPMK) homologs, AtIPK2α and AtIPK2β phosphorylates InsP6 to generate 4/6-InsP7 as the predominant PP-InsP species in vitro. Consistent with this, AtIPK2α and AtIPK2β act redundantly to control 4/6-InsP7 production in planta. Notably, activity of these IPK2 proteins is critical for heat stress acclimation in Arabidopsis. Our parallel investigations using the liverwort Marchantia polymorpha suggest that the PP-InsP synthase activity of IPK2 and role of IPK2 in regulating the heat stress response are conserved in land plants. Furthermore, we show that the transcription activity of heat shock factor (HSF) is regulated by IPK2 proteins, providing a mechanistic framework of IPK2-controlled heat stress tolerance in land plants. Collectively, our study indicates that IPK2-type kinases have played a critical role in transducing environmental cues for biological processes during land plant evolution.

KIDINS220 and InsP8 safeguard the stepwise regulation of phosphate exporter XPR1.

The wheat VIH2-3B, a functional PPIP5K controls the localization of fasciclin-like arabinogalactan protein.

Inositol pyrophosphates (PP‐InsPs) are highly phosphorylated signaling molecules that regulate diverse cellular processes, including phosphate homeostasis and energy metabolism across species. Despite extensive research on well‐characterized exhaustively phosphorylated PP‐InsPs, such as 5‐PP‐InsP5 (5‐InsP7) and 1,5‐(PP)2‐InsP4 (1,5‐InsP8), the functional relevance of less abundant not fully phosphorylated isomers, remains largely unknown. In this study, we synthesized all unsymmetric 5‐PP‐InsP4 isomers in enantiopure form and assigned their structures using 31P‐NMR analysis in combination with a chiral solvating agent. Additionally, we developed 18O‐labeled PP‐InsP4 standards for mass spectrometry in combination with capillary electrophoresis (CE‐MS), enabling the assignment of PP‐InsP4 in Arabidopsis thaliana under phosphate starvation. Our findings show that the previously detected, phosphate starvation‐induced root‐specific PP‐InsP4 isomer does not match any 5‐PP‐InsP4 isomer, contrary to previous suggestions, thus indicating an alternative phosphorylation pattern. Enzyme assays further demonstrate that Arabidopsis ITPK1 selectively phosphorylates [6‐OH]‐InsP5 and [3‐OH]‐InsP5 at the 5‐position, while other InsP5 isomers remain unchanged. This suggests that an unidentified enzymatic activity is involved in the formation of the elusive root PP‐InsP4 species. Our study provides a comprehensive framework for the synthesis, analysis, and functional investigation of PP‐InsP4, providing an entry point for future studies on their biochemical activity and their physiological roles.

ZusammenfassungInositolpyrophosphate (PP‐InsPs) sind hochphosphorylierte Signalmoleküle, die in verschiedenen Organismen zentrale zelluläre Prozesse wie die Phosphathomöostase und den Energiestoffwechsel regulieren. Während gut charakterisierte, vollständig phosphorylierte PP‐InsPs wie 5‐PP‐InsP5 (5‐InsP7) und 1,5‐(PP)2‐InsP4 (1,5‐InsP8) intensiv untersucht wurden, ist bislang kaum etwas über die Funktion wenig häufiger, nur partiell phosphorylierter Isomere bekannt. In dieser Studie wurden alle unsymmetrischen 5‐PP‐InsP4‐Isomere in enantiomeren‐reiner Form synthetisiert und mithilfe von 31P‐NMR‐Spektroskopie in Kombination mit einem chiralen Solvatisierungsreagenz strukturell zugewiesen. Zusätzlich entwickelten wir 18O‐markierte PP‐InsP4‐Standards für CE‐MS‐Analysen (Kapillarelektrophorese gekoppelt mit Massenspektrometrie), die die Identifizierung eines PP‐InsP4‐Isomers in Arabidopsis thaliana unter Phosphatmangelbedingungen ermöglichten. Unsere Ergebnisse zeigen, dass das unter Phosphatmangel beobachtete, wurzelspezifische PP‐InsP4‐Isomer entgegen früherer Annahmen keinem der synthetisierten 5‐PP‐InsP4‐Isomere entspricht. Dies weist auf ein alternatives Phosphorylierungsmuster hin. Enzymatische Untersuchungen belegen zudem, dass Arabidopsis ITPK1 bevorzugt [6‐OH]‐InsP5 und [3‐OH]‐InsP5 an der 5‐Position phosphoryliert, während andere InsP5‐Isomere unverändert bleiben. Dies legt nahe, dass eine bislang unbekannte enzymatische Aktivität an der Biosynthese des bislang nicht zugewiesenen PP‐InsP4‐Isomers beteiligt ist. Unsere Studie bietet ein umfassendes methodisches Fundament für die Synthese, Analyse und funktionelle Charakterisierung von PP‐InsP4 und eröffnet neue Perspektiven für die Erforschung ihrer biochemischen Eigenschaften und physiologischen Funktionen.

DELLA proteins integrate external and internal cues into a signaling network critical for land plant survival. In flowering plants, gibberellins (GAs) induce DELLA recognition to its receptor GID1, resulting in the polyubiquitylation and degradation of DELLA repressors. Notably, bryophytes lack the canonical GA receptor, despite the presence of genes encoding DELLA in these nonvascular land plants. Consequently, the underlying molecular mechanisms of DELLA regulation in bryophytes have remained largely elusive. Here, we report that MpVIH, responsible for the synthesis of inositol pyrophosphate messenger InsP8, negatively regulates DELLA in the liverwort Marchantia polymorpha to promote cell division and thallus development. Mechanistically, we elucidate that MpVIH-derived InsP8 binds to MpDELLA, promoting polyubiquitination and proteasomal degradation of the repressor. Lastly, our study provides evidence for the conservation of inositol pyrophosphate-controlled DELLA degradation in angiosperms. We, thus, identify a 'noncanonical' type of DELLA regulation by the inositol pyrophosphate messengers in land plants.

Phosphate (Pi) is a critical nutrient for plants and is often a limiting factor in food production, as many agricultural soils are limited in available Pi. Inositol pyrophosphates (PP-InsPs) are signaling molecules involved in Pi sensing and jasmonic acid (JA)-regulated plant defense. Here, we report that overexpression of 1,3,4-trisphosphate 5/6-kinase 1 (ITPK1) and the kinase domain of the dual-domain diphosphoinositol pentakisphosphate kinase 2 (VIP2KD) in Arabidopsis thaliana results in unique elevations in PP-InsPs, accompanied by altered leaf growth and senescence patterns, as well as delayed time to flowering. While plants overexpressing ITPK1 and VIP2KD (ITPK1 OX and VIP2KD OX) accumulated significantly lower levels of Pi, transcriptomic and qRT-PCR analysis revealed that these plants showed elevated expression of Pi starvation response genes. Our transcriptomic analysis also revealed ITPK1 OX and VIP2KD OX showed a significant enrichment in differentially expressed genes relating to plant defense and hypoxia. Of the two transgenic types, VIP2KD OX had significantly higher expression of more diverse plant defense-related differentially expressed genes and showed greater resistance to Trichoplusia ni compared to WT and ITPK1 OX plants. ITPK1 OX, although also having elevated PP-InsPs, was fed upon by insect larvae comparably to WT plants. Taken together, our data indicate the elevation of certain PP-InsPs may be a useful strategy for developing new traits in crop plants.

Cells experience strong variations in the consumption and availability of inorganic phosphate (Pi). Since Pi is an essential macronutrient but excess Pi has negative impacts on nucleotide hydrolysis and metabolism, its concentration must be maintained in a suitable range. Conserved storage organelles, acidocalcisomes, provide this buffering function. We used acidocalcisome-like yeast vacuoles to study how such organelles are set up to perform this task. Our combined in vitro and in vivo analyses revealed that their ATP-driven polyphosphate polymerase VTC converts cytosolic Pi into inorganic polyphosphates (polyP), which it transfers into the vacuole lumen. Luminal polyphosphatases immediately hydrolyse this polyP to establish a growing reservoir of vacuolar Pi. Product inhibition by this Pi pool silences the polyphosphatases, caps Pi accumulation, and favours vacuolar polyP storage. Upon cytosolic Pi scarcity, the declining inositol pyrophosphate levels activate the vacuolar Pi exporter Pho91 to replenish cytosolic Pi. In this way, acidocalcisome-like vacuoles constitute a feedback-regulated buffering system for cytosolic Pi, which the cells can switch between Pi accumulation, Pi release, and high-capacity phosphate storage through polyP.

Inositol phosphates control many central processes in eukaryotic cells including nutrient availability, growth, and motility. Kinetic resolution of a key modulator of their signaling functions, the turnover of the phosphate groups on the inositol ring, has been hampered by slow uptake, high dilution, and constraining growth conditions in radioactive pulse-labeling approaches. Here, we demonstrate a rapid (seconds to minutes) and nonradioactive labeling strategy of inositol polyphosphates through 18O-water in yeast, human cells, and amoeba, which can be applied in any media. In combination with capillary electrophoresis and mass spectrometry, 18O-water labeling simultaneously dissects the in vivo phosphate group dynamics of a broad spectrum of even rare inositol phosphates. The good temporal resolution allowed us to discover vigorous phosphate group exchanges in some inositol polyphosphates and pyrophosphates, whereas others remain remarkably inert. We propose a model in which the biosynthetic pathway of inositol polyphosphates and pyrophosphates is organized in distinct, kinetically separated pools. While transfer of compounds between those pools is slow, each pool undergoes rapid internal phosphate cycling. This might enable the pools to perform distinct signaling functions while being metabolically connected.

Inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8 are eukaryal signaling molecules that influence cell physiology, especially phosphate homeostasis. In fission yeast, 1,5-IP8 and 1-IP7 impact gene expression by acting as agonists of RNA 3'-processing and transcription termination. 1,5-IP8 is synthesized by position-specific kinases Kcs1 and Asp1 that convert IP6 to 5-IP7 and 5-IP7 to 1,5-IP8, respectively. Inositol pyrophosphatase enzymes Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase) are agents of inositol pyrophosphate catabolism in fission yeast. Whereas Asp1, Siw14, and Aps1 are individually inessential, double pyrophosphatase mutants asp1-H397A aps1∆ and siw14∆ aps1∆ display severe growth defects caused by overzealous 3'-processing/termination. By applying CE-ESI-MS to profile the inositol pyrophosphate content of fission yeast mutants in which inositol pyrophosphate toxicity is genetically suppressed, we elucidated the functional redundancies of the Asp1, Siw14, and Aps1 pyrophosphatases. Asp1, which exclusively cleaves the 1-β-phosphate, and Aps1, which prefers to cleave the 1-β-phosphate, play essential overlapping roles in guarding against the accumulation of toxic levels of 1-IP7. Aps1 and Siw14 together catabolize the inositol-5-pyrophosphates, and their simultaneous inactivation results in overaccumulation of 5-IP7. Cells lacking all three pyrophosphatases amass high levels of 1,5-IP8 and 1-IP7, with concomitant depletion of IP6. A genetic screen identified three missense mutations in the catalytic domain of Kcs1 kinase that suppressed inositol-1-pyrophosphate toxicosis. The screen also implicated the 3'-processing factor Swd22, the inositol pyrophosphate sensor Spx1, and the nuclear poly(A)-binding protein Nab2 as mediators of inositol-1-pyrophosphate toxicity.IMPORTANCEInositol pyrophosphates are key effectors of eukaryal cellular phosphate homeostasis. They are synthesized by kinases that add a β-phosphate to the 5- or 1-phosphate groups of IP6 and catabolized by three classes of pyrophosphatases that hydrolyze the β-phosphates of 5-IP7, 1-IP7, or 1,5-IP8. Whereas the fission yeast inositol pyrophosphatases-Asp1 (histidine acid phosphatase), Siw14 (cysteinyl phosphatase), and Aps1 (Nudix hydrolase)-are inessential for growth, Asp1/Aps1 and Aps1/Siw14 double mutations and Asp1/Siw14/Aps1 triple mutations elicit severe or lethal growth defects. By profiling the inositol pyrophosphate content of pyrophosphatase mutants in which this toxicity is genetically suppressed, we reveal the functional redundancies of the Asp1, Siw14, and Aps1 pyrophosphatases. Their synergies are manifested as excess accumulation of 1-IP7 upon dual inactivation of Asp1 and Aps1 or an excess of 5-IP7 in aps1∆ siw14∆ cells. In the absence of all three pyrophosphatases, cells accrue high levels of 1,5-IP8 and 1-IP7 while IP6 declines.

A recent paper reported that fungi use Nudix effectors to disrupt plant phosphate sensing by breaking down inositol pyrophosphate signals, worsening disease, although the exact mechanism remains unclear. This commentary discusses these groundbreaking results and asks whether these effectors affect shoot-root communication and plant nutrition-immunity crosstalk.

Millions of people are infected with Trypanosoma cruzi, and the current treatment is not satisfactory. Inositol pyrophosphates have been established as important signaling molecules. Our work demonstrates the presence of a phospholipase C-independent pathway for the synthesis of inositol pyrophosphates in T. cruzi. Furthermore, we demonstrate that this pathway starts with the synthesis of inositol monophosphates from glucose 6-phosphate or from inositol phosphoceramide, linking it to carbohydrate and sphingolipid metabolism. The essentiality of the pathway for the survival of T. cruzi infective stages makes it an ideal drug target for treating American trypanosomiasis.