Glycocalyx‐Driven Immunomodulation in Trypanosoma cruzi: Structures, Host Responses, Diagnostic and Therapeutic Targeting

ABSTRACT

Chagas disease, caused by Trypanosoma cruzi, remains an important neglected infection due to underdiagnosis, global spread resulting from increased migration of people infected with T. cruzi and concomitant organ transplantation and blood transfusion, and limited availability of chemotherapy. The parasite's surface is covered by a dense glycocalyx enriched in GPI‐anchored mucins, trans‐sialidases, and glycoinositolphospholipids (GIPLs), which collectively shape host–parasite interactions. Here we synthesize structural and biosynthetic features of key glycoconjugates and discuss how they modulate innate and adaptive immunity through pattern‐recognition receptors (e.g., TLR2/6 and TLR4), inhibitory lectins (e.g., Siglec‐E), extracellular vesicle signalling, and complement evasion mediated by TS‐like regulators (T‐DAF, TcCRP) and factor H recruitment. We highlight how strain‐ and stage‐specific glycan variation, including α‐Galp‐ and β‐Galf‐containing epitopes, influences immune recognition, pathogenesis, and persistence. Finally, we evaluate translational opportunities: parasite‐selective targeting of sugar‐nucleotide metabolism and glycosyltransferases, and development of defined glycoepitopes (glycotopes) as improved biomarkers for diagnosis and post‐treatment monitoring. Together, these insights position the T. cruzi glycocalyx as a central driver of immunomodulation and a promising source of therapeutic, vaccine and diagnostic targets.

Abbreviations

BT

bloodstream trypomastigotes

ChD

Chagas disease

CRP

complement regulatory protein

DAF

decay accelerating factor

DC

dendritic cells

Epi

epimastigotes

EV

extracellular vesicle

GIPL

glycoinositolphospholipid

glycotope

glycan‐dependent epitope

GPI

glycophosphatidylinositol

IFN‐γ

interferon‐γ

Ig

immunoglobulin

MAPK

mitogen‐activated protein kinase

MT

metacyclic trypomastigotes

Mϕ

macrophage

NETs

neutrophil extracellular traps

NK

natural killer cells

NO

nitric oxide

NS

nucleotide sugars

NST

NS transporters

PBMCs

human peripheral blood monocytes

ppαGlcNAcT

UDP‐GlcNAc:polypeptide‐O‐α‐N‐acetyl‐D‐glucosaminyltransferase

PRRs

pattern recognition receptors

SA

sialic acid (Neu5Ac when sourced from humans)

TCT

tissue culture trypomastigotes—that is a surrogate for BT in the laboratory

tGPI‐mucin

GPI‐anchored mucin‐like glycoproteins from TCT

THP‐1

human peripheral blood monocyte

TLR

Toll‐like receptor

TNF‐α

tumour necrosis factor‐α

UGM

UDP‐galactopyranose mutase (see figure legends for abbreviations for additional enzymes, sugars and modifications)

1. Introduction

Trypanosoma cruzi, a protozoan parasite, causes Chagas disease (ChD), which the Drugs for Neglected Diseases initiative lists among the 10 most neglected diseases worldwide. More than 6 million people are currently infected; fewer than 10% are diagnosed, and ~75 million live in areas at risk of infection [1, 2]. Currently, ChD continues to spread globally, as it is present in endemic countries in Latin America as well as the USA, Canada, Europe, Japan, and Australia. Estimates (2012) placed the annual global cost at US $627 million in healthcare expenses and 806,170 Disability‐Adjusted Life Years annually due to heart disease and other outcomes [3]. However, in Brazil alone, a recent study estimated the annual economic burden at US $11.44 billion (0.23% of GDP), with a lifetime cost of US $45,034 per patient [4].

Transmission occurs primarily via triatomine insects, but can also occur through blood transfusion, organ transplantation, congenital transmission, or oral ingestion of food/beverages contaminated with T. cruzi [1]. Clinically, ChD comprises an acute and a chronic phase. The acute phase is characterized by high parasitemia and is frequently asymptomatic or oligosymptomatic; when present, symptoms may include fever, anorexia, lymphadenopathy, lasting 4–8 weeks. Acute manifestations resolve in ~90% of cases, and ~60%–70% of infected individuals remain in the indeterminate form. Nevertheless, 30%–40% of patients progress to chronic disease; among these, ~30% develop Chagas cardiomyopathy, a severe outcome that can be fatal in up to two‐thirds of affected individuals. Chronic disease may also involve digestive complications such as megaesophagus and megacolon [5].

Only two drugs are currently available for treatment—benznidazole and nifurtimox—both discovered more than 50 years ago. They have substantial efficacy and safety limitations, including high toxicity, low solubility, and reduced efficacy [6, 7, 8]. Benznidazole cures ~30%–50% of acute‐phase patients and is largely ineffective in chronic infection. Treatment is often interrupted due to long regimens and adverse effects, and drug resistance has been reported [1, 7, 9, 10]. Because ChD causes significant comorbidities and vaccination is not available, there is an urgent need to develop new drugs and/or therapeutic combinations.

T. cruzi has four developmental stages: replicative epimastigotes and amastigotes, and infective non‐replicative metacyclic (MT) and bloodstream (BT) trypomastigotes. In mammals, infection begins when MT parasites present in triatomine insect faeces/urine enter through the bite during blood feeding. After invasion of any nucleated cell, trypomastigotes pass through the parasitophorous vacuole, escape to the cytosol, and differentiate into amastigotes. Amastigotes replicate for ~3–5 days before differentiating into BT forms. Following host‐cell rupture, BT disseminate via the bloodstream and infect neighbouring cells or distant tissues. In the insect vector, BT forms differentiate into epimastigotes in the midgut and subsequently into MT forms in the posterior digestive tract [11].

Across its life cycle, T. cruzi undergoes marked biochemical and morphological changes [12], including remodelling of its cell surface. The parasite is coated by a dense glycocalyx rich in glycolipids and glycoproteins (Figure 1) [13, 14]. These glycoconjugates are central to parasite–host interactions [15] and contribute to evasion and subversion of the mammalian immune system [16, 17, 18].

FIGURE 1.

Trypanosoma cruzi surface glycoconjugates recognition by antibodies produced by B‐cells. Schematic model of the trypomastigote surface with an emphasis on glycoconjugates. Active trans‐sialidase (aTS) molecules appear to reside in 10–150 nm nanodomains separate from their Tc‐mucin's targets, and sialylated (SA)‐Tc‐mucins are themselves mostly distinct and separate from their αGalp‐Tc‐mucin counterparts. Consequently, SA‐Tc‐mucins and aTS can be found in distinct microvesicles shed by parasites in blood (top left). GIPLs mainly containing Galf epitopes are presented on a variety of cores and are based on GIPLs from epimastigotes. Finally, terminal sialylation occurs on MASP O‐glycans and Tc85 N‐glycans, and α‐galactosylation also occurs on Tc85. Above, recognition of these various T. cruzi antigens induce expression of effector molecules by T cells, which activate B‐cells. These differentiate into plasma cells that produce lytic or non‐lytic antibodies that can recognize a range of molecules, including glycoantigens such as αGalp epitope (blue) and Galf (green), multiple Tc‐mucins (purple), TS (yellow), or MASPs (orange). As targets of natural antibodies, these antigens are candidates for vaccine development. Note that for clarity, the density of the glycocalyx is underestimated and drawings are not to scale. Abbreviations: 2‐AEP, 2‐ aminoethylphosphonate; InsP, phosphatidylinositol; EtNP, ethanolamine‐phosphate; GlcN, glucosamine; Man, mannose; Galf, β‐galactofuranose; αGal, α‐galactopyranose; SA, sialic acid; Tc‐85, surface glycoproteins of ~85 kDa; MASP, mucin‐associated surface proteins; GIPL, glycoinositolphospholipids; TS, trans‐sialidase.

Given the importance of the T. cruzi glycocalyx, this review addresses three questions: (1) What are the structures and functions of the major T. cruzi glycoconjugates involved in host immune responses? (2) How do these glycoconjugates shape parasite–host interactions, modulate immunity, and influence ChD pathogenesis and progression? (3) Can parasite‐specific glycans or enzymes in their biosynthetic pathways serve as therapeutic targets or diagnostic biomarkers? We integrate structural, biochemical, and immunological evidence to address these questions.

2. Composition of the T. cruzi Glycocalyx

T. cruzi epimastigotes express ~10⁶ copies/cell glycosylphosphatidylinositol (GPI)‐anchored mucin‐like glycoproteins (Tc‐mucins) and ~10⁷ copies/cell glycoinositolphospholipids (GIPLs), which together comprise upwards of 80% of the parasite plasma membrane beyond the bilayer (Figure 1) [19, 20, 21]. In trypomastigotes, additional surface glycoproteins—including active and inactive trans‐sialidase isoforms (aTS/iTS) of the Gp85/Trans‐sialidase superfamily [22, 23, 24], mucin‐associated surface proteins (MASPs) [25], surface proteases [26, 27], Disperse Gene Protein Family [28], together with neutral, sulfated and sialylated glycolipids [29, 30, 31]—appear as less abundant components of the coat. These factors are immunogenic and essential for recognition, adhesion, and activation of host signalling pathways that promote invasion [15]. Here, we focus on the best‐studied glycoconjugates implicated in immune evasion and subversion, and which contribute to lifelong infection [16, 17, 18].

2.1. Monosaccharides and Sugar Nucleotide Precursors

The monosaccharide composition of glycoconjugates differs among the so‐called ‘Tritryps’ (Trypanosoma cruzi, T. brucei , Leishmania spp.). While all three contain D‐mannose (Man), D‐GlcNAc, D‐glucosamine (GlcN), D‐glucose (Glc), and D‐galactopyranose (Galp), only T. cruzi and Leishmania major contain D‐galactofuranose (Galf), and only T. cruzi contains D‐xylose (Xyl), L‐rhamnopyranose (Rha), and L‐fucose (Fuc) [32]. These taxon‐specific features highlight opportunities for parasite‐selective targeting.

To assemble glycoconjugates, parasites either import monosaccharides or recover them through degradation of extracellular or intracellular sources. In most eukaryotes, sugars are interconverted and activated into high‐energy donors, such as nucleotide sugars (e.g., UDP‐Glc) or lipid‐linked intermediates (e.g., dolichol‐phosphate‐mannose) in the cytoplasm, but in Trypanosomes many of the reactions (Figure 2) occur in glycosomes [33, 34, 35]. Glycosomes are kinetoplastid peroxisome‐like vesicles that contain glycolytic enzymes including HK and PGI that are also components of the de novo pathways to the nucleotide sugars (Figure 2) [34, 36, 37].

FIGURE 2.

Scheme of the biosynthesis of UDP‐Glc, UDP‐Galp, UDP‐Galf, UDP‐GlcNAc, GDP‐Man and GDP‐Fuc in T. cruzi . The intermediates and enzymes involved in steps discussed in the text are boxed and the right arm (highlighted in ice blue) contain intermediates and enzymes of the hexosamine biosynthetic pathway. See text for origin of other nucleotide sugars via the broad specificity USP. Enzyme abbreviations (in italics) are: GALE, UDP‐glucose 4′‐epimerase; GALK, galactokinase; GFAT, glucosamine‐fructose‐6‐phosphate aminotransferase; GMD, GDP‐mannose dehydratase; GMER, GDP‐4‐dehydro‐6‐deoxy‐D‐mannose epimerase/reductase; GNA, glucosamine‐6‐phosphate N‐acetyltransferase; HK, hexokinase; MPGT, mannose phosphate guanyltransferase; PAGM, phospho‐N‐acetylglucosamine mutase; PGI, phospho‐glucose isomerase; PGM, phospho‐glucose mutase; PMI, phospho‐mannose isomerase; PMM, phospho‐mannose mutase; PAGM, phospho‐N‐acetylglucosamine mutase; UAP, UDP‐N‐acetylglucosamine pyrophosphorylase; UGP, UDP‐glucose pyrophosphorylase; UGM, UDP‐galactopyranose mutase; USP, UTP sugar‐1‐phosphate uridyltransferase. IDs of each gene present in the T. cruzi CL‐B Esmeraldo‐like reference genome are highlighted in red.

Bioinformatic and biochemical evidence indicates that UDP‐GlcNAc, the donor substrate for GlcNAc‐transferases, is synthesized in T. cruzi primarily via the canonical eukaryotic hexosamine biosynthetic pathway (Figure 2, right arm) [32]. Trypanosomatids may also phosphorylate GlcN directly via HK. Because GlcNAc and/or GlcN occur in most known trypanosomatid glycoconjugates, UDP‐GlcNAc biosynthesis represents a potentially broad‐spectrum therapeutic target—provided that parasite‐selective inhibitors can be developed. A major regulatory bottleneck in this pathway is GFAT, which catalyses the first committed step using glutamine as the amino donor. Consistent with its central role, glutamine analogues that inhibit GFAT impair proliferation, intracellular development, and metacyclogenesis in T. cruzi [38]. Downstream enzymes have been studied in more detail in T. brucei . For example, GNA is essential in bloodstream‐form parasites [39], and UAP is also essential, as demonstrated using conditional null mutants [40]. PGAM, which interconverts GlcNAc‐6‐P → GlcNAc‐1‐P, is highly conserved among Tritryps [41].

T. cruzi does not import Gal via its constitutive TcrHT1 transporter [42]. A major source derives from epimerization of UDP‐Glc to UDP‐Gal by TcGALE (Figure 2, left arm) [43]. In glycosomes, HK and glucokinase generate Glc‐6‐P [34, 44, 45], which TcPGM interconverts to Glc‐1‐P. TcPGM activity is supported by its rescue of a lethal phenotype in a S. cerevisiae pgm1Δ/pgm2Δ double mutant grown on galactose [46]. UGP forms UDP‐Glc with strict specificity for Glc‐1‐P in T. cruzi , unlike the human enzyme [47]. TcGALE converts UDP‐Glc → UDP‐Gal but not UDP‐GlcNAc → UDP‐GalNAc, unlike the human enzyme, which rationalizes the α‐GlcNAc initiation of O‐glycans in T. cruzi mucins [43]. Failed attempts to generate TcGALE null mutants and phenotypes of TcGALE ^+/− lines [48] suggest essentiality: defective galactose metabolism leads to a six‐ to nine‐fold reduction of Galp mucins and smaller reductions in Galf‐GIPLs. These findings strengthen the rationale for exploring galactose‐pathway enzymes as drug targets.

Although TcrHT1 does not transport galactose, epimastigotes can grow in low‐glucose medium supplemented with galactose [49], implying a compensatory metabolism. Two GALKs phosphorylate galactose to Gal‐1‐P (Figure 2) [50]. This and other sugar‐1‐P donors are converted to UDP‐Gal, UDP‐Glc, UDP‐Xyl, and UDP‐glucuronic via the action of a broad‐specificity USP [51]. In L. major , simultaneous UGP/USP loss depletes UDP‐Glc/UDP‐Gal, causing arrest and death [52]. LmUSP structural work reveals conserved PPi‐coordinating features among Tritryp USPs, including TcUSP, validating these enzymes as potential drug targets [53].

UGM converts UDP‐Galp → UDP‐Galf (Figure 2) and is absent in humans, implying selective therapeutic potential [54]. TcUGM, the first protozoan UGM crystallized [55], shares UDP‐binding features with eukaryotic rather than bacterial UGMs, which may allow for selective inhibitor design.

It is not known how nucleotide sugars (NS) exit glycosomes, but NS transporters (NSTs) translocate NS into the lumen of the endoplasmic reticulum and Golgi apparatus for glycosylation reactions. A family of 11 NSTs was predicted in T. cruzi , and TcNTS1 was characterized as a Golgi UDP‐GlcNAc transporter expressed in all life cycle stages (Figure 3) [56]. In addition, a Golgi‐localized Mn²⁺‐Ca²⁺/H⁺ exchanger (TcGDT1) was shown to be required for efficient protein glycosylation, host cell invasion, and intracellular replication, but did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by exogenous Mn²⁺, underscoring the importance of this transition metal for Golgi GTs [57].

FIGURE 3.

Scheme of the main nucleotide sugar and Mn⁺² transporters and Golgi glycosyltransferases in T. cruzi CL‐Brener Esmeraldo‐like genome. The nucleotide sugar [56] and Mn⁺² [57] transporters and Golgi glycosyltransferases from GT60 [58, 59], GT67 [60, 61], GT77 (Koeller et al., manuscript in preparation), and GT40 [60, 62] are listed according to their Esmeraldo‐like genome IDs (red) obtained from the Tritryp DB (https://tritrypdb.org/tritrypdb/app).

2.2. N‐Glycans

Trypanosomatid N‐glycosylation displays several unusual features. For example, the lipid‐linked N‐glycan donor uses shorter polyprenols (10–13 isoprene units) and lacks the 3 terminal Glc residues present in most eukaryotes [63]. Nevertheless, after transfer the Man₉GlcNAc₂‐glycan may undergo quality‐control monoglucosylation in the ER by the mis‐folding sensor UDP‐Glc:glycoprotein glucosyltransferase (TcGT1) [64]. Disruption of both TcGT1 alleles abolishes detectable interaction of the cysteine protease cruzipain with the foldase chaperone calreticulin. Cruzipain secretion is delayed primarily due to prolonged association with the ER chaperone BiP/Grp78. This is associated with partial defects in differentiation and mammalian cell invasion, though parasite growth remains largely unaffected.

Additional N‐glycan processing to Man₅GlcNAc₂ and other isoforms likely occurs in the Golgi, although the precise subcellular sites of complex‐type glycan formation remain unresolved [65]. Core GlcNAc 6‐sulfation [66] is highly immunogenic [26]. In trypomastigotes, complex N‐glycans vary and contain peripheral β‐GlcNAc, β‐Galf [67], α‐Fuc, β‐Galp (Figure 4), and α3‐linked Galp, and sialic acid (SA), like in Tc‐85 (Figure 1) [66, 68].

FIGURE 4.

Representative scheme of the main N‐ and O‐glycan structures in T. cruzi . N‐glycan biosynthesis initiates in the rough endoplasmic reticulum with the transfer of the Man₉GlcNAc₂‐glycan to the target protein. Additional N‐glycan processing to Man_5‐7GlcNAc₂ and other isoforms containing GlcNAc 6‐sulfation may take place at the Golgi and, for trypomastigotes, this will include complex N‐glycans containing β‐GlcNAc, β‐Gal, α‐Fuc as found in cruzipain, or α‐Gal (not shown) as found in Tc‐85 (shown in Figure 1). Mucin‐type O‐glycosylation initiates in the Golgi with an α‐GlcNAc residue linked to Ser or Thr. The GlcNAcα1‐O‐Thr/Ser core is sequentially modified by β‐Galp or β‐Galf residues producing stage‐ and strain‐dependent branching patterns as indicated. The Tc‐mucin O‐glycan structures are described for the epimastigote stage. Sialylation of the N‐ and O‐glycans occurs at the parasite surface by TS (not shown).

2.3. O‐Glycans

O‐glycans on mucins comprise a major fraction of the glycocalyx (Figure 1). In trypanosomatids, mucin‐type O‐glycosylation initiates with an α‐GlcNAc residue linked to serine (Ser) or threonine (Thr) (Figure 4) [69, 70], unlike mammalian mucins, which initiate with α‐N‐acetylgalactosamine (GalNAc) [71, 72], and in T. cruzi , a small fraction remains as a monosaccharide modification [73].

Our group identified two paralogs, TcOGNT1 and TcOGNT2 (CAZy GT60‐family) (Figure 3), that encode gatekeeper ppαGlcNAcTs that transfer α‐GlcNAc from UDP‐α‐GlcNAc to Ser/Thr residues of polypeptide acceptors. Mutations of catalytic‐site residues (DSH) abolish activity [58], consistent with an ortholog in Dictyostelium [74] and related ppαGalNAcTs in mammals [71]. TcOGNT2 contributes to differentiation, infectivity, and virulence [59, 75], and failure to generate null mutants supports essentiality. Thus, ppαGlcNAcTs emerge as rational chemotherapeutic targets.

The resulting GlcNAcα1‐O‐Thr/Ser core is sequentially modified in the Golgi by β‐Galp or β‐Galf residues producing stage‐ and strain‐dependent branching patterns (Figure 4) [76, 77, 78, 79, 80, 81]. The actual O‐glycan repertoire depends on which GTs are expressed and their efficiency, and the availability of UDP‐Galp and UDP‐Galf donors in the secretory pathway. The GTs reside in CAZy families [82] found in all Tritryps [60, 83, 84] and include members from GT67‐βGalpTs and GT40‐βGalfTs (Figure 3) [61, 85, 86, 87, 88]. Many of the GTs are encoded by multigene families consisting of dozens or more copies, making genetic analysis of their roles challenging. For example, the application of CRISPR‐Cas9 technology to disrupt βGalfT activity was effective towards 63% of the 65 coding genes [62], but remaining expression precluded a test of their roles.

Terminal β‐Galp residues can only be modified by α2,3‐linked SA from exogenous sialoglycoconjugates, a reaction mediated by surface aTS [89, 90, 91, 92, 93]. In addition, sialylated O‐glycans can be found in MASPs (Figure 1) [94]. Alternatively, O‐glycans may terminate with α‐Galp at O‐3 of β‐Galp residues. The Galα1‐3Galβ1‐4GlcNAc motif is prominent in tissue‐culture trypomastigotes (TCTs) and in BT forms, is highly immunogenic in humans, and is a major target of lytic anti‐α‐Gal antibodies abundant in chronic ChD sera (Figure 1) [95, 96]. Recently, Tcα3GalT (CAZy GT77‐family) was identified as a UDP‐Gal:Gal α3‐galactosyltransferase candidate. Tcα3GalT deletion reduces infectivity, delays parasite release, and impairs differentiation to trypomastigotes while lowering α‐Gal epitopes (Koeller et al., manuscript in preparation). These findings functionally connect O‐glycan biosynthesis to virulence and suggest pathway enzymes as therapeutic targets and glycotopes as diagnostic markers.

2.4. GPI Anchors and GIPLs

All Tc‐mucin GPI anchors share a conserved glycan core (Manα1‐2Manα1‐2Manα1‐6Manα1‐4GlcNα1‐6InsPO₄) [19, 77], although TCT anchors may include branched Gal_0‐4 substitutions [19]. Despite O‐glycan similarities between epimastigote and MT mucins, lipid composition varies: epimastigotes contain exclusively glycerol‐linked saturated fatty acids, whereas MT anchors are predominantly (70%) inositol‐phosphoceramides [77, 97]. Tc‐mucins from TCTs are called tGPI‐mucins and their lipids consist of alkyl‐acyl‐phosphatidylinositol structures and include unsaturated fatty acids (C18:1/C18:2) at sn‐2 (Figure 1) [19, 98, 99, 100].

GIPLs are GPI‐like structures lacking attached proteins (formerly termed lipopeptidophosphoglycan, LPPG) and are present in all T. cruzi stages and strains [14, 21, 101, 102, 103, 104, 105, 106]. Like canonical GPI anchors, GIPLs contain mostly the same core, but substitutions vary across strains, like β1 → 2/3‐linked Galf residues, aminoethylphosphonate, and/or ethanolamine‐phosphate motifs. The lipid moiety can be ceramide (sphinganine [d18:0] or sphingosine [d18:1] linked to lignoceric/palmitic acid) or alkyl‐acylglycerol (sn‐1‐O‐alkyl‐C16:0; sn‐2‐O‐acyl‐C16:0/C18:0) (Figure 1) [100, 105, 106, 107].

3. Relationship Between Mucins and Trans‐Sialidase

Throughout development, T. cruzi is coated by extensively O‐glycosylated Tc‐mucins that contribute to parasite survival, infectivity, and ChD pathogenesis [77, 91, 108, 109]. Tc‐mucins are highly O‐glycosylated polyanionic molecules (~60% carbohydrate/weight) enriched in Ser/Thr [110]. Polypeptide diversity is driven by a large gene repertoire (~850 per haploid genome), broadly grouped into TcMUC and TcSMUG families based on structural criteria [111, 112]. TcMUC genes are expressed in mammalian stages, whereas TcSMUG genes, referred to as gp35/50 [76, 113], predominate in insect stages [20, 108]. In TCTs, tGPI‐mucins are heterogeneous due to simultaneous expression of multiple TcMUC genes, which vary in length/sequence and in O‐glycan structure and/or extension (Figure 1) [108].

In insect stage MTs, gp35/50 can protect against proteolysis and favour oral infection [114]. Together with gp82 and gp90, gp35/50 contributes to the MT invasion repertoire: monoclonal antibody 10D8 (recognizing Galf‐O‐glycans) and purified native Tc‐mucins reduce infectivity [113, 115]. In general, sialylated or not, gp35/50 contributes to host‐cell recognition, probably by annexin A2, and signalling by intracellular Ca²⁺ mobilization [115, 116, 117, 118].

In TCTs, sialylated tGPI‐mucins contribute to attachment and invasion [90, 92]. Although the detailed O‐glycan structures of tGPI‐mucins remain incompletely characterized, a key difference relative to insect‐stage mucins is the expression of α1,3Gal epitopes (Figure 1) [96]. Biochemical studies proposed that sialylation reduces susceptibility to anti‐α‐Gal‐mediated lysis [21], implying interplay between intracellular α‐glycosylation and surface sialylation. However, in vivo microscopy indicates that TS and tGPI‐mucins occupy distinct membrane domains; only a minority of α‐galactosylated and sialylated O‐glycans co‐occur within the same tGPI‐mucin, suggesting at least two glycoforms that segregate at the surface [119, 120]. Moreover, TS and sialylated tGPI‐mucins are found in extracellular vesicles (EVs) rather than as soluble proteins.

Functionally, trypomastigotes have a reduced invasion capacity in Lec2 cells that express low amounts of SA [92], and monoclonal antibodies to SA residues on tGPI‐mucins inhibit host‐cell entry [90]. In addition, a remarkable application of CRISPR‐Cas9 technology to the aTS multigene family documented that aTS knockout parasites retain the ability to invade and escape from parasitophorous vacuoles in vitro, but show impaired differentiation from amastigotes to trypomastigotes and reduced parasite egress [121]. In vivo, aTS mutants failed to establish infection even in IFN‐γ knockout mice, supporting a more complex role for aTS in parasite–host interactions than previously recognized.

4. Host Immune Response Modulation by Trypanosoma cruzi

4.1. Innate Immune Response vs. T. cruzi Glycoconjugates

T. cruzi has evolved multiple strategies to evade and subvert innate and adaptive immunity, enabling bloodstream dissemination and long‐term persistence in vertebrate hosts [16, 122]. Early suppression or misdirection of protective responses during acute infection is therefore a key step towards establishment of chronic disease.

Phagocytes—particularly macrophages, neutrophils, and dendritic cells (DCs)—constitute the first line of defence [18, 123]. These cells detect pathogen‐associated molecular patterns (PAMPs) and damage‐associated molecular patterns (DAMPs) through receptors such as Toll‐like receptors (TLRs). TLR signalling contributes to phagocyte recruitment and activation at infection sites, promoting parasite control [124, 125]. Upon activation, macrophages produce pro‐inflammatory cytokines including interleukin (IL)‐12 and TNF‐α, which synergistically stimulate IFN‐γ production by NK cells [126]. IFN‐γ and TNF‐α trigger inducible nitric oxide synthase in macrophages and thereby promote NO production, an important effector mechanism for parasite killing [127].

Among parasite ligands (Table 1), the lipid moiety of tGPI‐mucins strongly induces IL‐12, TNF‐α, and NO production in IFN‐γ‐primed murine macrophages [19, 98, 99, 100]. The disruption of sugar rings in the glycan GPI part by periodate oxidation reduces cytokine production, corroborating the need for the carbohydrate portion too [98]. Furthermore, tGPI‐mucins induce serum amyloid A3 [130] and chemokines from murine macrophages [131, 132]. This activity may contribute to leukocyte recruitment and to inflammatory pathology such as chagasic myocarditis. In PBMCs, tGPI‐mucins also induce IL‐12, which is potentiated by IFN‐γ and CD40–CD40L interactions [133].

TABLE 1.

Stage‐specific glycoconjugates involved in modulation of host immune response by Trypanosoma cruzi.

Stage	Ligand	Ligand fraction	PRR(s)	Signalling pathway	Downstream immune effect	Host/model system	References
TCT	tGPI‐mucin	Unsaturated fatty acid chains or glycan of GPI	TLR2	Via MAPK cascade and NF‐kB signalling	Induce IL‐12, TNF‐α and NO production by IFN‐γ primed murine Mϕ	In vitro assays using inflammatory Mϕ from C3H/HeJ and C3H/HeN mice [98, 99, 100]; bone‐marrow and peritoneal Mϕ from WT and TLR2−/− mice [128]; inflammatory Mϕ from C57BL/6 or C3H/HeJ mice [129]	[19, 98, 99, 100, 128, 129]
	tGPI‐mucin or TCT	—	Unknown	Not described	Induce serum amyloid A3 expression and chemokines by Mϕ	In vitro assays using inflammatory Mϕ from C3H/HeJ [130] or C57BL/6 [131] mice	[130, 131]
	tGPI‐mucin	—	TLR2	Mediated by IFN‐γ production and requires CD4+ and CD8+ cells in FCA‐primed mice	Induce leukocyte recruitment of IL‐12 primed mice and chemokine MCP‐1	In vivo and in vitro assays using pleural cavity leukocytes and Mϕ, respectively, of WT, IFN−/−, TLR2−/− C57BL/6 mice	[132]
	tGPI‐mucin or TCT	—	Unknown	Dependent of CD40‐CD40L interaction and IFN‐𝛾	Induce IL‐12 by PBMCs	In vitro assays with PBMCs from blood of healthy donors and heart‐infiltrating T Cell Lines from chronic ChD	[133]
	TCT	Less or more sialylated strain	Siglec‐E	Not described	Reduce IL‐12 and suppression of T cell activation through BMDCs	In vitro assays with BMDCs plus T cells and CHO cells expressing or not Siglec‐E	[134]
	tGPI‐mucin or TCT	—	TLR2/6	Negative regulation of TLR9‐Myd88 signalling	Decrease IL‐12 production triggered by T. cruzi unmethylated CpG motifs in murine DCs	In vitro assays with bone marrow and peritoneal Mϕ [135] or splenic cells of infected animals [136] from WT, TLR2−/−, TLR9−/− or Myd88−/− C57BL/6 mice	[135, 136]
	BTs or T. cruzi antigens	Probable by GPI‐anchored molecules	TLR2/4	Reactive oxygen species‐dependent manner	Stimulate NETs release by human neutrophils modulating parasite infectivity/pathogenicity	In vitro assays with isolated human neutrophils from blood of healthy donors	[137]
	T‐DAF, TcCRP	—	—	Bind to C3b/C4b and accelerates decay of C3 convertases or assembly	Block complement activation to lyse T. cruzi	In vitro assays with human [138, 139], rabbit or guinea complement [140, 141] components and mouse, rabbit or human anti‐TCT [138, 139] anti‐T‐DAF [139] or anti‐CRP [140, 141] sera	[138, 139, 140, 141]
	Gp58/68, tGPI‐mucin	SA of tGPI‐mucin	—	Block interaction of factor B to parasite‐bound C3b or promoting conversion of C3b to iC3b	Reduce activation of alternative complement pathway	In vitro assays with human [142, 143], guinea pig or rat complement components [142]; normal [143, 144] or anti‐ T. cruzi human serum, anti‐ T. cruzi monoclonal antibodies [144] and sialylated or not TCTs [145]	[142, 143, 144, 145]
	TCTs	EVs	Unknown	Increased amounts of IL‐4 and IL‐10 synthesis	Promote severe cardiac pathology in mice, intense inflammation and high tissue parasitism	Ex vivo analyses of Balb/c mice inoculated or not with T. cruzi EVs prior parasite infection	[146]
Epis, MTs, amasti‐gotes	AgC10	—	CD62L/L‐selectin	Not described	Impairs IL‐12 and TNF‐α secretion by Mϕ	In vitro assays with human monocytes and J774‐G8 murine Mϕ cell line	[147]
		—	CD62L/L‐selectin	Reduction of transcription/expression of IL‐2 receptor and IL‐2 secretion in CD4+ and CD8+ cells	Inhibit proliferation of human T cells, and decrease production of IL‐2 and IFN‐γ by PBMCs	In vitro assays with purified human T cells and PBMCs [148, 149] or spleen cells and/or purified CD4+/CD8+ T cells of WT, iNOS−/− or Sell−/− C57Bl/6 mice [150]	[148, 149, 150]
Epis	GIPLs	Ceramide	Unknown	Not described	Induce apoptosis, decrease secretion of IL‐12, IL‐10 and TNF‐α, and expression of co‐stimulatory molecules in Mϕ	In vitro assays with primary Mϕ of Balb/c mice or J774‐G8 murine Mϕ cell line [151]; human Mϕ from PBMCs [152]	[151, 152]
	GIPLs (epi) or BTs	Ceramide of GIPLs	Unknown	Dependent of CD40L	Decrease secretion of TNF‐α, IL‐10 and IL‐12 and downregulation of activation and maturation markers of APC in human DC	In vitro assays with human DC from PBMCs	[152, 153]
	GIPLs	—	TLR4	Via TLR4/MD‐2 complex and NK‐kB activation	Induce neutrophil attraction and recruitment	Neutrophil ex vivo analyses of WT or TLR4−/− C57BL/10 mice inoculated or not with GIPLs and CHO/CD14 reporter cell line	[154]
	GIPLs	Ceramide	Unknown	Not described	Suppression of CD4+ and CD8+ T cells mitogenesis in vitro and lymph node T cell activation in vivo	In vitro assays with splenic CD4+ and CD8+ T cells of Balb/c mice and ex vivo lymph nodes analyses of WT Balb/c mice injected or not with GIPLs	[155]
	GIPLs	Ceramide	Unknown	Induced a rise in free [Ca2+]i and the dephosphorylation of nuclear factor of activated T cells 1 (NFAT1) and its translocation to the nucleus	Enhance CD3‐ and Th1‐mediated IL‐2 production	In vitro assays with T hybridoma cell line DO‐11.10 and splenic T cell of WT Balb/c mice	[156]
	GIPLs	Glycan or whole molecule	Unknown	Not described	B‐cell activation and Ig secretion and increase IL‐2‐induced NK cells proliferation	In vitro assays with splenic B cell of WT Balb/c mice [157] or CD3e transgenic (deficient of T and NK cells) mice [158]	[157, 158]

In contrast, some mucin‐like antigens expressed in insect forms and amastigotes can dampen macrophage activation. For instance, T. cruzi AgC10 impairs IL‐12 and TNF‐α secretion by LPS‐activated human monocytes or T. cruzi ‐infected macrophages via the CD62L/L‐selectin receptor [147]. In addition, the ceramide portion of GIPLs appears to decrease LPS‐induced secretion of IL‐12, IL‐10 and TNF‐α, expression of co‐stimulatory molecules (e.g., CD40), and induce apoptosis in human and murine macrophages, especially through IFN‐γ synergism [151, 152]. In conclusion, these data corroborate reports that insect‐stage Tc‐mucins and GIPLs are 100–1000‐fold less active than tGPI‐mucins in inducing pro‐inflammatory cytokines and NO in murine macrophages, and this is mainly explained by the lipid moiety of the GPI anchors [19, 98, 99, 100].

Activation of innate cells also shapes adaptive immunity. In the presence of PAMPs/DAMPs, DCs, macrophages, and neutrophils upregulate costimulatory molecules and cytokines that promote T cell activation [159]. IFN‐γ is central in this transition, affecting the development of CD4+ Th1 responses and CD8+ T cell immunity necessary to control parasite replication in acute infection. Among CD8+ T cell subsets, type‐1 cytotoxic T cells contribute to parasite control through the production of TNF‐α and IFN‐γ [160, 161].

T. cruzi infection can also impose tolerogenic programmes on antigen‐presenting cells. Based on in vitro assays using C57BL/6 WT mice, T. cruzi modulates DC biology with varying intensities depending on the parasite strain [162]. In human DCs, BT parasites and the ceramide portion of GIPLs can reduce TNF‐α, IL‐10, and IL‐12 production and mediate downregulation of MHC class I/II and CD40 co‐receptor, impairing antigen presentation [152, 153]. Similarly, sialylated TCT structures of the Tulahuen strain, through interaction with Siglec‐E, result in reduced IL‐12 secretion and suppression of T cell activation—likely CD8+ T cells—induced by murine bone marrow‐derived DCs (BMDCs) [134].

Several T. cruzi glycoconjugates (Table 1) act as TLR ligands, connecting the innate immune response to subsequent inflammation and Th1 response polarization. In murine macrophages, tGPI‐mucins act as TLR2/6 agonists and induce cytokine production via activation of MAPK cascades and NF‐κB signalling, promoting NO and pro‐inflammatory cytokine production [128, 129]. Furthermore, the tGPI‐mucins and TLR2 association can induce leukocyte recruitment in IL‐12‐primed mice, and the underlying mechanism involves priming of animals with Freund's complement adjuvant (FCA), which leads to the activation of CD4+ and CD8+ cells, which also produce IFN‐γ and directs the inflammatory response induced by tGPI‐mucins [132].

In addition, GIPLs as TLR4 agonists can induce murine neutrophil attraction and recruitment via TLR4/MD‐2 complexes and NF‐kB activation and are probably also responsible for the hypersusceptible T. cruzi infection in TLR4^−/− mice [154]. However, human neutrophils stimulated by BTs or soluble T. cruzi antigens, including GPI‐anchored molecules, can release neutrophil extracellular traps (NETs) via TLR2/4 in a reactive oxygen species–dependent manner, modulating parasite infectivity/pathogenicity without necessarily reducing viability [137]. In addition, THP‐1 macrophages infected with T. cruzi release increased levels of EVs compared with uninfected cells. These EVs can carry parasite proteins, including tGPI‐mucins, that interact with TLR2 and induce NF‐κB nuclear translocation, sustaining production of pro‐inflammatory cytokines such as TNF‐α, IL‐6, and IL‐1 [163, 164].

TLR cross‐talk may further modulate the balance between early inflammation and late adaptive responses. In murine DCs, TLR2/6 stimulation by tGPI‐mucins negatively regulates TLR9‐dependent IL‐12/IL‐23p40 production, which is triggered by unmethylated CpG motifs in T. cruzi DNA [135, 136]. This interaction may contribute to delayed development of parasite‐specific adaptive immunity and facilitate early ‘silent’ infection [136].

Finally, MTs of some strains (CL‐Brener, Gamba 05, MLCD88) but not others have been reported to resist humoral immunity by evading complement‐mediated lysis through lectin and alternative pathways [165, 166]. This activity involves multiple surface molecules that inhibit complement activation (Table 1). Trypomastigote decay‐accelerating factor (T‐DAF), an analog of human DAF, binds C3b and C4b and accelerates decay of C3 convertases, limiting amplification of classical and alternative pathways [138, 139]. Complement regulatory protein (TcCRP; gp160), a GPI‐anchored glycoprotein expressed in TCTs, also binds C3b/C4b and prevents C3 convertase assembly, thereby inhibiting complement activation [140, 141]. It has been suggested that higher TcCRP expression correlates with virulence in different strains [167]. T‐DAF and TcCRP are TS‐like glycoproteins that belong to the iTS family, but they lack lectin activity [139, 140]. Furthermore, gp58/68 reduces the alternative pathway by forming C3 convertase, blocking the interaction of factor B with parasite‐bound C3b [142]. High levels of SA on tGPI‐mucins can also recruit factor H, promoting conversion of C3b to iC3b and reducing alternative pathway activation [143, 144, 145]. Collectively, these mechanisms reduce C3 deposition and compromise complement‐mediated clearance, enabling persistence.

4.2. Adaptive Immune Response vs. T. cruzi Glycoconjugates

T lymphocytes are central to adaptive immunity against T. cruzi . In murine models, depletion of CD4+ and/or CD8+ T cells—by monoclonal antibody treatment or genetic disruption—results in increased tissue and blood parasitemia and failure to survive acute infection, demonstrating the essential contribution of these subsets [168, 169, 170, 171].

Several parasite glycoconjugates and glycoantigens can directly modulate T cell responses (Table 1). Because TS immunomodulation—particularly effects on T and B cells—has been reviewed extensively elsewhere [17, 24], we focus here on other carbohydrate antigens and glycotopes that shape immune recognition.

Many studies report that the parasite's AgC10 mucin inhibits the proliferation of human T lymphocytes, reduces transcription/expression of IL‐2 receptor chains in CD4+ and CD8+ cells, and decreases production of type 1 cytokines such as IL‐2 and IFN‐γ by PBMCs [148, 149]. Furthermore, the restriction of T cell proliferation, as well as IL‐2 secretion and IL‐2 mRNA induction in response to mitogens by AgC10 in murine spleen cells, involves the inhibition of the initial steps of T cell activation, such as the blockage of the transcription factors NFTA, AP‐1, and the activation of the IL‐2 promoter through the CD62L/L‐selectin, and appear to be independent of IFN‐γ or NO [150].

T. cruzi EVs originated from TCTs containing tGPI‐mucins and TS have been reported to promote severe cardiac pathology in mice, with intense inflammation, increased synthesis of IL‐4 and IL‐10, and high tissue parasitism with amastigote nests [146]. In this model, the infiltrate is enriched in CD4+ T cells and macrophages rather than CD8+ T cells, and NO levels are low [146].

The ceramide portion of GIPLs has been implicated in suppressing CD4+ and CD8+ T cell mitogenesis and regional lymph node T cell activation in vitro and in vivo, respectively, with loss of IL‐2 responsiveness [155]. Conversely, the same GIPL fragment, along with mitogenic antibodies, can enhance CD3‐ and Thy1‐mediated IL‐2 secretion and IL‐2 mRNA accumulation, indicating context‐dependent modulation of host signalling [156].

B cells and antibodies also contribute to parasite control (Figure 1). In mouse models, B cell‐deficient animals can survive longer than mice lacking CD4+ or CD8+ T cell function, suggesting that T cell immunity is especially critical for early control of infection. Nonetheless, B‐cell deficient mice eventually die during acute infection [171, 172]. In vitro, the oligosaccharide moiety of GIPLs, with appropriate cytokines, co‐stimulatory signals or IL‐2‐induced NK cells, can promote B cell activation and IgM and IgG secretion [157, 158]. In addition, GIPLs can also increase IL‐2‐induced NK cell proliferation [158]. Consistent with broad antigenic exposure, the Immune Epitope Database contains numerous entries for T. cruzi molecules and thousands of antibody‐binding epitopes identified in human infection and animal models, including glycoantigens such as gp160, gp90, T‐DAF, MASPs, TS, gp82, multiple Tc‐mucins, and the α‐Gal epitope [18].

For example, Galf is absent from human glycoconjugates but is found in bacteria, fungi, plants, and protozoa [173]. Because humans lack Galf, infected individuals can develop anti‐Galf antibodies [67], linking Galf biosynthesis to immunogenicity and suggesting possible diagnostic and therapeutic applications. In T. cruzi , Galf is found in GIPLs [102], GPI anchors, and mucin O‐glycans (Figure 1) [103, 174], but not in all strains.

Infective forms display additional immunodominant O‐glycan epitopes, including Galα1,3Galβ1,4GlcNAc and branched α‐Gal‐terminated structures (Figure 1) [96, 100]. Anti‐α‐Gal antibodies can trigger complement‐mediated killing of trypomastigotes [175], although parasites evade this response through shedding and altered epitope presentation.

Although not all antibodies are lytic, lytic antibodies can directly promote parasite killing via classical and alternative complement pathways [175, 176]. However, antibody responses alone generally fail to eliminate T. cruzi , allowing persistent infection, using adipose tissue, cardiac and skeletal muscles, and the nervous system as chronic infection reservoirs [16, 18].

5. Are Trypanosoma cruzi Glycans and Glycan‐Biosynthetic Enzymes Targets for Therapy and Diagnosis?

5.1. Inhibitors as Potential Agents Against T. cruzi

Efforts to disrupt T. cruzi metabolic pathways using small‐molecule inhibitors have largely focused on early carbohydrate metabolism, because interference at this level limits the production of sugar nucleotide donors required for biosynthesis of a range of glycan types. Multiple groups have pursued hexokinase inhibitors through in silico modelling and high‐throughput screening. Candidate scaffolds include bisphosphonates, nitrochromenes, sulfonamides, and glucosamine analogues. These molecules exploit structural differences within the glucose‐binding site or mimic PPi, a natural inhibitor of the parasite enzyme that does not inhibit human hexokinase [45, 177, 178, 179, 180].

A complementary strategy targets PGM, which acts upstream of UDP‐Glc synthesis (Figure 2). The small molecule ISFP10 selectively inhibits fungal PGM, like from Aspergillus fumigatus , with ~50‐fold selectivity relative to human PGM [181]. This selectivity reflects a conserved cysteine residue positioned near the sugar‐binding loop in pathogenic fungi [182]. TcPGM contains two additional cysteines near the PO₄‐binding loop not present in human or fungal PGMs [46]. These structural differences suggest that ISFP10‐derived scaffolds could achieve parasite‐specific inhibition, although no experimental validation exists yet in T. cruzi .

Inhibitors of UDP‐sugar interconversion enzymes also show promise. Flexible docking has identified GALE inhibitors in T. brucei , yielding low‐micromolar compounds based on a 2′‐carbamoyl‐[1,1′‐biphenyl]‐2‐carboxylate core [183]. Separately, a reported inhibitor of L. donovani UGM (PubChem6064500: (4E)‐4‐(4‐ethoxyphenyl)‐4‐[[3‐[(2‐methoxyphenyl)sulfamoyl]benzoyl]hydrazinylidene]butanoic acid) reduces promastigote growth, but displays comparable toxicity towards THP‐1 cells, which limits its translational potential [184].

Despite the importance of glycan‐dependent immunity, inhibitors targeting T. cruzi β‐GalfTs, α/β‐GalpTs, or α/β‐GlcNAcTs have not been described. Only a limited number of β‐GalfT inhibitors exist for mycobacteria [173, 185]. Oncology provides a conceptual precedent for glycosylation‐directed therapies: strategies such as glycan‐targeted vaccines, CAR‐T approaches, and small‐molecule glycosyltransferase inhibitors have advanced to phase 1–2 clinical trials, including SGN‐2FF, which reduces fucosylation of glycoproteins in solid tumours [186]. More broadly, glycosylation inhibitors probe glycan function and hold therapeutic potential in cancer, inflammation, and infectious diseases [187, 188]. Yet successful inhibition requires penetration into the cytosol and access to Golgi‐localized enzymes, and many compounds fail because of polarity and poor permeability. A recent cell‐based screen for inhibitors of O‐glycosylation in a mammalian cell line, designed to bypass this limitation, yielded effective compounds but their effects were apparently rendered by disrupting Golgi organization rather than directly inhibiting the GTs [189].

Given that TcOGNT1/2 represents putative chemotherapeutic targets within T. cruzi , a practical approach is to test existing cell‐permeable glycosylation inhibitors as lead scaffolds. Most inhibitors characterized to date target mammalian mucin‐type initiation by GALNTs [190] or cytosolic/nuclear OGT [191, 192]. This creates two mechanistic challenges for translational development: (i) T. cruzi initiates mucin O‐glycan biosynthesis with GlcNAc rather than GalNAc, and (ii) the linkage is α‐O‐GlcNAc rather than β‐O‐GlcNAc. Among known cell‐permeable structures, peracetylated N‐thioglycolyl‐D‐galactosamine (Ac5GalNTGc) inhibits mucin‐type O‐glycosylation by entering via the GalNAc salvage pathway and incorporating into Ser/Thr residues, thereby blocking O‐glycan extension [193, 194]. This inhibition reduces leukocyte selectin‐ligand expression and inflammatory recruitment [194]. Importantly, these findings raise the hypothesis that the Ac5GlcNTGc derivative—used as a negative control—could inhibit T. cruzi ppαGlcNAcTs.

5.2. Glycotopes for Diagnosis

In acute ChD, motile trypomastigotes can be detected in the blood by microscopy or culture, and parasite DNA can be detected by PCR, which currently offers the most sensitive early diagnostic method [195]. In chronic disease, diagnosis relies primarily on IgG serology, but no single assay achieves optimal sensitivity and specificity, necessitating the use of multiple tests [196]. Anti‐parasitic antibodies may better reflect treatment efficacy than direct parasite detection because sustained immune stimulation can maintain high antibody titres even with low parasite burden or circulating parasite antigens [197]. However, crude antigens from T. cruzi lysates are heterogeneous and can cross‐react, which limits diagnostic precision [198, 199].

Defined glycotopes provide attractive alternatives to crude parasite lysates. For instance, α‐Gal epitopes present on tGPI‐mucins elicit abundant and relatively specific antibodies in chronic patients [96, 175, 200] and can seroconvert following chemotherapy, supporting their use as biomarkers of treatment response [201, 202]. However, anti‐α‐Gal antibodies are produced in response to various stimuli and represent a significant component of the humoral immune repertoire in Old World monkeys, apes, and humans [203]. Given the multiple potential sources of α‐Gal antigens—namely, food, microbiota, and infectious agents [204]—one would expect host variability in anti‐α‐Gal titres. As a rule, anti‐α‐Gal antibody titres tend to rise with age, exhibiting low levels in infants and elevated levels in elderly individuals. This phenomenon suggests that the intestinal microbiota may play a pivotal role in providing the primary α‐Gal antigen immunization in humans [205]. It has been shown recently that these antibodies presented lower Galα1‐3Galβ1‐ avidity than anti‐α‐Gal antibodies from ChD individuals, suggesting that they were originally induced by a different (though structurally related) antigen [206]. Therefore, baseline anti‐α‐Gal antibodies in uninfected individuals will necessitate careful antigen design [203, 207, 208]. A chemiluminescence‐enzyme‐linked immunosorbent assay revealed that a synthesized neoglycoprotein containing Galα [1, 3]Galβ [1, 4]GlcNAcα‐BSA is recognized by pooled sera of chronic ChD patients ∼20‐fold more strongly than pooled normal human sera or shorter saccharide‐BSA versions [208].

In this context, β‐Galf‐containing epitopes, which are absent from humans, represent particularly promising diagnostic targets. Low baseline reactivity in uninfected sera may reduce cross‐reactivity and improve assay specificity [67, 209, 210, 211]. Together, α‐Galp and β‐Galf glycotopes have untapped potential to overcome limitations of lysate‐based assays and strengthen both diagnosis and post‐treatment monitoring.

6. Glycovaccines

Despite the clear economic rationale for a vaccine—estimated to provide net cost savings even at low efficacy—the field suffers from chronic underinvestment, with Chagas R&D receiving only a fraction of the funding allocated to other infectious diseases like HIV [212, 213]. Over a century of research has explored various approaches, from whole parasites killed by various methods, sub‐cellular fractions or extracts, purified or recombinant proteins, attenuated live parasites and, more recently, to viral vectors and DNA–RNA vaccines [197, 214, 215, 216]. Yet achieving sterilizing immunity remains elusive [217]. Major hurdles include the parasite's complex life cycle, its diverse genetic lineages, and sophisticated immune evasion mechanisms, such as entering a dormant or ‘persister’ state [197, 218]. Furthermore, scientific divergence persists regarding whether immune exhaustion or excessive pro‐inflammatory responses drive chronic pathogenesis, complicating the definition of a protective ‘balanced’ response [209, 217].

A promising frontier involves targeting the parasite's glycocalyx, specifically the α‐Gal glycotopes found on surface‐expressed mucins [215]. Research demonstrates that α‐Gal‐based glycovaccines can elicit high levels of protective lytic antibodies, providing up to 99.9% reduction in parasite load in experimental models [209, 215]. Additionally, the inclusion of Shed Acute Phase Antigen repeats in trans‐sialidase (TS) vaccines has been shown to modulate the host response towards a more balanced, anti‐inflammatory profile by increasing IL‐10 production [216]. The potential of mRNA platforms cannot be applied to glycotopes, but this and combination approaches such as vaccine‐linked chemotherapy can be expected to overcome inherent limitations of monotherapy [197, 217]. For example, vaccine‐linked chemotherapy improved cardiac structure and function in a mouse model of chronic ChD, suggesting a long‐lasting protective effect [219].

7. Conclusions

Collectively, evidence across structural glycobiology and immunology indicates that the T. cruzi glycocalyx is not merely a protective coat but an active interface that orchestrates immune activation, immune suppression, and complement resistance in a stage‐ and strain‐dependent manner. GPI‐anchored mucins, trans‐sialidase‐associated functions, and GIPLs can trigger inflammatory pathways while simultaneously engaging inhibitory circuits and reshaping antigen‐presenting cell function, thereby promoting parasite persistence and contributing to chronic pathology. From a translational perspective, parasite‐restricted features—such as Galf metabolism and distinct glycosylation initiation steps—support the pursuit of selective inhibitors of sugar‐nucleotide biosynthesis and glycosyltransferases, although cell permeability and compartment access remain key hurdles. In parallel, defined α‐Galp and β‐Galf glycotopes offer a rational path towards more specific serologic assays, improved biomarkers of therapeutic response, and glycovaccines. Future progress will depend on resolving incompletely characterized glycan structures in infective stages, integrating in vivo validation of enzymatic targets, and standardizing glycotope‐based platforms across diverse clinical settings.

Author Contributions

N.H., C.M.W. and C.M.K. designed and implemented the review, performed a literature search, and wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. All the authors designed and produced illustrations and the table.

Funding

Work in our laboratories was funded by grants from Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ) (CMK grant E‐26/210.361/2022); the National Council for Scientific and Technological Development (CNPq) (CMK grant 416128/2024‐4); the National Institutes of Health, USA (CMW grant R21 AI123161) and National Institute of Science and Technology (INCT) (NH and CMK grant 408756/2024‐INCT_2024).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.