Infection Of Human Cells Research Articles

Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly

Nanodecoy systems based on analogues of viral cellular receptors assembled onto fluid lipid-based membranes of nano/extravescicles are potential new tools to complement classic therapeutic or preventive antiviral approaches. The need for lipid-based membranes for transmembrane receptor anchorage may pose technical challenges along industrial translation, calling for alternative geometries for receptor multimerization. Here we developed a semisynthetic self-assembling SARS-CoV-2 nanodecoy by multimerizing the biotin labelled virus cell receptor -ACE2- ectodomain onto a poly-avidin nanoparticle (NP) based on the Avidin-Nucleic-Acid-NanoASsembly-ANANAS. The ability of the assembly to prevent SARS-CoV-2 infection in human lung cells and the affinity of the ACE2:viral receptor-binding domain (RBD) interaction were measured at different ACE2:NP ratios. At ACE2:NP = 30, 90 % SARS-CoV-2 infection inhibition at ACE2 nanomolar concentration was registered on both Wuhan and Omicron variants, with ten-fold higher potency than the monomeric protein. Lower and higher ACE2 densities were less efficient suggesting that functional recognition between multi-ligand NPs and multi-receptor virus surfaces requires optimal geometrical relationships. In vivo studies in mice showed that the biodistribution and safety profiles of the nanodecoy are potentially suitable for preventing viral infection upon nasal instillation. Viral receptor multimerization using ANANAS is a convenient process which, in principle, could be rapidly adapted to counteract also other viral infections.

Antiviral effects of micafungin against pteropine orthoreovirus, an emerging zoonotic virus carried by bats

Bat-borne emerging zoonotic viruses cause major outbreaks, such as the Ebola virus, Nipah virus, and/or beta coronavirus. Pteropine orthoreovirus (PRV), whose spillover event occurred from fruits bats to humans, causes respiratory syndrome in humans widely in South East Asia. Repurposing approved drugs against PRV is an effective tool to confront future PRV pandemics. We screened 2,943 compounds in an FDA-approved drug library and identified eight hit compounds that reduce viral cytopathic effects on cultured Vero cells. Real-time quantitative PCR analysis revealed that six of eight hit compounds significantly inhibited PRV replication. Among them, micafungin used clinically as an antifungal drug, displayed a prominent antiviral effect on PRV. Secondly, the antiviral effects of micafungin on PRV infected human cell lines (HEK293T and A549), and their transcriptome changes by PRV infection were investigated, compared to four different bat-derived cell lines (FBKT1 (Ryukyu flying fox), DEMKT1 (Leschenault's rousette), BKT1 (Greater horseshoe bat), YUBFKT1 (Eastern bent-wing bats)). In two human cell lines, unlike bat cells that induce an IFN-γ response pathway, an endoplasmic reticulum stress response pathway was commonly activated. Additionally, micafungin inhibits viral release rather than suppressing PRV genome replication in human cells, although it was disturbed in Vero cells. The target of micafungin's action may vary depending on the animal species, but it must be useful for human purposes as a first choice of medical care.

Mechanism of Ellagic Acid-Induced Autophagy in Cis-Diamminedichloroplatinum(II)-Resistant Ovarian Cancer Cells

This study investigated the effect of ellagic acid on autophagy in cisplatin-resistant human epithelial ovarian cancer cells. After the autophagy double-labeled mred fluorescence protein, green fluorescence protein, light chain 3 carrying adenovirus infected cisplatin-resistant human epithelial ovarian cancer cells, the changes of autophagy flux and autophagy-related proteins were detected. Cell counting kit-8 assay was employed to detect the viability of cisplatin-resistant human epithelial ovarian cancer cells. The results showed that the number of autophagosomes and autolysosomes in the ellagic acid group increased significantly in comparison with the in cisplatin-resistant human epithelial ovarian cancer group. With the increase in ellagic acid concentration, light chain 3-II/I ratio and Beclin1 expression increased, p62 expression and cell viability decreased, and caspase-3 activated, in cisplatin-resistant human epithelial ovarian cancer cells. Conversely, 3-methyladenine inhibited ellagic acid-induced autophagy and activated caspase-3 and enhanced the cell viability. Similarly, chloroquine significantly increased cell survival rate, light chain 3-II/I ratio, and activated caspase-3, but decreased p62 expression in the ellagic acid group. In conclusion, ellagic acid can induce autophagy to inhibit the proliferation of cisplatin-resistant human epithelial ovarian cancer cells.

Integrated bioinformatics approaches to investigate alterations in transcriptomic profiles of monkeypox infected human cell line model

BackgroundThe recent re-emergence of the monkeypox (mpox) epidemic in nonendemic regions has raised concerns regarding a potential global outbreak. The mpox virus (MPV) is a smallpox-like virus belonging to the genus Orthopoxvirus (family: Poxviridae). Although studies suggest that MPV infection suppresses the Toll-like receptor-3- and tumor necrosis factor-α-related signaling pathways, whether MPV regulates other immune-related pathways remains unclear. MethodsIn this study, two distinct temporal patterns were used for establishing an MPV-infected human immortal epithelial cancer cell line (HeLa). These two durations 2 and 12 h of incubation were selected to identify the coregulated genes and pathways affected by MPV infection. ResultsThe use of the Gene Ontology framework, Kyoto Encyclopedia of Genes and Genome database, and MetaCore software yielded valuable insights. Specifically, various pathways were found to be enriched in HeLa cells infected with MPV for 2 and 12 h. These pathways included Notch, CD40, CD95, hypoxia-inducible factor-1-α, interleukin (IL)− 1, IL-6, phosphoinositide 3-kinase, nuclear factor-κB, mitogen-activated protein kinase, and oxidative stress–induced signalling pathways. Clusters and pathways of metabolism and viral replication cycles were significantly associated with the 2-hour infection group. This association was identified based on the regulation of genes such as HSPG2, RHPN2, MYL1, ASPHD2, CA9, VIPR1, SNX12, MGC2752, SLC25A1, PEX19, and AREG. Furthermore, clusters and pathways related to immunity and cell movement were found to be associated with the 12-hour infection group. This association was identified based on the regulation of genes such as C1orf21, C19orf48, HRK, IL8, GULP1, SCAND2, ATP5C1, FEZ1, SGSH, TACC2, CYP4X1, MMP1, CPB1, P2RY13, WDR27, PRPF4, and ENDOD1. ConclusionsThis study can improve our understanding of the mechanisms underlying the pathophysiology and post-infection sequelae of mpox. Our findings provide valuable insights into the various modes of MPV infection.

Autophagy gene-dependent intracellular immunity triggered by interferon-γ.

Interferon-γ (IFNγ) is a critical mediator of cell-intrinsic immunity to intracellular pathogens. Understanding the complex cellular mechanisms supporting robust interferon-γ-induced host defenses could aid in developing new therapeutics to treat infections. Here, we examined the impact of autophagy genes in the interferon-γ-induced host response. We demonstrate that genes within the autophagy pathway including Wipi2, Atg9, and Gate-16, as well as ubiquitin ligase complex genes Cul3 and Klhl9 are required for IFNγ-induced inhibition of murine norovirus (norovirus hereinafter) replication in mouse cells. WIPI2 and GATE-16 were also required for IFNγ-mediated restriction of parasite growth within the Toxoplasma gondii parasitophorous vacuole in human cells. Furthermore, we found that perturbation of UFMylation pathway components led to more robust IFNγ-induced inhibition of norovirus via regulation of endoplasmic reticulum (ER) stress. Enhancing or inhibiting these dynamic cellular components could serve as a strategy to control intracellular pathogens and maintain an effective immune response.

TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

Dual spatially resolved transcriptomics for human host–pathogen colocalization studies in FFPE tissue sections

Technologies to study localized host–pathogen interactions are urgently needed. Here, we present a spatial transcriptomics approach to simultaneously capture host and pathogen transcriptome-wide spatial gene expression information from human formalin-fixed paraffin-embedded (FFPE) tissue sections at a near single-cell resolution. We demonstrate this methodology in lung samples from COVID-19 patients and validate our spatial detection of SARS-CoV-2 against RNAScope and in situ sequencing. Host–pathogen colocalization analysis identified putative modulators of SARS-CoV-2 infection in human lung cells. Our approach provides new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes in FFPE samples.

Protective efficacy of Toxoplasma gondii infected cells-derived exosomes against chronic murine toxoplasmosis

Exosomes were isolated from T. gondii infected human hepatoblastoma cells using the exosome isolation kit and characterized by electron microscopy and Western blotting. Exosomes adsorbed to alum adjuvant were evaluated as a potential immunizing agent against murine chronic toxoplasmosis compared to excretory secretory antigens (ESA)-alum. Mice were immunized at days 1, 15 and 29. The levels of IgG, IFN-γ, IL-4 and IL-10, CD4+ and CD8+ T cells were determined using sandwich enzyme-linked immunosorbent assay (sandwich ELISA) at days 14, 28 and 56 of the experiment. Then mice were infected orally with 10 cysts of T. gondii. The protective efficacy of the antigens were evaluated by counting the brain cysts and measuring the aforementioned humoral and cellular parameters 60 days post infection. The results showed that alum increased the protective efficacy of the exosomes. Immunization with exosome-alum induced both humoral and mixed Th1/Th2 cellular immune responses. Exosome-alum gave higher levels of the humoral and cellular parameters, compared to ESA-alum. After challenge infection, exosome-alum significantly reduced the brain cyst burden by 75 % while ESA-alum gave 42 % reduction and evoked higher humoral and cellular immune responses. Therefore, the possibility of using T. gondii infected cells-derived exosome-alum as a vaccine is a new perspective in toxoplasmosis.

Comparative Transcriptome Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 on Bovine Rectoanal Junction Cells and Human Colonic Epithelial Cells during Initial Adherence.

Shiga toxin-producing Escherichia coli (STEC) are notorious foodborne pathogens, capable of causing severe diarrhea and life-threatening complications in humans. Cattle, acting as both primary reservoirs and asymptomatic carriers of STEC, predominantly harbor the pathogen in their rectoanal junction (RAJ), facilitating its transmission to humans through contaminated food sources. Despite the central role of cattle in STEC transmission, the molecular mechanisms governing STEC's adaptation in the RAJ of the asymptomatic reservoir host and its subsequent infection of human colonic epithelial cells, resulting in diarrhea, remain largely unexplored. This study aims to uncover these complicated dynamics by focusing on the STEC O157:H7 serotype within two distinct host environments, bovine RAJ cells and human colonic epithelial cells, during initial colonization. We employed comparative transcriptomics analysis to investigate differential gene expression profiles of STEC O157:H7 during interactions with these cell types. STEC O157:H7 was cultured either with bovine RAJ cells or the human colonic epithelial cell line CCD CoN 841 to simulate STEC-epithelial cell interactions within these two host species. High-throughput RNA sequencing revealed 829 and 1939 bacterial genes expressed in RAJ and CCD CoN 841, respectively. After gene filtering, 221 E. coli O157:H7 genes were upregulated during initial adherence to CCD CoN cells and 436 with RAJ cells. Furthermore, 22 genes were uniquely expressed with human cells and 155 genes with bovine cells. Our findings revealed distinct expression patterns of STEC O157:H7 genes involved in virulence, including adherence, metal iron homeostasis, and stress response during its initial adherence (i.e., six hours post-infection) to bovine RAJ cells, as opposed to human colonic epithelial cells. Additionally, the comparative analysis highlighted the potential role of some genes in host adaptation and tissue-specific pathogenicity. These findings shed new light on the potential mechanisms of STEC O157:H7 contributing to colonize the intestinal epithelium during the first six hours of infection, leading to survival and persistence in the bovine reservoir and causing disease in humans.

Investigating the Molecular Mechanisms Underlying Early Response to Inflammation and Helicobacter pylori Infection in Human Gastric Epithelial Cells.

Helicobacter pylori is a leading cause of chronic gastric inflammation, generally associated with gastritis and adenocarcinoma. Activation of the NF-κB pathway mainly contributes to the inflammatory phenotype observed in H. pylori infection in humans and experimental models. Since the gastric epithelium undergoes rapid turnover, inflammation and pathogenicity of H. pylori result from early phase and chronically activated pathways. In the present study we investigated the early host response to H. pylori in non-tumoral human gastric epithelial cells (GES-1). To dissect the pathogen-specific mechanisms we also examined the response to tumor necrosis factor (TNF), a prototypical cytokine. By analyzing the activation state of NF-κB signaling, cytokine expression and secretion, and the transcriptome, we found that the inflammatory response of GES-1 cells to H. pylori and TNF results from activation of multiple pathways and transcription factors, e.g., NF-κB and CCAAT/enhancer-binding proteins (CEBPs). By comparing the transcriptomic profiles, we found that H. pylori infection induces a less potent inflammatory response than TNF but affects gene transcription to a greater extent by specifically inducing transcription factors such as CEBPβ and numerous zinc finger proteins. Our study provides insights on the cellular pathways modulated by H. pylori in non-tumoral human gastric cells unveiling new potential targets.

SARS-CoV-2 infection of human lung epithelial cells induces TMPRSS-mediated acute fibrin deposition

Severe COVID-associated lung injury is a major confounding factor of hospitalizations and death with no effective treatments. Here, we describe a non-classical fibrin clotting mechanism mediated by SARS-CoV-2 infected primary lung but not other susceptible epithelial cells. This infection-induced fibrin formation is observed in all variants of SARS-CoV-2 infections, and requires thrombin but is independent of tissue factor and other classical plasma coagulation factors. While prothrombin and fibrinogen levels are elevated in acute COVID BALF samples, fibrin clotting occurs only with the presence of viral infected but not uninfected lung epithelial cells. We suggest a viral-induced coagulation mechanism, in which prothrombin is activated by infection-induced transmembrane serine proteases, such as ST14 and TMPRSS11D, on NHBE cells. Our finding reveals the inefficiency of current plasma targeted anticoagulation therapy and suggests the need to develop a viral-induced ARDS animal model for treating respiratory airways with thrombin inhibitors.

Esculentoside a Prevents Cell Damage Induced by Carbapenem-Resistant Klebsiella Pneumoniae Infection

We have investigated the role of Esculentoside A in the progression of carbapenem-resistant Klebsiella pneumoniae infection in Human MLE-12 cells. The results of the studies show that Esculentoside A enhances the viability of carbapenem-resistant Klebsiella pneumoniae-infected MLE-12 cells and suppresses cell apoptosis. EsA also suppresses mitochondrial damage and oxidative stress in lung epithelial cells caused by carbapenem-resistant Klebsiella pneumoniae infections. The data further confirmed that Esculentoside A could relieve mitochondrial damage, apoptosis, and oxidative stress in carbapenem-resistant Klebsiella pneumonia-infected lung epithelial cells by regulating the mitogen-activated protein kinase pathway.

Salmonella Derby adaptation to swine and simultaneous attenuation for humans go through decay of Salmonella Pathogenicity Island I.

We report the identification, within the global population of Salmonella Derby, of a lineage highly diffused and expanding in swine while being significantly under-represented in humans which carries stop mutations in the Salmonella Pathogenicity Island 1 (SPI-1) genes sipA and hilC. Single-cell analysis of invasion, vacuolar load, and cytosolic hyper-replication show that these mutations attenuate S. Derby virulence in human intestinal cells. Identification of this lineage follows the previous detection of another lineage over-represented in swine compared with humans which carries a loss-of-function mutation in hilD, the major SPI-1 regulator, responsible for reduced infection of human cells. Notably, this hilD-mutated lineage represents a sub-population of the wider sipA and hilC-mutated lineages identified in this study, indicating that SPI-1 is subjected to genetic decay in this part of the Salmonella Derby population adapted to swine. Virulence assessment on swine intestinal cells showed drastically lower levels for both wild-type and mutant S. Derby compared with human cells. Still, S. Derby with impaired sipA and hilC showed further attenuation also in swine cells compared with the wild type. These data suggest that invasion of swine intestinal epithelium is not needed by S. Derby to circulate in pigs, leading to the decay of SPI-1, which in turn determines attenuation for humans. Accordingly, a further S. Derby lineage under-represented in humans compared with pigs was found carrying truncated or missing SPI-1 genes, indicating that the S. Derby population is undergoing converging evolution in its adaptation to swine while attenuating for humans through loss of SPI-1 function.IMPORTANCEThis study integrated population data with in vitro assessment of virulence phenotypes to unveil that a considerable part of the global population of Salmonella Derby is evolving to enhance its host adaptation to the swine host and that this evolution is simultaneously increasing its attenuation for humans. The study shows that the fixation of deleterious mutations in SPI-1 has a role in this process. This evidence indicates that SPI-1 has a key role for S. Derby virulence in humans but not for its circulation in swine. The results show that genes generally considered essential for Salmonella pathogenesis do not play the same key role for all Salmonella serovars or lineages and/or all hosts. The study helps in understanding the molecular mechanisms underlying the ecology and host adaptation of Salmonella showing that the adaptation process can vary for different types of Salmonella and hosts.

Differential RNA editing landscapes in host cell versus the SARS-CoV-2 genome

The SARS-CoV-2 pandemic was defined by the emergence of new variants formed through virus mutation originating from random errors not corrected by viral proofreading and/or the host antiviral response introducing mutations into the viral genome. While sequencing information hints at cellular RNA editing pathways playing a role in viral evolution, here, we use an invitro human cell infection model to assess RNA mutation types in two SARS-CoV-2 strains representing the original and the alpha variants. The variants showed both different cellular responses and mutation patterns with alpha showing higher mutation frequency with most substitutions observed being C-U, indicating an important role for apolipoprotein B mRNA editing catalytic polypeptide-like editing. Knockdown of select APOBEC3s through RNAi increased virus production in the original virus, but not in alpha. Overall, these data suggest a deaminase-independent anti-viral function of APOBECs in SARS-CoV-2 while the C-U editing itself might function to enhance genetic diversity enabling evolutionary adaptation.

Heparin Precursors with Reduced Anticoagulant Properties Retain Antiviral and Protective Effects That Potentiate the Efficacy of Sofosbuvir against Zika Virus Infection in Human Neural Progenitor Cells.

Zika virus (ZIKV) infection during pregnancy can result in severe birth defects, such as microcephaly, as well as a range of other related health complications. Heparin, a clinical-grade anticoagulant, is shown to protect neural progenitor cells from death following ZIKV infection. Although heparin can be safely used during pregnancy, it retains off-target anticoagulant effects if directly employed against ZIKV infection. In this study, we investigated the effects of chemically modified heparin derivatives with reduced anticoagulant activities. These derivatives were used as experimental probes to explore the structure-activity relationships. Precursor fractions of porcine heparin, obtained during the manufacture of conventional pharmaceutical heparin with decreased anticoagulant activities, were also explored. Interestingly, these modified heparin derivatives and precursor fractions not only prevented cell death but also inhibited the ZIKV replication of infected neural progenitor cells grown as neurospheres. These effects were observed regardless of the specific sulfation position or overall charge. Furthermore, the combination of heparin with Sofosbuvir, an antiviral licensed for the treatment of hepatitis C (HCV) that also belongs to the same Flaviviridae family as ZIKV, showed a synergistic effect. This suggested that a combination therapy approach involving heparin precursors and Sofosbuvir could be a potential strategy for the prevention or treatment of ZIKV infections.

Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

Ebola Virus Uses Tunneling Nanotubes as an Alternate Route of Dissemination.

Ebola virus (EBOV) disease is marked by rapid virus replication and spread. EBOV enters the cell by macropinocytosis and replicates in the cytoplasm, and nascent virions egress from the cell surface to infect neighboring cells. Here, we show that EBOV uses an alternate route to disseminate: tunneling nanotubes (TNTs). TNTs, an actin-based long-range intercellular communication system, allows for direct exchange of cytosolic constituents between cells. Using live, scanning electron, and high-resolution quantitative 3-dimensional microscopy, we show that EBOV infection of primary human cells results in the enhanced formation of TNTs containing viral nucleocapsids. TNTs promote the intercellular transfer of nucleocapsids in the absence of live virus, and virus could replicate in cells devoid of entry factors after initial stall. Our studies suggest an alternate model of EBOV dissemination within the host, laying the groundwork for further investigations into the pathogenesis of filoviruses and, importantly, stimulating new areas of antiviral design.

Rottlerin impairs early and late steps of Toxoplasma gondii infection in human trophoblast cells and villous explants

Congenital toxoplasmosis, caused by the opportunistic protozoan parasite T. gondii, can cause stillbirths, miscarriages and fetal abnormalities, as well as encephalitis and chorioretinitis in newborns. Available treatment options rely on antiparasitic drugs that have been linked to serious side effects, high toxicity and the development of drug-resistant parasites. The search for alternative therapeutics to treat this disease without acute toxicity for the mother and child is essential for the advancement of current therapeutic procedures. The present study aimed to unravel the mode of the anti-T. gondii action of Rottlerin, a natural polyphenol with multiple pharmacological properties described. Herein, we further assessed the antiparasitic activity of Rottlerin against T. gondii infection on the human trophoblastic cells (BeWo cells) and, for the first time, on human villous explants. We found that non-cytotoxic doses of Rottlerin impaired early and late steps of parasite infection with an irreversible manner in BeWo cells. Rottlerin caused parasite cell cycle arrest in G1 phase and compromised the ability of tachyzoites to infect new cells, thus highlighting the possible direct action on parasites. An additional and non-exclusive mechanism of action of Rottlerin involves the modulation of host cell components, by affecting lipid droplet formation, mitochondrial function and upregulation of the IL-6 and MIF levels in BeWo cells. Supporting our findings, Rottlerin also controlled T. gondii proliferation in villous explants with low toxicity and reduced the IL-10 levels, a cytokine associated with parasite susceptibility. Collectively, our results highlighted the potential use of Rottlerin as a promising tool to prevent and/or treat congenital toxoplasmosis.