ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi

Bruno Rafael Barboza; Janaina Macedo-da-Silva; Lays Adrianne Mendonça Trajano Silva; Vinícius de Morais Gomes; Deivid Martins Santos; Antônio Moreira Marques-Neto; Simon Ngao Mule; Claudia Blanes Angeli; Juliana Borsoi; Carolina Borsoi Moraes; Cristiane Moutinho-Melo; Martina Mühlenhoff; Walter Colli; Suely Kazue Nagashi Marie; Lygia da Veiga Pereira; Maria Julia Manso Alves; Giuseppe Palmisano

doi:10.1371/journal.pntd.0012454

. 2024 Sep 25;18(9):e0012454. doi: 10.1371/journal.pntd.0012454

ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi

Bruno Rafael Barboza ¹, Janaina Macedo-da-Silva ¹, Lays Adrianne Mendonça Trajano Silva ¹, Vinícius de Morais Gomes ¹, Deivid Martins Santos ¹, Antônio Moreira Marques-Neto ¹, Simon Ngao Mule ¹, Claudia Blanes Angeli ¹, Juliana Borsoi ², Carolina Borsoi Moraes ³, Cristiane Moutinho-Melo ^4,⁵, Martina Mühlenhoff ⁶, Walter Colli ⁷, Suely Kazue Nagashi Marie ⁸, Lygia da Veiga Pereira ², Maria Julia Manso Alves ⁷, Giuseppe Palmisano ^1,^9,^*

Editor: Susan Madison-Antenucci¹⁰

¹GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

²Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, São Paulo, Brazil

³Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil

⁴Laboratory of Vaccine Development, Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

⁵Laboratory of Immunological and Antitumor Analysis, Department of Antibiotics, Bioscience Center, and Keizo Asami Immunopathology Laboratory, Federal University of Pernambuco, Recife, Brazil

⁶Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany

⁷Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil

⁸Laboratory of Molecular and Cellular Biology (LIM 15), Department of Neurology, School of Medicine, University of São Paulo, São Paulo, Brazil

⁹School of Natural Sciences, Macquarie University, Sydney, New South Wales, Australia

¹⁰Wadsworth Center, UNITED STATES OF AMERICA

The authors have declared that no competing interests exist.

^✉

* E-mail: palmisano.gp@usp.br

Roles

Bruno Rafael Barboza: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Janaina Macedo-da-Silva: Data curation, Formal analysis, Methodology, Software, Visualization, Writing – review & editing

Lays Adrianne Mendonça Trajano Silva: Formal analysis, Methodology, Visualization, Writing – review & editing

Vinícius de Morais Gomes: Formal analysis, Methodology, Visualization, Writing – review & editing

Deivid Martins Santos: Formal analysis, Methodology, Visualization, Writing – review & editing

Antônio Moreira Marques-Neto: Formal analysis, Investigation, Methodology, Visualization, Writing – review & editing

Simon Ngao Mule: Formal analysis, Investigation, Methodology, Visualization, Writing – review & editing

Claudia Blanes Angeli: Data curation, Methodology

Juliana Borsoi: Investigation, Methodology, Visualization, Writing – review & editing

Carolina Borsoi Moraes: Investigation, Methodology, Visualization, Writing – review & editing

Cristiane Moutinho-Melo: Data curation, Methodology

Martina Mühlenhoff: Investigation, Methodology, Resources, Writing – review & editing

Walter Colli: Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Suely Kazue Nagashi Marie: Investigation, Methodology, Resources, Visualization, Writing – review & editing

Lygia da Veiga Pereira: Investigation, Methodology, Resources, Writing – review & editing

Maria Julia Manso Alves: Formal analysis, Investigation, Methodology, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing

Giuseppe Palmisano: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Susan Madison-Antenucci: Editor

PMCID: PMC11466412 PMID: 39321148

Abstract

Glycosylation is one of the most structurally and functionally diverse co- and post-translational modifications in a cell. Addition and removal of glycans, especially to proteins and lipids, characterize this process which has important implications in several biological processes. In mammals, the repeated enzymatic addition of a sialic acid unit to underlying sialic acids (Sia) by polysialyltransferases, including ST8Sia2, leads to the formation of a sugar polymer called polysialic acid (polySia). The functional relevance of polySia has been extensively demonstrated in the nervous system. However, the role of polysialylation in infection is still poorly explored. Previous reports have shown that Trypanosoma cruzi (T. cruzi), a flagellated parasite that causes Chagas disease (CD), changes host sialylation of glycoproteins. To understand the role of host polySia during T. cruzi infection, we used a combination of in silico and experimental tools. We observed that T. cruzi reduces both the expression of the ST8Sia2 and the polysialylation of target substrates. We also found that chemical and genetic inhibition of host ST8Sia2 increased the parasite load in mammalian cells. We found that modulating host polysialylation may induce oxidative stress, creating a microenvironment that favors T. cruzi survival and infection. These findings suggest a novel approach to interfere with parasite infections through modulation of host polysialylation.

Author summary

Glycosylation, the process of adding and removing sugar molecules from proteins, lipids, and small RNAs, plays a crucial role in numerous biological processes. Trypanosoma cruzi, the parasite responsible for Chagas disease affecting 6 to 8 million people globally, significantly alters the sialylation of host cell glycoproteins. In mammals, the enzyme ST8Sia2 is involved in forming polysialic acid (polySia), primarily studied in the nervous system, yet its role in infectious diseases remains poorly understood. In our study, we found that T. cruzi infection reduces the expression of ST8Sia2 in host cells, leading to decreased levels of polySia. This reduction also affects key proteins such as NCAM1 and SCN5A. Furthermore, experiments involving the removal of polySia, chemical inhibition of ST8Sia2, or genetic silencing of the enzyme resulted in increased intracellular parasites numbers. We propose that altering host polysialylation may induce oxidative stress, thereby favoring T. cruzi survival and infection. Our findings suggest that inhibiting ST8Sia2 enhances T. cruzi infection, providing new insights into Chagas disease mechanisms and highlighting the important role of host polysialylation.

Introduction

Glycans are important regulators in different biological processes, with prominent roles in cell adhesion [1], cell activation and signaling [2,3], modulation of immune responses [4,5], cell death [6], and initiation of tumor progression and metastases [7]. The addition and removal of glycans (mono, oligo and polysaccharides) to proteins, lipids [8] and small RNAs [9] is performed by an array of glycosyltransferases and glycosidases, that act in a concerted way to remodel the cellular glycome [10,11]. Glycosylation is one of the most structurally and functionally complex co- and/or post-translational modifications that occur in cells [12,13]. Glycans attached to glycoconjugates, commonly expressed on the surface of cells, exhibit enormous variability and structural complexity, contributing to their respective biological activities [14].

The majority of glycan chains are composed of monosaccharides with five- or six-carbons, with the striking exception of sialic acid (Sia), which belongs to a large family of sugars with a nine-carbon atom backbone and typically found attached to the terminal position of glycans [15]. The glycosidic linkage between a Sia monomer to another underlying Sia residue generates homo-oligo/polymer structures from di- to polysialic acid (polySia) with different α2,4/8/9 intersialyl linkages [16,17]. The biosynthesis of α2,8-linked polySia is catalyzed by ST8Sia polysialyltransferases (polySTs) [18]. In mammals, polysialylation is catalyzed by Golgi-resident polySTs: ST8Sia2 and ST8Sia4 [16,19]. Recently, ST8Sia3 was reported as a polyST capable of autopolysialylation [20]. Structurally, polySia is characterized by repeated Sia monomers with a degree of polymerization ranging from 8 to 400 sialic acid molecules [21]. The presence of polySia is confined to a restricted set of glycoproteins, with the neural cell adhesion molecule 1 (NCAM1) as the best-studied polySia-carrier in mammals [22].

The biological relevance of polySia in mammals has been extensively explored in the brain, highlighting its role in the migration of neuronal precursor cells [23], synaptic neuronal plasticity [19], and axonal outgrowth [24]. The involvement of polySia has also been demonstrated in the development of other organs, including the liver [25], kidney [26], placenta [27], and heart [28]. Beyond the considerable number of studies that have provided important information about the role of polysialylation in the nervous system, the participation of polySTs and polySia in the cardiac system has been less explored. Recently, NCAM and polysialylation have been shown to play critical roles in the cardiac conduction system [29]. Additionally, in ST8Sia2 knockout mice, polysialylation deficiency compromised the action potential and the voltage-gated sodium channel functions in atrial cardiomyocytes [30], experimentally evidencing that modulation in polysialylation is sufficient to alter the complex developmental biology and function of cardiomyocytes.

Chagas disease, also known as American trypanosomiasis, is an anthropozoonosis caused by the protozoan T. cruzi that affects between 6 and 8 million individuals worldwide, being considered a neglected tropical disease in the world [31,32]. Clinically, CD can be characterized by two main successive phases, the acute and the chronic phases [33]. Among the clinical manifestations of Chagas disease, Chronic Chagas Cardiomyopathy (CCC), presents itself as the most severe manifestation of the disease and affects 1/3 of infected individuals. CCC comprises four main syndromes: heart failure, arrhythmias, thromboembolism and anginal manifestations that can simultaneously occur in the same individual [34,35].

In the course of host infection, T. cruzi uses a wide variety of mechanisms to escape from host immune surveillance and establish a successful infection, with emphasis on Trans-sialidases (TS) [36,37] which catalyze direct Sia transfer between macromolecules, a novel enzymatic activity that was earlier demonstrated [38]. During host cell adhesion and invasion, these enzymes provide an elegant mechanism in which T. cruzi specifically captures α2,3-sialic acid from host cells and covers its own surface molecules, mainly mucins, as reviewed in [37,39,40]. Of note, T. cruzi parasites are incapable of synthesizing Sia [41,42]. In fact, parasite cells transfer Sias from host cell-surface, the internal surface of the phagolysosome of host cells and from serum sialoglycoproteins to trypanosome cell-surface glycoconjugates [36,43,44].

Previous studies reported that in T. cruzi infection, possible disorders of host aberrant glycosylation are due to impairment of glycoprotein sialylation. In 1986, Libby and colleagues [45] demonstrated in vitro that T. cruzi trypomastigotes modify the surface of rat myocardial and human vascular endothelial cells by desialylation. According to Marin-Neto and colleagues [46], the loss of Sias on the surface of endothelial cells can promote an increase in platelet aggregation and, consequently, microvascular thrombosis. More recently, it was demonstrated that CD43, a sialoglycoprotein expressed at high levels on leucocytes and previously reported as a natural receptor for T. cruzi trans-sialidase [47], plays a critical role in the pathogenesis of acute chagasic cardiomyopathy. In this study, a reduced cardiac parasite load in CD43 knockout mice [48] was observed, suggesting the involvement of CD43 in the development of Chagas cardiomyopathy.

Considering the clinical implications of Chagas cardiomyopathy and the current gaps in understanding the involvement of cardiac glycosylation in susceptibility and/or resistance to T. cruzi infection, exploring the interplay between glycosylation modulation and possible changes in the pathophysiology of Chagas cardiomyopathy presents an compelling avenue for investigation. This study unveils novel molecular mechanisms exploited by T. cruzi to facilitate successful host cell infection. Employing a blend of in silico and experimental tools, our investigation assessed the impact of host polysialylation modulation during experimental T. cruzi infection. Our results demonstrate that T. cruzi infection notably reduces ST8Sia2 expression and substrate polysialylation. Moreover, chemical and genetic inhibition of ST8Sia2 enhances parasite burden within host cells. Additionally, we found that modulation of host polysialylation induces oxidative stress, establishing a conducive microenvironment for T. cruzi survival and infection. In summary, our findings underscore the pivotal role of host polysialylation in T. cruzi infection dynamics.

Results

Modulation of genes involved in host N-glycosylation machinery by T. cruzi is accompanied by downregulation of ST8Sia2 polysialyltransferase

In this study, we first performed an in-silico analysis on publicly available transcriptome data obtained from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) infected with T. cruzi Y strain [49] to map the differential expression of glycan-related genes (glycogenes) (S1 Fig). A total of 39 glycogenes, including α-glucosidases, fucosyltransferases, galactosyltransferases, glucosyltransferases, mannosidases, mannosyltransferases, n-acetylglucosaminyltransferases, sialidases, and sialyltransferases were modulated in infected hiPSC-CM (S1A Fig). Among the regulated glycogenes, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8Sia2) was downregulated in T. cruzi-infected hiPSC-CM (S1B–S1E Fig), being more pronounced at 24 and 48 hours post-infection (h.p.i.) (S1D Fig). A previous study also showed that polysialylation deficiency compromises the functionality of atrial cardiomyocytes in ST8Sia2 knockout mice [30]. Taken together, these findings strongly suggested that in hiPSC-CM, T. cruzi parasite modulates the expression of glycogenes, and manipulates host polysialylation machinery by downregulating ST8Sia2.

T. cruzi infection modulates hiPSC-CM ST8Sia2 expression and compromises host polysialylation

In order to investigate the impact of host polysialylation upon T. cruzi infection, we differentiated human pluripotent stem cells (hiPSC) into cardiomyocytes (hiPSC-CM), and the efficiency of differentiation on hiPSC-CM was determined by the expression of troponin T and α-actinin, using immunofluorescence microscopy (S2A Fig), and by the percentage of positive cells stained with anti-troponin T (TNNT2+) antibodies analyzed by flow cytometry (S2B Fig). Then, we evaluated the levels of polysialylation in hiPSC-CM infected with T. cruzi (Fig 1). T. cruzi parasites were efficiently detected 48 h.p.i. (Fig 1A and 1B), as described previously [50]. Corroborating our initial data, we observed a 70% downregulation of ST8Sia2 mRNA in T. cruzi-infected hiPSC-CM using qRT-PCR (Fig 1C). Furthermore, ST8Sia2 protein levels are also reduced in infected hiPSC-CM (Fig 1D and 1H).

Fig 1 — **(A-G)** hiPSC-CM cells were seeded in 24-well microplates (2 x 10⁵ cells/well) and infected with T. *cruzi* trypomastigotes (Y strain) at ratio of 1:5 (hiPSC-CM:trypomastigotes), follow by 48 h incubation at 37°C. hiPSC-CM cells incubated with medium alone (Uninfected) were used as negative control; **(A)** Representative immunofluorescence images of T. *cruzi-*infected hiPSC-CM. Cell nuclei were stained with DAPI (scale bars = 10 μm). White arrows are representative of T. *cruzi* amastigotes inside hiPSC-CM cells; **(B)** Representative graphs of the percentage of T. *cruzi-*infected hiPSC-CM (left), and quantification of the number of intracellular parasites (amastigotes) per infected cell (right); **(C)** Relative expression of mRNA *ST8Sia2* measured by qRT-PCR in T. *cruzi-*infected hiPSC-CM. The Ct values of the target transcripts were normalized to the relative expression of *GAPDH* as endogenous control, and the relative expression of *ST8Sia2* transcripts was quantified by the 2^-ΔΔ Ct method. Each bar represents the mean of three independent experiments performed in triplicate; **(D-H)** Representative Western blotting quantification of ST8Sia2 **(D)**, polySia **(E)**, NCAM1 **(F)**, and SCN5A **(G)** in T. *cruzi-*infected hiPSC-CM cells. The protein levels were analyzed by Western blotting of RIPA cell lysates (15 μg protein) performed under reducing conditions. Results are presented as arbitrary densitometry units (AU). After normalization to the corresponding β-actin content (endogenous control), data were plotted as the ratio between the values obtained in infected and uninfected cells in their respective endogenous controls. Each bar represents the mean of three independent experiments performed in triplicate; **(H)** Representative Western blotting images of levels of ST8Sia2 **(D)**, polySia **(E)**, NCAM1 **(F)**, and SCN5A **(G)** in T. *cruzi-*infected hiPSC-CM cells. Results are expressed as mean ± SEM. Significant differences compared to the uninfected cells are shown by (*) p < 0.05, (**) p < 0.001, and (***) p <0.0001; ns = not significant.

Since polysialylation is a post-translational modification restricted to a set of glycoproteins [16], including the Sodium channel protein type 5 subunit alpha (SCN5A) of the primary cardiac isoform of the voltage-gated sodium channel (Na_V1.5), and neural cell adhesion molecule 1 (NCAM1) [51], both involved in modulating cardiac functions [29,52], both molecules were focused herein. Following the reduction in ST8Sia2 levels observed in Fig 1, reduced polySia levels were also detected in T. cruzi-infected hiPSC-CM (Fig 1E and 1H). No significant modulation in NCAM1 levels was observed between hiPSC-CM infected and non-infected with T. cruzi (Fig 1F and 1H), as expected since the cardiomyocyte model used in this study did not express PSA-NCAM, the polysialylated form of NCAM1 (S2C Fig). Accordingly, no signal was detected using a specific antibody for PSA-NCAM in non-infected (S2C Fig; lane 1) and infected (S2C Fig; lane 2) hiPSC-CM. As controls, the efficiency of immunostaining was demonstrated in TE671 cells (S2C Fig; lane 3), a human rhabdomyosarcoma cell previously described as positive for NCAM and PSA-NCAM [53] specificity of the antibody towards host polysialylation was demonstrated using T. cruzi trypomastigotes (S2C Fig; lane 4). In addition, we detected a reduction in SCN5A levels in T. cruzi-infected hiPSC-CM (Fig 1G and 1H). These data suggested that infection of hiPSC-CM with T. cruzi leads to the downregulation of ST8Sia2, affecting polySia transfer to specific substrates such as SCN5A, thereby reducing their abundance.

T. cruzi infection reduces the expression of host polysialylated target molecules

In order to broaden the approach to the effect of T. cruzi infection on the host polysialylation machinery, it became interesting to investigate the impact on polysialylation in SH-SY5Y cells, a human cell line of neuroblastoma with high expression of ST8Sia2. The infection efficiency of SH-SY5Y cells with T. cruzi was evaluated after 48 h.p.i by quantifying internalized parasites and determining the percentage of infected cells (Fig 2A and 2B). Next, we investigated the impact of T. cruzi infection on the polysialylation of SH-SY5Y cells (Fig 2C, 2D and 2H). Similar to the findings observed on T. cruzi-infected hiPSC-CM, we detected that ST8Sia2 (Fig 2C and 2H), polySia (Fig 2D and 2H), and SCN5A (Fig 2E and 2H) levels were significantly reduced in T. cruzi-infected SH-SY5Y cells. We also observed reduced levels of NCAM1 (Fig 2F and 2H) in SH-SY5Y cells infected with T. cruzi. Furthermore, there was a significant reduction in the abundance of PSA-NCAM (Fig 2G and 2H). Thus, these findings reinforce our hypothesis that the host cell polysialylation is modulated during the course of T. cruzi infection.

Fig 2 — **(A)** Representative immunofluorescence images of T. *cruzi-*infected SH-SY5Y cells. SH-SY5Y cells were seeded in 24-well microplates (5 x 10⁵ cells/well) and infected with T. *cruzi* trypomastigotes (Y strain) at ratio of 1:5 (SH-SY5Y:trypomastigotes) at 37°C. 48 h.p.i SH-SY5Y cells incubated with medium alone (Uninfected) was used as negative control. Cell nuclei were stained with DAPI (scale bars = 10 μm). White arrows are representative of T. *cruzi* amastigotes inside in SH-SY5Y cells; **(B)** Representative graphs of the percentage of T. *cruzi-*infected hiPSC-CM (left), and quantification of the number of intracellular parasites (amastigotes) per infected cell (right); **(C-H)** Western blotting quantification of ST8Sia2 levels **(C),** polysialic acid (polySia) **(D)**, SCN5A **(E)**, NCAM1 **(F)** and PSA-NCAM **(G)** in T. *cruzi-*infected SH-SY5Y cells. SH-SY5Y cells were seeded in 6-well microplates (1 x 10⁶ cells/well) and infected with T. *cruzi* as in A. 48 h.p.i, the proteins levels were analyzed by Western blotting of RIPA cell lysates (15 μg protein) performed under reducing conditions. Results are presented as arbitrary densitometry units (AU). After normalization to the corresponding β-actin content (endogenous control), data were plotted as the ratio between the values obtained in infected and uninfected cells in their respective endogenous controls. Each bar represents the mean of three independent experiments performed in triplicate; **(H)** Representative Western blot images of ST8Sia2 **(C)**, polySia **(D)**, SCN5A **(E)**, NCAM1 **(F)**, and PSA-NCAM **(G)** in T. *cruzi-*infected SH-SY5Y cells. Results are expressed as mean ± SEM. Significant differences compared to the uninfected cells are shown by (*) p < 0.05, (**) p < 0.001, and (****) p < 0.0001.

Furthermore, we evaluated the abundance of ST8Sia2 and polySia in T. cruzi-infected SH-SY5Y cells using immunofluorescence microscopy approach. Corroborating the data presented in Fig 2, we observed a significant reduction in the abundance of ST8Sia2 (Fig 3A and 3C) and polySia (Fig 3B and 3D) in T. cruzi-infected SH-SY5Y cells, when compared to non-infected cells. Next, we quantified the polySia content in the supernatant of SH-SY5Y cells infected with T. cruzi using a chromatographic method with fluorescence-based detection. For this assay, SH-SY5Y cells infected and non-infected with T. cruzi were incubated with endo-neuraminidase (EndoN), an enzyme that specifically cleaves α2,8-linked sialic acid polymers [16]. We observed that the polySia levels from the supernatant of T. cruzi-infected SH-SY5Y cells were significantly reduced compared to non-infected SH-SY5Y cells (Fig 3E), reinforcing our findings that host polysialylation is differentially modulated upon T. cruzi infection. In addition, we treated SH-SY5Y cells with EndoN to remove polySia and evaluated whether it would have an impact on T. cruzi infection, adopting the workflow shown in Fig 4A. Our results showed that removal of polySia in SH-SY5Y cells increased the number of intracellular parasites 48 h.p.i (Fig 4B and 4C), reinforcing that host polysialylation has a vital role during T. cruzi infection.

Fig 3 — **(A,B)** Representative images of ST8Sia2 **(A)** and PolySia **(B)** levels in T. *cruzi-*infected SH-SY5Y cells. SH-SY5Y cells were seeded in 24-well microplates (5 x 10⁵ cells/well) and infected with T. *cruzi* trypomastigotes (Y strain) at ratio of 1:5 (SH-SY5Y:trypomastigotes) at 37°C. After 48 h.p.i, T. *cruzi-*infected SH-SY5Y cells were stained with specific anti-ST8Sia2 **(A)** and anti-polySia **(B)** antibodies, and labeling for the targets was visualized by immunofluorescence microscopy. SH-SY5Y cells incubated with medium alone (Medium) was used as negative controls. Cell nuclei were stained with DAPI (scale bars = 10 μm); **(C,D)** Quantification of ST8Sia2 **(C)** and polySia **(D)** fluorescence intensity in T. *cruzi-*infected SH-SY5Y cells using the calculation for corrected total cell fluorescence (CTCF) as explained in Material and Methods section. Each dot represents the CTCF read out from one cell. A total of 50 SH-SY5Y cells per condition (infected and uninfected) were quantified. The values are expressed as Mean CTCF ± standard error of the mean (SEM); **(E)** polySia levels measured in T. *cruzi*-infected and non-infected SH-SY5Y cells supernatants. SH-SY5Y cells were seeded in T75-flasks (5 x 10⁵ cells/mL) and infected with T. *cruzi* trypomastigotes (Y strain) at ratio of 1:5 (SH-SY5Y:trypomastigotes) at 37°C. After 48 h.p.i, T. *cruzi-*infected SH-SY5Y cells were treated with EndoN [0.5 μg/ml] for 1h at 37°C, and polySia levels were measured in supernatants using UHPLC. The same procedure was applied to non-infected SH-SY5Y cells. The results are expressed in pg/mL. Significant differences compared to the uninfected are shown by (****) p < 0.0001.

Fig 4 — **(A)** Experimental workflow adopted to investigate the effect of enzymatic removal of polySia in T. *cruzi-*infected SH-SY5Y cells by EndoNeuraminidase (EndoN) [0.5 μg/ml]. SH-SY5Y cells were seeded in 24-well microplates (5 x 10⁵ or 5 x 10⁴ cells/well) and treated with EndoN [0.5 μg/ml] for 24h before infection, followed by addition of T. *cruzi* trypomastigotes (Y strain) at ratio of 1:5 (SH-SY5Y:trypomastigotes). After 24 h.p.i, T. *cruzi-*infected SH-SY5Y cells were submitted to another treatment with EndoN [0.5 μg/ml] for an additional 24h. Medium alone (Medium) were used as negative control; **(B)** Quantification of the number of intracellular parasites (amastigotes) per infected cell. Cell nuclei were stained with DAPI and following the experimental approach shown in B, amastigotes were counted in a total of 100 infected SH-SY5Y cells. Results are expressed as mean ± standard error of the mean (SEM) performed in triplicates; **(C)** Representative immunofluorescence images of T. *cruzi-*infected SH-SY5Y cells following the strategy adopted in the experimental workflow presented in B. Cell nucleus were stained with DAPI (scale bars = 10 μm). White arrows are representative of T. *cruzi* amastigotes inside in SH-SY5Y cells. Significant differences compared to the T. *cruzi-*infected SH-SY5Y treated with medium alone (Medium) are shown by (****) p < 0.0001; ns: not significant. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/), and BioRender.com under academic license.

Chemical and genetic Inhibition of ST8Sia2 increases the number of parasites internalized in SH-SY5Y cells

The impact of T. cruzi infection on the modulation of host polysialylation was demonstrated here by different experimental approaches in Figs 1–4. Thereafter, we evaluated whether chemical and genetic inhibition of ST8Sia2 would have an impact on host cell infection by T. cruzi. For this, we used cytidine 5′-monophosphate (CMP), a molecule previously reported as a pharmacological inhibitor of ST8Sia2 [54], and the siRNA-directed approach. Initially, we demonstrated that chemical or genetic inhibition of ST8Sia2 in SH-SY5Y cells with CMP or siRNA ST8Sia2, respectively, does not affect cell viability (S3A and S3B Fig). Then, SH-SY5Y cells were treated with CMP and infected with T. cruzi according to the workflow shown in Fig 5A. The percentage of infected cells, and the number of internalized parasites was determined after labeling them at 48 h post-infection (Fig 5B and 5C). We demonstrated that chemical inhibition of ST8Sia2 with CMP in SH-SY5Y cells increased the number of intracellular parasites (Fig 5C and 5F). We also demonstrated that the percentage of infected cells and the number of intracellular parasites in untreated cells (culture medium only) or GMP-treated cells did not differ significantly (Fig 5C and 5F), demonstrating that the inhibition of ST8Sia2 favors infection by T. cruzi. Along the same purpose, we used a siRNA-directed approach to silence ST8Sia2 according to the workflow presented in Fig 5A. Transfection efficiency was evaluated by the relative expression of the ST8Sia2 transcript by qRT-PCR (S3C Fig) according to the workflow shown in S3D Fig. Additionally, we validated the ST8Sia2 silencing strategy in SH-SY5Y cells by visualizing and quantifying polySia levels using immunofluorescence microscopy (S3E Fig) and UHPLC approach (S3F Fig), respectively. Our results demonstrated that genetic silencing of ST8Sia2 in SH-SY5Y cells also increased the percentage of infected cells (Fig 5D), and the load of internalized parasites (Fig 5E and 5G). Taken together, these findings reinforce our hypothesis that ST8Sia2 inhibition favors T. cruzi infection.

Fig 5 — **(A)** Experimental workflow adopted to investigate the effect of chemical and genetic inhibition of ST8Sia2 in T. *cruzi-*infected SH-SY5Y cells using 0.5 mM of CMP. SH-SY5Y cells were seeded in 24-well microplates (5 x 10⁵ or 5 x 10⁴ cells/well), and previously treated with CMP [0.5 mM] or siRNA *ST8Sia2* [100 nM] for 24h before infection, followed by infection with T. *cruzi* trypomastigotes (Y strain) at ratio of 1:5 (SH-SY5Y:trypomastigotes). After 24 h.p.i, T. *cruzi-*infected SH-SY5Y cells were treated with CMP [0.5 mM] or siRNA *ST8Sia2* [100 nM] for an additional 24h. Guanosine 5’-monophosphate (GMP), siRNA negative control (NTC), and/or medium alone (Medium) were used as negative controls for chemical or genetic inhibition of *ST8Sia2*. In all controls used in experiments with siRNA was added Lipofectamine RNAiMAX; **(B)** Representative graphs of the percentage of T. *cruzi-*infected SH-SY5Y treated with CMP; **(C)** Quantification of the number of intracellular parasites (amastigotes) per infected cell treated with CMP. Cell nuclei were stained with DAPI and following the experimental approach shown in A, amastigotes were counted in a total of 100 infected SH-SY5Y cells. **(D)** Representative graphs of the percentage of T. *cruzi-*infected SH-SY5Y treated with siRNA *ST8Sia2* (siST8Sia2); **(E)** Quantification of the number of intracellular parasites (amastigotes) per infected cell. Cell nuclei were stained with DAPI and following the experimental approach shown in A, amastigotes were counted in a total of 100 infected SH-SY5Y cells. Results are expressed as mean ± standard error of the mean (SEM) performed in triplicates; **(F,G)** Representative immunofluorescence images of T. *cruzi-*infected SH-SY5Y cells treated with CMP **(F)**, and T. *cruzi-*infected SH-SY5Y treated with siRNA *ST8Sia2* **(G)**, following the strategy adopted in the experimental workflow presented in A. Cell nuclei were stained with DAPI (scale bars = 10 μm). White arrows are representative of T. *cruzi* amastigotes inside in SH-SY5Y cells. Significant differences compared to the T. *cruzi-*infected SH-SY5Y treated with medium alone (Medium) are shown by (***) *p < 0*.0005, (****) p < 0.0001; ns: not significant. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/), and BioRender.com under academic license.

Silencing of ST8Sia2 in T. cruzi-infected SH-SY5Y cells intensifies oxidative stress

Herein, we have utilized different experimental techniques and strategies to demonstrate the impact of T. cruzi infection on the modulation of host cell polysialylation. However, insights into a possible mechanism where the effect of polySia could influence the cell survival of T. cruzi remain unknown. It has been previously demonstrated that in T. cruzi infection, the parasite requires signaling provided by reactive oxygen species (ROS) from macrophages to establish a successful infection [55]. In HeLa cells, oxidative stress and DNA damage are activated during T. cruzi infection. Cho et al. [56] reported that sialic acid deficiency is associated with oxidative stress. In this study, it was observed that lower levels of sialic acid increase oxidative stress.

Considering these evidence collectively, we hypothesize that the impact on host polysialylation, and consequently, the increase in parasite survival could be related to the imbalance of oxidative stress. To confirm our hypothesis that there is an potential interplay between the disruption in polysialylation machinery and a generation of a status of oxidative stress, we used the strategy of ST8Sia2 siRNA-directed silencing (S3D Fig), and monitored oxidative stress in the presence or absence of Antimycin A and/or N-acetyl cysteine, both molecules previously reported as inducer [57] or inhibitor [58] of oxidative stress, respectively.

When measuring cytosolic and mitochondrial ROS levels in SH-SY5Y siST8Sia2 cells, we observed a significant reduction in cytosolic (Fig 6A) and mitochondrial (Fig 6D) ROS production, compared to the baseline levels detected in SH-SY5Y cells from the control group (NTC). We also demonstrated that in response to treatment with antimycin A, SH-SY5Y siST8Sia2 cells exhibited higher levels of cytosolic (Fig 6B) and mitochondrial (Fig 6E) ROS. On the other hand, no significant change was observed in cytosolic (Fig 6C) and mitochondrial (Fig 6F) ROS levels in SH-SY5Y siST8Sia2 cells treated with N-acetyl cysteine, compared to the levels detected in SH-SY5Y NTC cells. These results provide for the first time that modulation of host polysialylation can influence oxidative stress.

Under conditions of oxidative stress, alterations in intracellular calcium ion (Ca²⁺) levels are frequently observed [59]. Elevated intracellular Ca²⁺ concentration can modulate nitric oxide (NO) production, culminating in the generation of oxidants, thereby amplifying Ca²⁺ levels and subsequently exacerbating oxidative stress [60]. It has been documented that in conditions of oxidative stress, the concurrent decrease in mitochondrial membrane potential (ΔΨm) coupled with heightened Ca²⁺ production signify potential mitochondrial dysfunction and oxidative stress [61]. Subsequently, mitochondrial membrane potential and mitochondrial Ca²⁺ levels were assessed in SH-SY5Y siST8Sia2 cells. We observed that silencing of ST8Sia2 led to dysregulation in mitochondrial membrane potential (Fig 6G), concomitant with a marked elevation in Ca²⁺ production (Fig 6H), as compared to baseline levels detected in SH-SY5Y cells from the control group (NTC). Taken together, these findings strongly suggest that the reduced levels of ST8Sia2 alter the baseline levels of factors implicated in oxidative stress, notably ROS and Ca²⁺ intracellular, potentially heightening oxidative stress reactivity upon exposure to secondary stimuli such as Antimycin A.

Additionally, a bottom-up large-scale mass spectrometry-based proteomics approach was employed to identify and quantify proteins differentially regulated in T. cruzi-infected ST8Sia2-silenced SH-SY5Y cells (siST8Sia2 Ty). A total of 851 proteins were identified in this analysis, of which 762 were quantified across all biological replicates (Fig 7A, left flowchart, S1 and S2 Tables), and 24 proteins were quantified exclusively (Fig 7A, right flowchart) in T. cruzi-infected siST8Sia2 SH-SY5Y cells and T. cruzi-infected SH-SY5Y cells incubated with culture medium alone (Fig 7A). A clear separation into two large groups was observed based on the Principal Component Analysis (PCA) (Fig 7B).

Fig 7 — SH-SY5Y cells were seeded in 6-well microplates (1 x 10⁶ cells/well), and previously treated with siRNA *ST8Sia2* [100 nM] for 24h before infection, followed by infection with T. *cruzi* trypomastigotes (Y strain) at ratio of 1:5 (SH-SY5Y:trypomastigotes). After 24 h.p.i, T. *cruzi-*infected SH-SY5Y cells were treated with siRNA *ST8Sia2* [100 nM] for an additional 24h. Medium alone (Medium) was used as negative control for genetic inhibition of *ST8Sia2*. In all controls used in experiments with siRNA was added Lipofectamine RNAiMAX. **(A)** Proteins identified in all biological replicates or exclusively in one of the evaluated groups: cells silenced for *ST8sia2* (siST8sia2, n = 3) and medium-treated with Lipofectamine RNAiMAX (Medium, n = 3); **(B)** Principal component analysis (PCA) of SH-SY5Y cells silenced for *ST8sia2* (siST8sia2, green) and Medium-treated control (Medium, grey), both infected with T. *cruzi;* **(C)** Chord plot showing the proteins involved in biological pathways and processes related to the different stress responses; **(D)** Volcano plot illustrating the differentially regulated proteins in the T. *cruzi-*infected siST8si2 SH-SY5Y vs. Medium comparison, with upregulated proteins shown in orange, downregulated proteins in blue, and unregulated proteins in grey (p-value < 0.05); **(E)** Boxplot of differentially regulated stress response proteins in T. *cruzi*-infected SH-SY5Y Cells with *ST8Sia2* Silencing. Significant differences compared to T. *cruzi*-infected siST8Sia2 SH-SY5Y cells with T. *cruzi*-infected SH-SY5Y cells treated with medium (negative control for genetic silence of *ST8Sia2* are indicated by p <0.0001 (****); p <0.001 (***); p <0.01 (**); p < 0.05 (*).

A total of 68 proteins were regulated between T. cruzi-infected siST8Sia2 SH-SY5Y cells and T. cruzi-infected SH-SY5Y cells incubated with culture medium alone (S3 Table). In the Chord plot shown in Fig 7C, we mapped in the total dataset the expression of proteins involved in pathways and biological processes related to the stress response. We identified 62 proteins whose associated with pathways and biological processes related to oxidative stress response, stress granule assembly, and abiotic and biotic stress (Fig 7C). These proteins showed a modulation in T. cruzi-infected ST8Sia2-silenced (siST8Sia2 Ty) compared to non-silenced (Medium Ty) SH-SY5Y cells (Fig 7C). After applying statistical test, among the differentially regulated proteins (Fig 7D), we identified a set of proteins involved in oxidative stress such as the upregulation of proteolipid protein 2 (PLP2, Q04941) (Fig 7D and 7E) and protein S100-A6 (P06703) (Fig 7D and 7F), and the downregulation of serine/threonine-protein kinase PAK 2 (Q13177) (Fig 7D and 7G) and DNA mismatch repair protein MSH2 (P43246) (Fig 7D and 7H) in T. cruzi-infected siST8Sia2 SH-SY5Y cells. Collectively, these results reinforce our hypothesis that the infection rate of T. cruzi in host cells is enhanced by the downregulation of ST8Sia2 and the reduction in polysialylation, which is linked to increased oxidative stress that creates a microenvironment permissive to infection.

Discussion

Different studies have extensively explored the manipulation of host cell glycosylation in infectious processes since many pathogens use this post-translational modification to ensure a successful infection [62,63]. In the case of T. cruzi, the role of Sia in the infection has been extensively explored, with a focus on the transfer of this sugar from host α-2,3-sialylated glycans to the surface of the parasite catalyzed by trans-sialidase [37,40]. Also, knockout mice for CD43 [48], a sialoglycoprotein previously reported as a natural receptor for T. cruzi trans-sialidase [47], showed reduced cardiac parasite complications [48], reinforcing that manipulation of host glycosylation could directly or indirectly interfere in the T. cruzi infection. However, the impact of T. cruzi infection on host glycosylation machinery has not been explored to date.

Herein, we described the modulation of host cell N-glycosylation machinery in experimental T. cruzi infection, and, providing for the first time, evidence of downregulation of ST8Sia2, a host polysialyltransferase. Using an initial in-silico approach, different genes encoding enzymes involved in the protein glycosylation pathways, was suggested to be up (e.g. Neuraminidase 1—Neu1, and α-1,3-mannosyltransferase—ALG3, or down (ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 –ST8Sia2, and α-Glucosidase 2—GANAB) regulated in the course of infection in human cardiomyocytes (S1 Fig). Among these (glyco)genes, ST8Sia2 showed a sustained downregulation over different time points (S1 Fig), which should lead at the end to a decrease in the polySia content of glycoproteins, as described in the literature [64].

Consistent with our initial observations, we found experimentally a significant reduction in ST8Sia2 mRNA levels (Fig 1C) and polysialic content (Fig 1E and 1H) in human induced-pluripotent stem cells derived cardiomyocyte (hiPSC-CM) after 48 h of T. cruzi infection, although a less expressive decrease at the protein level of ST8Sia2 was observed at this time point (Fig 1D and 1H). Similar results, regarding decrease of ST8Sia2 expression (Fig 2C and 2H) and polySia (Fig 2D and 2H) were obtained with SH-SY5Y, a human neuroblastoma cell line, after 48 h of T. cruzi infection. A direct correlation between the expression of ST8Sia2 and the reduction in polySia levels has already been well demonstrated in previous studies that investigated the relevance of polysialylation in the nervous system [65,66].

Although polySia is added to N- and O-glycosylation structures, its presence is confined to a restricted set of glycoproteins, emphasizing NCAM1, which is the main and best-studied carrier of polySia in mammals [22], and in few other proteins, such as sodium voltage-gated channel alpha subunit 5 (SCN5A). Then, to better understand the impairment of host polysialylation in T. cruzi infection, we investigated whether NCAM1 and SCN5A would be differentially modulated in hiPSC-CM and SH-SY5Y during infection. In hiPSC-CM, NCAM1 levels did not differ between uninfected or infected cells (Fig 1F and 1H), in accordance with the observation that this cultured-cells do not express PSA-NCAM, the polysialylated form of NCAM1. On the other hand, the abundance of polySia in SCN5A, the primary cardiac isoform of the voltage-gated sodium channel (Na_v 1.5), was significantly reduced in hiPSC-CM- infected cells (Fig 1G and 1H). Interestingly, it was shown that differential sialylation in the primary cardiac isoform of Na_v 1.5 channel generates more depolarized potentials in atrial and ventricular cardiomyocytes, suggesting that cardiac contractility may be modulated by changes in sialic acids associated with the channels [51,67]. Furthermore, in ST8Sia2 knockout mice, polysialylation deficiency compromises action potential and Nav functions in atrial and ventricular cardiomyocytes [29,30]. Altogether, these results support our hypothesis that T. cruzi infection manipulates the polysialylation machinery of the host, compromising the expression of ST8Sia2, polySia, and SCN5A, which are targets of polysialylation, and thus may alter the complex biology of cardiomyocytes.

Similar to our findings with T. cruzi-infected hiPSC-CM, we detected reduced levels of ST8Sia2 and polySia in T. cruzi-infected human neuroblastoma (SH-SY5Y) cells, in addition to lower polysialylation of PSA-NCAM, expressed by this cell line (Fig 2G and 2H), or SCN5A (Fig 2E and 2H). Metabolic differences between the cell lines employed may explain the lower levels in the polysialylated SCN5A and NCAM1, as well as in ST8Sia2 expression observed in SH-SY5Y in relation to hiPSC-CM cells (Figs 1 and 2). Nevertheless, both results reinforce our hypothesis that the polysialylation machinery is modulated during T. cruzi infection. Although two different polysialyltransferases (ST8Sia2 and ST8Sia4) are involved in the synthesis of polySia chains, it was previously demonstrated that SH-SY5Y expresses high levels of ST8Sia2 mRNA, but it is almost devoid of ST8Sia4 mRNA [68] and expresses polySia bound to the two transmembrane proteoforms of NCAM, NCAM140, and NCAM180. Indeed, all expressed NCAM proteoforms are polysialylated [69], and at least, ST8Sia2 is related to polySia modulation of glycoproteins in cells infected by T. cruzi. Previous studies have demonstrated that post-translational modifications (PTMs) can profoundly affect protein stability and expression [70,71]. Consequently, we hypothesize that decreased levels of polysialylation may compromise the stability of NCAM protein and, as a result, its expression.

Although the role of NCAM is relatively little explored in the heart, its expression is known to be differentially regulated during the cardiac system development [72]. In the fetal heart, NCAM is expressed in the myocardium and ventricular conduction system, but in the adult heart, its expression is restricted to the ventricular conduction system [73]. Additionally, NCAM1 deletion compromises the expression of genes essential for the coordination of rhythmic contractions of the conduction system in a subpopulation of ventricular cardiomyocytes [29]. Considering that in chronic Chagas cardiomyopathy, the blockade of the ventricular conduction system by the high and persistent parasitism in the cardiac tissue leads to ventricular dysfunction, which can result in heart failure [34, 74, 75], it is tempting to speculate that somehow, the dysfunction in the ventricular conduction system frequently seen in chronic chagasic cardiomyopathy may result partly from manipulation of the polysialylation machinery that compromises PSA-NCAM and/or SCN5A expression.

Interestingly, both chemical or genetic inhibition of ST8Sia2 by CMP or siRNA ST8Sia2, respectively, increase the number of intracellular parasites in SH-SY5Y cells measured 48 h.p.i (Fig 5). This fact strongly suggests that during the T. cruzi infection, modulation of host polysialylation may be a strategy adopted by the parasite to ensure success in establishing the infection, either by affecting the invasion step (e.g. by decreasing the repulsive negative charges of surface molecules with time) or by affecting the intracellular cycle of the parasite. However, the mechanism, as yet remains elusive. Since the highest changes in the levels of ST8Sia2 were observed from 24 h.p.i. (S1B–S1E Fig). In this context, it is interesting to note that both, quantity and quality, of polySia synthetized by ST8SIA2 or ST8SIA4 are similar, but the resulted polySia-NCAMs exhibit different attractive and repulsive properties, which may affect biological functions [76]. To date, no study has explored the possible involvement of polysialylation in T. cruzi infection. However, previous studies reported that in Chagas disease, possible disorders of aberrant glycosylation are due to modifications in the sialylation of glycoproteins. As demonstrated before by Libby et al in 1986, T. cruzi trypomastigotes modify the surface of myocardial and vascular endothelial cells by desialylation [45].

In the present study, we demonstrate that T. cruzi infection leads to downregulation of ST8Sia2, compromising the levels of NCAM1 and SCN5A, both molecules previously reported as polysialylated, thereby providing a favorable environment for intracellular parasite replication and survival. To date, a possible mechanism explaining the modulation of host polysialylation during T. cruzi infection has not been demonstrated. Here, we show that the silencing of ST8Sia2 and disruption of host polysialylation modulate oxidative stress in SH-SY5Y cells (Fig 6).

A previous study reported that the removal of sialic acids in human endothelial cells modulates signaling mediated by Nuclear Factor E2-related factor 2 (Nrf2), promoting mitochondrial ROS accumulation [77]. Goswmami and Konet [78] experimentally demonstrated that the induction of oxidative stress in human platelets causes desialylation of platelet glycoproteins. Sialic acid has the ability to protect against oxidative damage by inducing a significant increase in the levels of antioxidant components, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), thereby reducing the ROS production [79].

It has been demonstrated that the endogenous sialidase Neuraminidase 1 (NEU1) can cleave polySia [80]. The reduction of NEU1 increases the expression of target genes associated with Nrf2-mediated signaling, including Heme oxygenase-1 (HO-1), directly implicating sialic acid in the regulation of Nrf2 signaling, a pathway involved in maintaining the redox homeostasis [77]. Furthermore, it has been demonstrated that the overexpression of HO-1 reduces T. cruzi parasitism both in vitro and in vivo infection [81]. As a result, higher levels of NEU1 may reduce polySia levels, increase oxidative stress, and promote parasite survival. Considering the impact of host cell polysialylation on T. cruzi infection presented here and the existence of a possible interplay between the downregulation of polysialylation and the modulation of the stress response, a proteomic approach was used to identify differentially regulated proteins involved in the stress response pathway in SH-SY5Y siST8Sia2 cells (ST8Sia2 gene silenced) infected by T. cruzi (Fig 7).

Our data demonstrated the upregulation of PLP2 and S100A6 in T. cruzi-infected siST8Sia2 SH-SY5Y cells. It has been previously demonstrated that the modulation of PLP2 levels in mice and human fibroblasts favors stress response [82]. Upregulation of S100A6 has been shown in Hep-2 cells, an epithelial human cell line, under stress conditions [83]. Another study reported that the increase in S100A6 expression, combined with its ligands such as heat shock proteins, modulates the cellular stress response [84]. Additionally, Zhou et al. [85] demonstrated that overexpression of S100A6 in host cells favors infection by Toxoplasma gondii, the etiological agent of toxoplasmosis (PMID: 34950858).

Proteomic analysis of T. cruzi-infected siST8Sia2 SH-SY5Y cells revealed the downregulation expression of PAK2 and MSH2 proteins. Supporting our hypotheses, previous studies have shown that under oxidative stress conditions, reduced levels of PAK2 were detected in cardiomyocytes [86]. In the same line, it was demonstrated that the depletion of PAK2 in cardiomyocytes compromises the establishment of a redox response under stress conditions [87], reinforcing that the reduced levels of PAK2 favors the generation of oxidative stress [88] The interplay between reduced MSH2 levels and the oxidative stress response has also been reported in human cells [89]. Due to this, our findings point towards a correlation between diminished levels of ST8Sia2 and increased susceptibility of host cells to induce oxidative stress, leading to elevated levels of ROS that could create an advantageous milieu for T. cruzi parasite replication.

In summary, the present study demonstrates the impairment of the host polysialylation machinery during infection by T. cruzi. We have shown that T. cruzi infection promotes the differential regulation of ST8Sia2 and polySia in host cells (Fig 8) and suggests a possible involvement of oxidative stress response during infection. Using different experimental approaches, we also showed a reduction in NCAM and SCN5A, both targets of polysialylation, which strongly suggests that ventricular conduction dysfunction frequently observed in chronic Chagas cardiomyopathy may be due to the remodeling of the polysialylation of these molecules.

Fig 8 — The findings in this study suggest an impact on host polysialylation during T. *cruzi* infection. Under conditions of homeostasis, ST8Sia2 catalyzes the transfer of Neu5Ac monomers from cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) to glycoproteins within the Golgi complex, forming a polymer of α-2,8-glycosidic linkages between Neu5Ac monomers known as polySia. This phenomenon, termed polysialylation, assumes pivotal roles across a spectrum of cellular functions. The addition of polySia into molecules such as NCAM1 and SCN5A influences cardiac contractility and neural communication, exemplifying its multifaceted significance. In this study, we show that host polysialylation impact T. *cruzi* infection dynamics. Our investigation has unveiled that T. *cruzi* infection diminishes the expression/abundance of the ST8Sia2 enzyme, compromising polySia formation, and its subsequent addition to polysialylated target molecules, including SCN5A and NCAM1. Consequently, the attenuation of ST8Sia2 and polySia levels may influence the host cell susceptibility to generate an oxidative stress environment that may favor T. *cruzi* infection, as polysialylation modulation is accompanied by an increase in the number of internalized parasites and susceptibility to oxidative stress. It is plausible to posit that the modulation of host polysialylation by the parasite serves as a determinant in the pathogenesis of Chagas disease. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

Furthermore, our findings show that reduced levels of ST8Sia2 heighten host cells susceptibility to oxidative stress upon T. cruzi infection. This suggests that downregulation of ST8Sia2 disrupts polySia production, diminishing polysialylation and enhancing ROS generation, potentially fostering a conducive environment to T. cruzi parasite replication. Importantly, pharmacological and genetic inhibition of ST8Sia2 in infected cells demonstrated an increase in intracellular parasite numbers, highlighting a possible functional role for manipulating polysialylation during T. cruzi infection. Of note, our study does not provide a comprehensive mechanistic understanding, but will help inform future studies aimed at elucidating the functions of polySia in infectious diseases, with a focus on T. cruzi infection and Chagas disease pathogenesis. Furthermore, studying different Discrete Typing Units (DTUs) can provide a comprehensive view on the role of ST8Sia2 in strain-specific pathogenic mechanisms of T. cruzi.

Materials and methods

Public RNA-seq data reanalysis

The fastq files were downloaded from the https://sra-explorer.info/ platform with the BioProject accession number PRJNA532430 [49]. The ‘FastQC’ module was used to report the quality reads, followed by the trimmed of adapters using TrimGalore! set to the single-end library. The TrimGalore! output sequences were aligned to the human reference genome hg38 using the HISAT2 platform [90]. A count table was generated using the FeatureCounts [91] algorithm. The differently regulated genes were analyzed by the limma, Glimma, edgeR, and Homo.sapiens packages applying a cut-off of |log2FC|>1 and a p-adjusted value <0.05 (Benjamini-Hochberg). Statistical data of the glycogenes of interest were filtered and the chord_dat() function, available in the GOplot package, was used to plot the chord plot. Volcano plot and heatmap graphics were made using Perseus software.

Human cell culture

Human induced-pluripotent stem cells (hiPSC) were differentiated into Human induced-pluripotent stem cells derived cardiomyocyte (hiPSC-CM) on Geltrex (Thermo Fischer, Waltham, MA, USA) coated plates in Cardiomyocytes-Plating Medium (Pluricell Biotech), using an established differentiation protocol [92]. All experiments were carried out between day 30–35 of differentiation. The human neuroblastoma SH-SY5Y cells (ATCC-CRL-2266) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco, Life Technologies, Carlsbad, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum, and 1% penicillin/streptomycin. All cells line were maintained in a humidified 5% CO₂ atmosphere at 37°C.

T. cruzi culture and infection assay

T. cruzi trypomastigote (Y strain) was routinely maintained by infection in monkey kidney epithelial cells (LLC-MK2) in Roswell Park Memorial Institute 1640 Medium (RPMI) supplemented with 2% fetal bovine serum in a humidified 5% CO₂ atmosphere at 37°C, according to previously described experimental procedures [93]. Five days after infection, the trypomastigotes released in the supernatants were purified using a DEAE-cellulose chromatography column. Therefore, the cells were washed in phosphate-buffered saline (PBS) by centrifugation at 10.000 × g for 12 min, and resuspended in adequate culture medium for each human cell line used. The total number of parasites/ml was counted in a Neubauer chamber. hiPSC-CM and/or SH-SY5Y cells were seeded at a density of 5x10⁵ cells/well in 24-well or 6-well microplates and infected with purified trypomastigotes at the rate of 5:1 (trypomastigotes:hiPSC-CM) in a volume of 500 μL per well [49]. After 3 h of incubation, the media was replaced with fresh media to remove the non-internalized parasites. The impact of T. cruzi infection on host polysialylation was evaluated at 48 h post-infection (h.p.i). In all experiments, an uninfected group was subjected to the same experimental conditions, except for incubation with T. cruzi.

Immunofluorescence assay

hiPSC-CM or T. cruzi-infected SH-SY5Y cells distributed in round glass coverslips (13 mm) contained in 24-well microplates with flat bottom were fixed with PBS/4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized with 0.1% Triton X-100 (Sigma, Chemical Company, St. Louis, MO, USA) for 15 min at RT. For evaluation the efficiency of differentiation on hiPSC-CM, coverslips were blocked with PBS/2% Bovine Serum Albumin (BSA) solution, and incubated with primary antibodies: mouse anti-cardiac troponin T (HyTest clone 4T19/2) and rabbit anti-α-actinin (Millipore) for 30 min at RT. After washing four times with PBS, the cells were incubated for 1 h at RT with the secondary antibodies: goat anti-rabbit immunoglobulin G (IgG) Alexa Fluor 488 and/or goat anti-mouse IgG Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The coverslips were washed four times and prepared for visualization by adding one drop of mounting medium (Slow Fade Gold Antifade; Thermo Fisher Scientific) with 4’,6-diamidino-2- phenylindole (DAPI). For evaluation of the ST8Sia2 and polySia levels in T. cruzi-infected SH-SY5Y cells, the cells seeded on coverslips, fixed and permeabilized were blocked with PBS/2% Bovine Serum Albumin (BSA) solution, and incubated with primary antibodies: ST8Sia2 monoclonal antibody (Catalog WH0008128M1, Sigma-Aldrich, St. Louis, MO, USA) and polySia-specific monoclonal antibody (mouse IgG2a, clone 735) for 30 min at RT. After washing four times with PBS, the cells were incubated for 1 h at RT with the secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, Massachusetts, USA) or Alexa Fluor 594 goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The coverslips were washed four times and prepared for visualization by adding one drop of mounting medium (Slow Fade Gold Antifade; Thermo Fisher Scientific) with 4’,6-diamidino-2- phenylindole (DAPI). To determine the number of parasites internalized in SH-SY5Y cells, the cells seeded on coverslips, fixed and permeabilized as above, were prepared for visualization by adding a drop of mounting medium (Slow Fade Gold Antifade; Thermo Fisher Scientific) containing 4’,6-diamidino-2-phenylindole (DAPI), used for imaging, which was performed using a Conventional Fluorescence Microscope (Zeiss). polySia-specific monoclonal antibody (mouse IgG2a, clone 735) were kindly provided by Dr. Martina Mühlenhoff (Institute of Clinical Biochemistry, Hannover Medical School, Germany).

Quantification of immunofluorescence

Fluorescence microscopy images were analyzed by ImageJ software, version 1.49v (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/) to evaluate ST8Sia2 and polySia-stained host cells. Using the calculation for corrected total cell fluorescence (CTCF) = integrated density–(area of selected cell × mean fluorescence of background readings), as described by Gachet-Castro et al. [94]. The fluorescence intensity of each cell was calculated. Subsequently, graphs were plotted with the fluorescence average values using GraphPad Prism 9.0 program (GraphPad Software, Inc., San Diego, CA).

Total cell lysates

hiPSC-CM and SH-SY5Y cells were infected with T. cruzi trypomastigotes at the rate of 5:1 (trypomastigotes:hiPSC-CM) for 48 h. After 48 h.p.i, the cells were washed four times with PBS to remove extracellular parasites, and lysed with radioimmunoprecipitation assay (RIPA) buffer [20 mM Tris pH 7.2, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)] containing a cocktail of protease (cOmplete, Sigma-Aldrich, St. Louis, MO, USA) and phosphatase (PhosStop, Sigma-Aldrich) inhibitors. The cell lysates were kept on ice for 10 min, centrifuged at 14.000 x g for 10 min at 4°C to pellet cell debris. Supernatants were collected, and the total protein was measured using the Kit Pierce BCA Protein Assay (Thermo Scientific) according to the manufacturer’s instructions. In all experiments, an uninfected group was subjected to the same experimental conditions. All Samples were resuspended in sample buffer for analysis by SDS-PAGE. The protein extract from human rhabdomyosarcoma cell line TE671 in sample buffer [2% sodium dodecyl sulfate, 10% glycerol, 0.002% bromophenol blue, 0.0625 M Tris-HCl pH 6.8, 5% 2-mercaptoethanol] were gifts from Dr. Martina Mühlenhoff (Institute of Clinical Biochemistry, Hannover Medical School, Germany).

Immunoblotting assay

Proteins extracted from total cell lysate (15μg of proteins) were resolved by SDS-PAGE and transferred to PVDF membranes, which were directly incubated with blocking buffer (5% BSA in Tris-buffered saline (TBS) at 0.05% Tween-20 (TBS/T) for 1 h. Subsequently, membranes were washed three times with TBS/T solution and incubated overnight with the primary antibodies: anti-ST8Sia2, anti-polySia, anti-NCAM1, anti-PSA-NCAM, anti-SCN5A, and Anti-ACTB, and washed three times with TBS/T. Then, the membrane was incubated with the respective secondary antibodies for 1 h at RT, followed by the incubation with a secondary antibody for 1h at room temperature. The immunoreactive bands were detected with the ChemiDoc XRS Imaging System equipment and protein quantification was performed using the ImageJ software. polySia-specific monoclonal antibody (mouse IgG2a, clone 735) and NCAM1-specific monoclonal antibody (mouse IgG1, clone 123C3) were kindly provided by Dr. Martina Mühlenhoff (Institute of Clinical Biochemistry, Hannover Medical School, Germany). All antibodies used in this study were tested for cross-reactivity against T. cruzi trypomastigote cell lysates.

Drugs treatment and chemical ST8Sia2 inhibition

SH-SY5Y cells were pretreated with 0.5 mM cytidine 5’-monophosphate (CMP, Sigma-Aldrich) or culture medium (negative control) 24 h before infection and 24 h post-infection with T. cruzi to evaluate the impact of pharmacological inhibition of ST8Sia2 activity during infection and replication of the parasite. After incubation with CMP for 24 h, the CMP-containing medium was replaced with fresh medium, and the cells were infected with T. cruzi trypomastigotes in a 5:1 ratio (trypomastigotes:hiPSC-CM). 24 h post-infection, the treatment with 0.5 mM CMP was repeated for another 24 h. Guanosine 5′-monophosphate (GMP), with no effect on ST8Sia2 inhibition, was used as a control. In all experiments, a group of infected cells (negative control of ST8Sia2 inhibition) was subjected to the same experimental conditions, except for incubation with pharmacological inhibitor.

siRNA-Directed Inhibition of ST8Sia2

Predesigned siRNAs for the ST8Sia2 transcripts (siST8Sia2) (hs.Ri.ST8Sia2.13.2; hs.Ri.ST8Sia2.13.3) were purchased from Integrated DNA Technologies (IDT, Coralville, IA). Transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA). Lipofectamine RNAiMAX:siST8Sia2 Mix (0.6 μL Lipofectamine:100 nM siRNAs) was formed in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 20 min and added to SH-SY5Y cells in antibiotic-free medium for overnight transfection at 37°C and 5% CO₂. After incubation, fresh Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco®, Life Technologies, Carlsbad, CA, USA), supplemented with 2% heat-inactivated fetal bovine serum was added, and the cells were infected with T. cruzi trypomastigotes in a 5:1 ratio (trypomastigotes:hiPSC-CM). 24 h post-infection, T. cruzi-infected SH-SY5Y cells were transfected again with Lipofectamine RNAiMAX:siST8Sia2 Mix (0.6 μL Lipofectamine:100 nM siRNAs) in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) antibiotic-free medium for an additional 24 h, at 37°C and 5% CO₂. siRNA negative control (NTC) and medium alone (Medium) were used as negative controls. Transfection efficiency was evaluated by the relative expression of the ST8Sia2 using Quantitative real-time reverse transcription PCR (qRT-PCR).

Quantitative real-time reverse transcription PCR (qRT-PCR)

hiPSC-CM were infected with T. cruzi trypomastigotes (Y strain) at the rate of 10:1 or 5:1 (trypomastigotes:host cell). Total RNA from these samples was extracted using TRIzol according to the manufacturer’s instructions, and the RNA was converted into cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). qRT-PCR was performed in a final volume of 10 μL using Maxima SYBR Green qPCR Master Mix (Thermo Scientific) and the quantification was done by QuantStudio 3 Real-Time PCR System (Applied Biosystems). The cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 60 s. The relative expression of the transcript was quantified using the ΔΔCt method [95] and normalized to β-actin expression. The specificity of amplification was determined using melting curve analysis. The following PCR primers were used: β-actin (F- CATGTACGTTGCTATCCAGGC, R- CTCCTTAATGTCACGCACGAT), and ST8Sia2 (F- CGTTCGCGGATACTGGCT, R- GCCAGAAGCCGTAGAGGTAG).

MTT assay

SH-SY5Y cells were seeded in 96-well microplates (1 × 105 cells/well) and treated with cytidine 5’-monophosphate (CMP; 62.5–500 μM) or siRNA ST8Sia2 (50–150 nM) for 24 and/or 48 h at 37°C. Guanosine 5’-monophosphate (GMP), siRNA negative control (NTC), and/or medium alone were used as negative controls for cell death. The cell viability of the cells was determined after the reduction of MTT (Sigma-Aldrich) to produce formazan crystals [96]. The procedure was performed as described by Barboza et al. [3]. Mitochondrial activity was expressed as a percentage after comparison with the absorbance of unstimulated SH-SY5Y cells.

Quantification of polysialic acid

For quantification of polysialic acid (polySia), SH-SY5Y cells infected with T. cruzi, as well uninfected cells, were incubated with iEndo-N (inactive Endo-N-acetylneuraminidase) for 1h at 37°C. In some experiments, polySia quantification was performed in SH-SY5Y cells with silenced ST8Siaa2 (siST8Sia2). After the incubation time, 30 μg of protein from each sample were immobilized on PVDF membrane and submitted to direct hydrolysis of the sialic acid with 2M acetic acid for two hours at 80° C. Hydrolyzed Sia were derivatized with 1,2-Diamino-4,5-methylenedioxybenzene (DMB; P/N D4784) obtained from Sigma-Aldrich and separated in reversed-phase chromatography with fluorescence detection using a Vanquish UPLC (Thermo Fisher). iEndo-N was a gift from Dr. Martina Mühlenhoff (Institute of Clinical Biochemistry, Hannover Medical School, Germany).

Measurement of cytosolic and mitochondrial ROS levels

Cytosolic and mitochondrial ROS levels were quantified using flow cytometry with the probes: dihydroethidium (DHE) (Sigma-Aldrich) and MitoSox Red (Thermo Fisher Scientific), respectively, as previously described [97]. SH-SY5Y siST8Sia2 cells plated in 6-well microplates (1 x 10⁶ cells/well) were treated or untreated with antimycin A (50 μM) or N-acetyl cysteine (5mM) for 4 hours to induce or inhibit oxidative stress, respectively. Negative control siRNA (NTC) was used as a negative control for ST8Sia2 silencing. Lipofectamine RNAiMAX was added in all controls used in experiments with siRNA. Subsequently, cells were washed three times with phosphate-buffered saline (PBS) and incubated with DHE for 40 minutes or with 5 μM of MitoSox Red for 10 minutes at 37°C and 5% CO₂. After incubation, cells were washed three times with PBS and centrifuged at 300 x g for 7 min at 26°C, and then transferred to cytometry tubes. Fluorescence intensity was analyzed on the LSR Fortessa cell analyzer flow cytometer (BD Biosciences) with excitation at 488nm and emission at 620nm. A minimum of 500.000 events was collected. Six experiments were performed for statistical analysis.

Measurement of Mitochondrial Calcium Ion (Ca²⁺) Concentration

Changes in mitochondrial Ca²⁺ ion were measured using the Rhod-2 probe (Thermo Fisher Scientific, Waltham, MA, USA). SH-SY5Y siST8Sia2 cells plated in 6-well microplates (1 x 10⁶ cells/well) were incubated with 5 μM Rhod-2 plus pluronic acid F-127 (5 μM; Sigma-Aldrich, St. Louis, MO, USA) and bovine serum albumin (30μg/mL; Sigma-Aldrich) at 37°C and 5% CO₂ for 60 minutes, as previously described [97]. Negative control siRNA (NTC) was used as a negative control for ST8Sia2 silencing. Lipofectamine RNAiMAX was added in all controls used in experiments with siRNA. After incubation, cells were washed three times with PBS, centrifuged at 300 x g for 7 min at 26°C, and then transferred to cytometry tubes. Maximum fluorescence was measured using tubes with cells incubated with ionomycin (1 μM) for 2 minutes, and minimum fluorescence was measured using tubes with cells incubated with EDTA (8mM) for 2 minutes. Fluorescence intensity was analyzed on the LSR Fortessa cell analyzer flow cytometer (BD Biosciences) with excitation at 395nm and emission at 525nm. A minimum of 500.000 events was collected. Next, mitochondrial Ca²⁺ concentration was calculated with the following equation: Ca²⁺ mitochondrial = Kd (Fsample—Fmin) / Fmax—Fsample, where F represents the fluorescence value and Kd was 390nM. Six experiments were performed for statistical analysis.

Determination of Mitochondrial Transmembrane Potential (ΔΨm)

Changes in ΔΨm were measured using the MitoTracker Red CMXRos probe (Thermo Fisher Scientific). SH-SY5Y siST8Sia2 cells plated in 6-well microplates (1 x 106 cells/well) were incubated with 100nM MitoTracker Red CMXRos at 37°C with 5% CO2 for 30 minutes, as previously described [97]. Negative control siRNA (NTC) was used as a negative control for ST8Sia2 silencing. Lipofectamine RNAiMAX was added in all controls used in experiments with siRNA. After incubation, cells were washed three times with PBS and centrifuged at 300 x g for 7 min at 26°C, and then transferred to cytometry tubes. Fluorescence intensity was analyzed on the LSR Fortessa cell analyzer flow cytometer (BD Biosciences) with excitation at 488 nm and emission at 620nm. A minimum of 500.000 events was collected. Six experiments were performed for statistical analysis.

Sample preparation for Mass-Spectrometry Based-Proteomics

T. cruzi-infected ST8Sia2 SH-SY5Y cells (siST8Sia2) were lysed in 8M urea containing protease using a sonicator for three cycles at 10% amplitude. T. cruzi-infected SH-SY5Y cells treated with culture medium alone (Medium) were used as negative control for ST8Sia2 silencing. Following extraction, a 1 μL aliquot of each sample was taken for protein quantification using the Qubit method. We used 30 μg of each sample for the digestion protocol. Ammonium bicarbonate (Ambic) was added to a final concentration of 50 mM, and the proteins were reduced with 10 mM DTT (DL-Dithiothreitol, Sigma-Aldrich) for 45 min at 30°C, alkylated with 40 mM iodoacetamide (Sigma-Aldrich) for 30 min at room temperature in the dark. The solution was diluted with 50 mM Ambic to 1M Urea, and the proteins were digested with trypsin at a ratio of 1:50 (μg trypsin/μg protein) for 16 hours at 30°C. The reaction was stopped with 1% formic acid (pH < 2) and desalted using C18 columns (StageTips).

Nano-LC-MS/MS Mass spectrometry analysis

The peptides were analyzed by nano-high performance liquid chromatography coupled with tandem mass spectrometry (nano-LC-MS/MS) using a nanoLC Easy-LTQ Orbitrap Velos-ETD (Thermo Scientific). The nanoLC gradient was from 0% to 30% solvent B (solvent A: 0.1% formic acid; solvent B: 90% acetonitrile, 0.1% formic acid) over 80 minutes at a flow rate of 300 nL/min. Precursor ions were selected based on data-dependent acquisition in positive mode. The 400–1600 m/z MS1 scans were recorded in the Orbitrap mass analyzer at a resolution of 120,000, with a target AGC of 3 × 10⁶ and a maximum injection time of 100 ms. The MS/MS spectra of the 20 most abundant precursor ions per scan were obtained through collision-induced dissociation (CID) with Orbitrap acquisition at a resolution of 30,000. Precursor ions were selected using an isolation window of 1.2 Da and fragmented by CID, with a maximum injection time of 150 ms.

Database search and proteomics data analysis

Raw data were searched with Proteome Discoverer software (version 3.0) using the Sequest search engine. The Swiss-Prot Homo sapiens database was concatenated with the unreviewed Trypanosoma cruzi database downloaded in March 2024. Trypsin was used as the cleavage enzyme and the search was limited to full cleavage; methionine oxidation and cysteine carbamidomethylation were defined as variable and fixed modifications, respectively. Other search criteria included: 2 missed cleavages; mass tolerance of 20 ppm (precursor ions), 0.5 Da (fragment ions), and minimum peptide length of 7 amino acids. The False Discovery Rate (FDR) was set to 1% for identifying peptide spectrum matches (PSMs), peptides, and proteins. Protein quantification was performed using the label-free quantification module. Functions for data transformation and filtering were applied using Perseus software. Proteins quantified in all biological replicates (out of a total of three replicates per group) were considered for differential regulation analysis (p-value < 0.05) by applying a t-test. Proteins considered exclusive were quantified in the three biological replicates of a group and none or one biological replicate of the group in the comparison. The chord plot was built by the GOplot package (10.1093/bioinformatics/btv300), available in Bioconductor. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [98] partner repository with the dataset identifier PXD053813 (Reviewer account details: Username: reviewer_pxd053813@ebi.ac.uk | Password: ULuzdFYhDYMe).

Statistical analysis

Initially, the results were tested for normal distribution and homogeneous variance. Once the normal distribution and homogeneous variance was confirmed, the One-way ANOVA test was applied. In the absence of normal distribution and heterogeneous variance, the Kruskal-Wallis test was applied. The statistical evaluation of the differences between the means of the groups were determined by the one-way analysis of variance, followed by the Bonferroni multiple comparison test and, in the non-parametric test, the comparisons between the groups were carried out by the Dunn’s multiple comparison test. For all statistical analyzes, the Prism 9.0 program (GraphPad Software, La Jolla, CA, USA) was used.

Supporting information

S1 Fig. T. cruzi infection modulates genes involved in host N-glycosylation machinery.

Bioinformatics analysis of Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) infected with Trypanosoma cruzi (Y strain). (A) Chordplot indicating the modulation of N-glycosylation machinery transcripts at 24 h (inner), 48 h (middle), and 72 h (outer) post-infection (h.p.i) with T. cruzi (infected vs non-infected; INFvsUNF). The R GOplot package was used to build chord plot. Red and blue colors indicate upregulated and downregulated transcripts, respectively; (B-E) Volcano plot (left graph) and Violin plot (right graph) of transcripts encoding enzymes involved in the N-glycosylation pathway that were differentially regulated in T. cruzi-infected hiPSC-CM, focusing on the modulation of the ST8Sia2 transcripts after 0 h (B), 24 h (C), 48 h (D), and 72h (E) post-infection. The logFC (INFvsUNF) indicates transcripts upregulated in orange and downregulated in dark blue.

(TIF)

pntd.0012454.s001.tif^{(2.6MB, tif)}

S2 Fig. Human pluripotent stem cells (hiPSC) are efficiently differentiated into cardiomyocytes (hiPSC-CM), and do not express PSA-NCAM.

(A) Representative immunofluorescence images of hiPSC-CM confirming the presence of α-actinin and troponin T (sarcomeric proteins). Cell nucleus were stained with DAPI (scale bars = 10 μm). (B) Histogram of Troponin T (TNNT2+) staining the differentiation of hiPSC into hiPSC-CM. (C) Representative western blot images of PSA-NCAM levels in uninfected hiPSC-CM (lane 1), T. cruzi-infected hiPSC-CM (lane 2), TE671 cells (lane 3), and T. cruzi trypomastigote (lane 4). hiPSC-CM cells were seeded in 6-well microplates (1 x 10⁶ cells/well) and infected with T. cruzi trypomastigotes (Y strain) in a ratio of 1:5 (SH-SY5Y:trypomastigotes) for 48 h at 37° C. Protein extracts from TE671 cell lysates (in Laemmli buffer) and T. cruzi trypomastigote (in RIPA buffer) were used as a positive and negative control for PSA-NCAM, respectively. 15 μg of protein extracts were used to analyze the PSA-NCAM levels by Western blotting using a specific anti-PSA-NCAM antibody.

(TIF)

pntd.0012454.s002.tif^{(2.1MB, tif)}

S3 Fig. Chemical and genetic inhibition of ST8Sia2 in SH-SY5Y cells does not affect cell viability.

(A,B) SH-SY5Y cells were seeded in 96-well microplates (1 × 10⁵ cells/well), and treated with cytidine 5’-monophosphate (CMP; 62.5–500 μM) or siRNA ST8Sia2 (50–150 nM) for 24 and/or 48 h. Guanosine 5’-monophosphate (GMP), siRNA negative control (NTC), and/or medium alone were used as negative controls for chemical or genetic inhibition of ST8Sia2. In all controls used in experiments with siRNA was added Lipofectamine RNAiMAX. Following 24 and 48 h incubation with CMP (A) or 24 h incubation with siRNA ST8Sia2 (B), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; 50 μg/mL) was added to the cells, and mitochondrial activity was estimated by MTT reduction and expressed as a percentage calculated from the ratio between the absorbance of stimulated and non-stimulated SH-SY5Y cells; (C) Relative expression of ST8Sia2 mRNA measured by qRT-PCR in SH-SY5Y cells treated with siRNA ST8Sia2 (siST8Sia2), following the experimental workflow presented in D. siRNA negative control (NTC) were used as negative control for genetic silencing of ST8Sia2. The Ct values of the target transcripts were normalized to the relative expression of GAPDH as endogenous control, and the relative expression of ST8Sia2 transcripts was quantified by the 2^-ΔΔ Ct method. Each bar represents the mean of three independent experiments performed in triplicate; (D) Experimental workflow adopted to investigate the effect of genetic inhibition of ST8Sia2 in SH-SY5Y cells using 100 nM of siRNA ST8Sia2. SH-SY5Y cells were seeded in 24-well microplates (5 x 10⁴ cells/well), and treated with siRNA ST8Sia2 [100 nM] for 24h, followed by resting time of 24 h additional. After this resting time, siST8Sia2 SH-SY5Y cells were restimulated with siRNA ST8Sia2 [100 nM] for an additional 24h. siRNA negative control (NTC) were used as negative control. In all controls used in experiments with siRNA was added Lipofectamine RNAiMAX; (E) Representative images of polySia levels in siST8Sia2 SH-SY5Y cells. SH-SY5Y cells were transfected with 100 nM ST8Sia2 siRNA using the same experimental strategy as presented in D. Following the completion of the ST8Sia2 silencing protocol, siST8Sia2 SH-SY5Y cells were stained with a specific anti-polySia (E) antibody, and the labeling for the target was visualized by immunofluorescence microscopy. siRNA negative control (NTC), and/or medium alone were used as negative controls for genetic inhibition of ST8Sia2.Cell nuclei were stained with DAPI (scale bars = 50 μm); (F) polySia levels measured in siST8Sia2 SH-SY5Y cells supernatants. SH-SY5Y cells were transfected with 100 nM ST8Sia2 siRNA using the same experimental strategy as presented in D. After the completion of the ST8Sia2 silencing protocol, siST8Sia2 SH-SY5Y cells were treated with EndoN [0.5 μg/ml] for 1h at 37°C, and polySia levels were measured in supernatants using UHPLC. The same procedure was applied to siRNA negative control (NTC), and/or medium alone SH-SY5Y cells. The results are expressed in pg/mL. Significant differences compared to the siST8Sia2 SH-SY5Y cells with siNTC SH-SY5Y cells are shown by (*) p < 0.05, (**) p < 0.001, and (****) p < 0.0001; ns: not significant. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

(TIF)

pntd.0012454.s003.tif^{(2.9MB, tif)}

S1 Table. Total proteins identified and quantified in the T. cruzi-infected SH-SY5Y cells with and without ST8sia2 gene silencing.

SH-SY5Y cells were seeded in 6-well microplates (1 x 10⁶ cells/well), and previously treated with siRNA ST8Sia2 [100 nM] for 24h before infection, followed by infection with T. cruzi trypomastigotes (Y strain) at ratio of 1:5 (SH-SY5Y:trypomastigotes). After 24 h.p.i, T. cruzi-infected SH-SY5Y cells were treated with siRNA ST8Sia2 [100 nM] for an additional 24h. Then, the cells were lysed in 8M urea containing protease inhibitors using a sonicator for three cycles at 10% amplitude, reduced, alkylated and digested. The tryptic peptides were analysed by nanoflow liquid chromatography coupled to mass spectrometry to map the differentially proteomic profile in response to T. cruzi infection. A triplicate for each condition was run. The total proteins identified in the two conditions along with their quantitative values in each replicate.

(XLSX)

pntd.0012454.s004.xlsx^{(206.6KB, xlsx)}

S2 Table. Proteins identified and quantified in all replicates of the two conditions under study, SH-SY5Y with and without ST8Sia2 gene silencing (siST8Sia2) infected with T. cruzi.

The total proteins identified in all replicates of the two conditions along with their quantitative values.

(XLSX)

pntd.0012454.s005.xlsx^{(82.9KB, xlsx)}

S3 Table. Differentially regulated proteins considering all replicates of the two conditions under study, SH-SY5Y with and without ST8Sia2 gene silencing (siST8Sia2) infected with T. cruzi.

(XLSX)

pntd.0012454.s006.xlsx^{(14.7KB, xlsx)}

S1 Data. Raw images data.

(PDF)

pntd.0012454.s007.pdf^{(92.5MB, pdf)}

S2 Data. Raw quantification data.

(XLSX)

pntd.0012454.s008.xlsx^{(87KB, xlsx)}

Acknowledgments

We thank Celia Ludio Braga at the Department of Biochemistry, Institute of Chemistry, University of São Paulo, Brazil for helping with the parasite cell culture. We thank the Vaccine Development Laboratory, Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Brazil, for providing the flow cytometry platform (LSR Fortessa cell analyzer flow cytometer, BD Biosciences).

Data Availability

All raw data required to replicate this study are deposited in PRIDE data repository (https://www.ebi.ac.uk/pride/) with the dataset identifier PXD053813 and within the manuscript and its supporting information files.

Funding Statement

We are grateful for the financial support provided by the São Paulo Research Foundation (FAPESP), grants processes n° 2018/18257-1 (GP), 2018/15549-1 (GP), 2020/04923-0 (GP), 2022/09915-0 (BRB), 2021/00140-3 (JMDS), 2022/00796-9 (LAMTS), 2021/00507-4 (VdMG), 2021/14179-9 (DMS), 2021/14751-4 (SNM), 2020/02988-7 (SKNM), 2023/02096-7 (CMM); by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (“Bolsa de Produtividade”) (SKNM, CMM, and GP); by Fundação Faculdade de Medicina (FFM-SKNM); by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (AMMN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0012454.r001

Decision Letter 0

Walderez O Dutra

18 Jan 2024

Dear Dr. Palmisano,

Thank you very much for submitting your manuscript "ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Walderez O. Dutra, PhD.

Section Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

Section Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Yes

-Is the study design appropriate to address the stated objectives? Yes but see Summary and General Comments

-Is the population clearly described and appropriate for the hypothesis being tested? N/A

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? Yes

-Were correct statistical analysis used to support conclusions? Yes

-Are there concerns about ethical or regulatory requirements being met? No

Reviewer #2: experiments well planned

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan? Yes

-Are the results clearly and completely presented? Yes

-Are the figures (Tables, Images) of sufficient quality for clarity? Yes

Reviewer #2: excelent figures and images . Very clear graphs .

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented? Yes but see Summary and General Comments

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?Yes

-Is public health relevance addressed? Yes

Reviewer #2: interesting and objectives conclusions. Maybe could speculate a little bit about the mechanism involved at the modulation.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: see Summary and General Comments

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This is an interesting paper that proposes that polysialic acid (synthesised by ST8Sia2) inhibits host cell invasion (and/or propagation?) by T. cruzi and that this is why T. cruzi infection of cells reduces ST8Sia2 expression. The data presented are well described but there are some missing controls, and the paper would benefit from more critical assessment of significance.

The authors should address the following points:

1. While the phenomena described are certainly noteworthy, it is difficult to understand an overall hypothesis as to why the phenomenon of T. cruzi infection of host cells causing downregulation of ST8Sia2 would influence an in vivo infection. Thus, the downregulation of ST8Sia2 in an individual cell would be post-invasion and unlikely(?) to influence cell invasion by more parasites, especially as it would take many hours for pre-existing polysialic acid to be turned over by the invaded host cell. The authors should help the reader understand how they think the T. cruzi invasion induced downregulation of ST8Sia2 expression could assist host infection and parasite survival.

2. While the data presented using immunofluorescent microscopy data appear sound, they are difficult to assess. For example, in Fig 1A what % of host cells are infected and with what number (average and SD and median) of amastigotes?

3. The reduction in ST8Sia2 mRNA is clear (Fig 1B) but the reduction in ST8Sia2 protein is slight (Fig 1B) (presumably because it has a long half-life – are there any data in the literature from global turnover studies?) bringing into question the reduction in polysialic acid as measured by Western (1D and G).

4. The data are clearer in Fig 2 using SH-SY5Y cells instead of iPSc derived cells. However, SH-SY5Y cells are neuroblastoma cells (selected for their propensity to express polysialic acid, presumably(?)) but does infection of neurones occur in natural T. cruzi infections? What is the physiological basis for choosing this cell line?

5. Also in Fig. 2 B the variance (by eye) of signals to b-actin look wider than those on the plots. For example, the third uninfected sample b-actin signal is much stronger than the other two yet the ST8Sia2, PSA-NCAM and poly-Sia and NCAM1 signals look to be lower than the other two. All signals relative to b-actin in infected cells certainly look lower. However, if a large proportion of cells are infected (? – see point 1) why not perform this (for St8Sia2 and NCAM) by quantitative proteomics, an area of expertise in the author’s lab?

6. Is the reduction in NCAM due to reduced protein stability or reduction in rate of protein synthesis? A pulse-chase experiment might resolve this issue.

7. The data in Fig 3 with SH-SY5Y cells using anti-ST8Sia2 and anti-PSA immunofluorescence and EndoN-releases PSA measures are striking and seem to be mich more profound that the data in Fig 2 – why is that? Further, the uninfected micrograph seems to have a small number od cells with strong signal but most at background level closer to the signals seen in the infected cells micrograph (again what 5 cells are infected with how many amastigotes?)

8. In Fig 3, where did the pre-existing PSA of the infected cells go to?

9. The removal of cell surface PSA with EndoN makes the cells more easily invaded and/or allows that parasites to propagate better than in control cells. Since the only parameter reported is average number of amastigotes per cell it is impossible to judge which. Again, % infected cells and parasites/cell with SD are needed to assess the data.

10. The data in Fig 5 are fascinating in principle but they lack important controls. We are asked to assume that CMP and ST8Sia2 siRNA do indeed reduce cell surface PSA (for some reason, that data is not presented using the techniques in the prior experiments) and that this, and this alone, is the reason for the increase in cell infection [Again, % infected cells and parasites/cell with SD are needed to assess the data, not just average amastigotes per cell]. Note: this reviewer is sceptical that exogenously added CMP would provide selective inhibition of Golgi enzyme ST8Sia2 (or even be taken up by cells at all). The authors should design experiments (perhaps more using EndoN?) where they can correlate extent of PSA reduction with infection efficiency.

Reviewer #2: The work of Barboza et al. contains important information regarding the modulation of trypanosoma cruzi of host machinery during infection and will be a great contribution in the area if published by PNTD.

The authors performed fine experiments associating infection, enzyme expression and acceptors of the transferred sugars, showing an inverse relationship between enzymatic activity and host polysialylation and infection load.To reach more general and conceptual conclusions, I will address a few points.

1- It was very convincing to use two different lines of cells to see the effect of the infection and the phenotype of the cell, it would also be interesting to see two polar strains of t cruzi, one more and one less invasive to determine if there is a correlation with the presence of the enzymatic activity of ST8Sia2 and the products detected.5CN5A.

2- Decreased RNAI expression of ST8sia2 produced an increase in infectivity of T cruzi, would the opposite situation of overexpression of the enzyme produce a decrease in infection?

3-The authors do not work on or speculate what is happening in the modulation by the parasite. Parasitic extracts, fixed parasites, alteration of parasite surface, blockade of invasion by antibodies have impact on the enzymatic activity of ST8Sia2

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Reviewer #2: No

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PLoS Negl Trop Dis. 2024 Sep 25;18(9):e0012454. doi: 10.1371/journal.pntd.0012454.r002

Author response to Decision Letter 0

17 Jul 2024

Attachment

Submitted filename: Reviewer_comments_final version.pdf

pntd.0012454.s009.pdf^{(124.1KB, pdf)}

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0012454.r003

Decision Letter 1

Susan Madison-Antenucci

13 Aug 2024

Dear Dr. Palmisano,

We are pleased to inform you that your manuscript 'ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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PLOS Neglected Tropical Diseases

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Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: this is ok

Reviewer #3: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: this is ok

Reviewer #3: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: this is ok

Reviewer #3: (No Response)

**********

Editorial and Data Presentation Modifications?

Reviewer #2: this is ok

Reviewer #3: (No Response)

**********

Summary and General Comments

Reviewer #2: the authors have improved the manuscript.

Reviewer #3: This is an interesting article with data that appropriately supports the manuscript's conclusions. Prior reviewer comments were appropriately addressed.

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Reviewer #3: No

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0012454.r004

Acceptance letter

Susan Madison-Antenucci

3 Sep 2024

Dear Dr. Palmisano,

We are delighted to inform you that your manuscript, "ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. T. cruzi infection modulates genes involved in host N-glycosylation machinery.

(TIF)

pntd.0012454.s001.tif^{(2.6MB, tif)}

S2 Fig. Human pluripotent stem cells (hiPSC) are efficiently differentiated into cardiomyocytes (hiPSC-CM), and do not express PSA-NCAM.

(TIF)

pntd.0012454.s002.tif^{(2.1MB, tif)}

S3 Fig. Chemical and genetic inhibition of ST8Sia2 in SH-SY5Y cells does not affect cell viability.

(TIF)

pntd.0012454.s003.tif^{(2.9MB, tif)}

S1 Table. Total proteins identified and quantified in the T. cruzi-infected SH-SY5Y cells with and without ST8sia2 gene silencing.

(XLSX)

pntd.0012454.s004.xlsx^{(206.6KB, xlsx)}

S2 Table. Proteins identified and quantified in all replicates of the two conditions under study, SH-SY5Y with and without ST8Sia2 gene silencing (siST8Sia2) infected with T. cruzi.

The total proteins identified in all replicates of the two conditions along with their quantitative values.

(XLSX)

pntd.0012454.s005.xlsx^{(82.9KB, xlsx)}

S3 Table. Differentially regulated proteins considering all replicates of the two conditions under study, SH-SY5Y with and without ST8Sia2 gene silencing (siST8Sia2) infected with T. cruzi.

(XLSX)

pntd.0012454.s006.xlsx^{(14.7KB, xlsx)}

S1 Data. Raw images data.

(PDF)

pntd.0012454.s007.pdf^{(92.5MB, pdf)}

S2 Data. Raw quantification data.

(XLSX)

pntd.0012454.s008.xlsx^{(87KB, xlsx)}

Attachment

Submitted filename: Reviewer_comments_final version.pdf

pntd.0012454.s009.pdf^{(124.1KB, pdf)}

Data Availability Statement

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PERMALINK

ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi

Bruno Rafael Barboza

Janaina Macedo-da-Silva

Lays Adrianne Mendonça Trajano Silva

Vinícius de Morais Gomes

Deivid Martins Santos

Antônio Moreira Marques-Neto

Simon Ngao Mule

Claudia Blanes Angeli

Juliana Borsoi

Carolina Borsoi Moraes

Cristiane Moutinho-Melo

Martina Mühlenhoff

Walter Colli

Suely Kazue Nagashi Marie

Lygia da Veiga Pereira

Maria Julia Manso Alves

Giuseppe Palmisano

Roles

Abstract

Author summary

Introduction

Results

Modulation of genes involved in host N-glycosylation machinery by T. cruzi is accompanied by downregulation of ST8Sia2 polysialyltransferase

T. cruzi infection modulates hiPSC-CM ST8Sia2 expression and compromises host polysialylation

Fig 1. T. cruzi infection modulates the abundance of ST8Sia2, SCN5A, and polysialylation on hiPSC-CM.

T. cruzi infection reduces the expression of host polysialylated target molecules

Fig 2. T. cruzi infection modulates the polysialylation on SH-SY5Y cells and affects the abundance of polysialylated molecules.

Fig 3. T. cruzi infection reduces ST8Sia2 and PolySia levels in SH-SY5Y cells.

Fig 4. Removal of polySia in SH-SY5Y cells favors T. cruzi infection by increasing the number of internalized parasites.

Chemical and genetic Inhibition of ST8Sia2 increases the number of parasites internalized in SH-SY5Y cells

Fig 5. Chemical and genetic inhibition of ST8Sia2 polysialyltransferase activity in SH-SY5Y cells favors T. cruzi proliferation.

Silencing of ST8Sia2 in T. cruzi-infected SH-SY5Y cells intensifies oxidative stress

Fig 6. Silencing of ST8Sia2 in SH-SY5Y cells modulates oxidative stress.

Fig 7. Proteomic analysis of SH-SY5Y cells with ST8sia2 gene silencing (siST8sia2) and Lipofectamine RNAiMAX-treated control (Medium), both infected with T. cruzi (Ty).

Discussion

Fig 8. Potential mechanism of host polysialylation modulation during T. cruzi infection.

Materials and methods

Public RNA-seq data reanalysis

Human cell culture

T. cruzi culture and infection assay

Immunofluorescence assay

Quantification of immunofluorescence

Total cell lysates

Immunoblotting assay

Drugs treatment and chemical ST8Sia2 inhibition

siRNA-Directed Inhibition of ST8Sia2

Quantitative real-time reverse transcription PCR (qRT-PCR)

MTT assay

Quantification of polysialic acid

Measurement of cytosolic and mitochondrial ROS levels

Measurement of Mitochondrial Calcium Ion (Ca2+) Concentration

Determination of Mitochondrial Transmembrane Potential (ΔΨm)

Sample preparation for Mass-Spectrometry Based-Proteomics

Nano-LC-MS/MS Mass spectrometry analysis

Database search and proteomics data analysis

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Walderez O Dutra

Roles

Author response to Decision Letter 0

Decision Letter 1

Susan Madison-Antenucci

Roles

Acceptance letter

Susan Madison-Antenucci

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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