Disruption of Active Trans-Sialidase Genes Impairs Egress from Mammalian Host Cells and Generates Highly Attenuated Trypanosoma cruzi Parasites

Burle-Caldas, Gabriela de Assis; Santos, Nailma S. A. dos; Castro, Julia; Mügge, Fernanda L. B.; Grazielle-Silva, Viviane; Oliveira, Antônio Edson Rocha; Pereira, Milton; Reis-Cunha, João Luís; Coqueiro-dos-Santos, Anderson; Gomes, Dawidson Assis; Bartholomeu, Daniella Castanheira; Moretti, Nilmar Silvio; Schenkman, Sérgio; Gazzinelli, Ricardo T.; Teixeira, Santuza Maria Ribeiro

doi:10.1128/mbio.03478-21

Cited by 11 publications

(11 citation statements)

References 55 publications

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“…Results determined that sequences within the genome corresponding to TS-GI were 16 genes, while the identity between them was from 86.9% to 100%. 58 In the present study, we expanded the number of sequences analyzed to a higher number of strains, confirming that the identity between TS-GI sequences is around 92%. This identity value within the species is similar to the value obtained for TS-GII TSA-1 antigen, for which sequence variability was analyzed to determine its potential use in vaccines.…”

Section: Discussionsupporting

confidence: 74%

“…In another recent paper that used the last‐generation method to obtain long reads, which allows high coverage and base call accuracy, the CL genome was re‐sequenced. Results determined that sequences within the genome corresponding to TS‐GI were 16 genes, while the identity between them was from 86.9% to 100% 58 . In the present study, we expanded the number of sequences analyzed to a higher number of strains, confirming that the identity between TS‐GI sequences is around 92%.…”

Section: Discussionsupporting

confidence: 71%

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The high identity of the Trypanosoma cruziGroup‐I of trans‐sialidases points them as promising vaccine immunogens

Pacini,

Perdomini,

Bulfoni Balbi

et al. 2023

Proteins

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Trans‐sialidase (TS) superfamily of proteins comprises eight subgroups, being the proteins of Group‐I (TS‐GI) promising immunogens in vaccine approaches against Trypanosoma cruzi. Strikingly, TS‐GI antigenic variability among parasite lineages and their influence on vaccine development has not been previously analyzed. Here, a search in GenBank detects 49 TS‐GI indexed sequences, whereas the main infecting human different parasite discrete typing units (DTU) are represented. In silico comparison among these sequences indicate that they share an identity above 92%. Moreover, the antigenic regions (T‐cell and B‐cell epitopes) are conserved in most sequences or present amino acid substitutions that scarcely may alter the antigenicity. Additionally, since the generic term TS is usually used to refer to different immunogens of this broad family, a further in silico analysis of the TS‐GI‐derived fragments tested in preclinical vaccines was done to determine the coverage and identity among them, showing that overall amino acid identity of vaccine immunogens is high, but the segment coverage varies widely. Accordingly, strong H‐2K, H‐2I, and B‐cell epitopes are dissimilarly represented among vaccine TS‐derived fragments depending on the extension of the TG‐GI sequence used. Moreover, bioinformatic analysis detected a set of 150 T‐cell strong epitopes among the DTU‐indexed sequences that strongly bind human HLA‐I supertypes. In all currently reported experimental vaccines based on TS‐GI fragments, mapping these 150 epitopes showed that they are moderately represented. However, despite vaccine epitopes do not present all the substitutions observed in the DTUs, these regions of the proteins are equally recognized by the same HLAs. Interestingly, the predictions regarding global and South American population coverage estimated in these 150 epitopes are similar to the estimations in experimental vaccines when the complete sequence of TS‐GI is used as an antigen. In silico prediction also shows that a number of these MHC‐I restricted T‐cell strong epitopes could be also cross‐recognized by HLA‐I supertypes and H‐2Kb or H‐2Kd backgrounds, indicating that these mice may be used to improve and facilitate the development of new TS‐based vaccines and suggesting an immunogenic and protective potential in humans. Further molecular docking analyses were performed to strengthen these results. Taken together, different strategies that would cover more or eventually fully of these T‐cell and also B‐cell epitopes to reach a high level of coverage are considered.

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Section: Discussionsupporting

confidence: 74%

Section: Discussionsupporting

confidence: 71%

The high identity of the Trypanosoma cruziGroup‐I of trans‐sialidases points them as promising vaccine immunogens

Pacini,

Perdomini,

Bulfoni Balbi

et al. 2023

Proteins

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“…Unfortunately, there is no available vaccine for CD and little funding is available to support the development of a vaccine. Nevertheless, several antigens or live attenuated and even CRISPR-edited parasites have been tested as vaccine candidates in experimental models 55 . Among the vaccine candidates different groups have used the TS, ASP-2, Tc24, cruzipain, members of the TcG family and other antigens 10 – 21 .…”

Section: Discussionmentioning

confidence: 99%

ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease

et al. 2023

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Immunization with the Amastigote Surface Protein-2 (ASP-2) and Trans-sialidase (TS) antigens either in the form of recombinant protein, encoded in plasmids or human adenovirus 5 (hAd5) confers robust protection against various lineages of Trypanosoma cruzi. Herein we generated a chimeric protein containing the most immunogenic regions for T and B cells from TS and ASP-2 (TRASP) and evaluated its immunogenicity in comparison with our standard protocol of heterologous prime-boost using plasmids and hAd5. Mice immunized with TRASP protein associated to Poly-ICLC (Hiltonol) were highly resistant to challenge with T. cruzi, showing a large decrease in tissue parasitism, parasitemia and no lethality. This protection lasted for at least 3 months after the last boost of immunization, being equivalent to the protection induced by DNA/hAd5 protocol. TRASP induced high levels of T. cruzi-specific antibodies and IFNγ-producing T cells and protection was primarily mediated by CD8+ T cells and IFN-γ. We also evaluated the toxicity, immunogenicity, and efficacy of TRASP and DNA/hAd5 formulations in dogs. Mild collateral effects were detected at the site of vaccine inoculation. While the chimeric protein associated with Poly-ICLC induced high levels of antibodies and CD4+ T cell responses, the DNA/hAd5 induced no antibodies, but a strong CD8+ T cell response. Immunization with either vaccine protected dogs against challenge with T. cruzi. Despite the similar efficacy, we conclude that moving ahead with TRASP together with Hiltonol is advantageous over the DNA/hAd5 vaccine due to pre-existing immunity to the adenovirus vector, as well as the cost-benefit for development and large-scale production.

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“…Other successful CRISPR/Cas9-mediated genome editing methods used in T. cruzi involve transfection of in vitro transcribed sgRNA(s), either with constitutive expression of Cas9 (Burle-Caldas et al, 2022;Marcelino et al, 2023;Marek et al, 2021;Peng et al, 2014) or delivered as an RNP complex with recombinant Cas9 (Saenz-Garcia et al, 2021;Soares Medeiros et al, 2017). In the latter cases, the transient presence of the sgRNA (as well as Cas9 when it is transfected as RNP complex) is considered beneficial.…”

Section: Discussionmentioning

confidence: 99%

Gene editing of putative cAMP and Ca²⁺‐regulated proteins using an efficient cloning‐free CRISPR/Cas9 system in Trypanosoma cruzi

Chiurillo,

Ahmed,

González

et al. 2023

J Eukaryotic Microbiology

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Trypanosoma cruzi, the agent of Chagas disease, must adapt to a diversity of environmental conditions that it faces during its life cycle. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca2+) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, host cell invasion and cell bioenergetics. Although the use of CRISPR/Cas9 technology prompted reverse genetics approaches for functional analysis in T. cruzi, it is still necessary to expand the toolbox for genome editing in this parasite, as for example to perform multigene analysis. Here we used an efficient T7RNAP/Cas9 strategy to tag and delete three genes predicted to be involved in cAMP and Ca2+ signaling pathways: a putative Ca2+/calmodulin‐dependent protein kinase (CAMK), Flagellar Member 6 (FLAM6) and Cyclic nucleotide‐binding domain/C2 domain‐containing protein (CC2CP). We endogenously tagged these three genes and determined the subcellular localization of the tagged proteins. Furthermore, the strategy used to knockout these genes allows us to presume that TcCC2CP is an essential gene in T. cruzi epimastigotes. Our results will open new venues for future research on the role of these proteins in T. cruzi.

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Disruption of Active Trans-Sialidase Genes Impairs Egress from Mammalian Host Cells and Generates Highly Attenuated Trypanosoma cruzi Parasites

Cited by 11 publications

References 55 publications

The high identity of the Trypanosoma cruziGroup‐I of trans‐sialidases points them as promising vaccine immunogens

The high identity of the Trypanosoma cruziGroup‐I of trans‐sialidases points them as promising vaccine immunogens

ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease

Gene editing of putative cAMP and Ca²⁺‐regulated proteins using an efficient cloning‐free CRISPR/Cas9 system in Trypanosoma cruzi

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Disruption of Active Trans-Sialidase Genes Impairs Egress from Mammalian Host Cells and Generates Highly Attenuated Trypanosoma cruzi Parasites

Cited by 11 publications

References 55 publications

The high identity of the Trypanosoma cruziGroup‐I of trans‐sialidases points them as promising vaccine immunogens

The high identity of the Trypanosoma cruziGroup‐I of trans‐sialidases points them as promising vaccine immunogens

ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease

Gene editing of putative cAMP and Ca2+‐regulated proteins using an efficient cloning‐free CRISPR/Cas9 system in Trypanosoma cruzi

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Gene editing of putative cAMP and Ca²⁺‐regulated proteins using an efficient cloning‐free CRISPR/Cas9 system in Trypanosoma cruzi