The Need for Community Standards to Enable Accurate Comparison of Glycoproteomics Algorithm Performance

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.

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Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication

PLoS Pathogens ◽

10.1371/journal.ppat.1009399 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009399

Author(s):

Srinivasan Ramakrishnan ◽

Linn M. Unger ◽

Rodrigo P. Baptista ◽

Teresa Cruz-Bustos ◽

Roberto Docampo

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Cell Invasion ◽

Critical Role ◽

Protein Glycosylation ◽

Growth Defect ◽

Mammalian Host ◽

Host Cell Invasion ◽

Intracellular Replication ◽

Parasite Life Cycle

Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.

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Faculty Opinions recommendation of The IL-12 Response of Primary Human Dendritic Cells and Monocytes to Toxoplasma gondii Is Stimulated by Phagocytosis of Live Parasites Rather Than Host Cell Invasion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725957291.793518980 ◽

2016 ◽

Author(s):

Frank Seeber

Keyword(s):

Dendritic Cells ◽

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Host Cell Invasion ◽

Human Dendritic Cells

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Faculty Opinions recommendation of Aspergillus fumigatus CalA binds to integrin α5β1 and mediates host cell invasion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727157720.793531362 ◽

2017 ◽

Author(s):

Leah Cowen ◽

Teresa O’Meara

Keyword(s):

Aspergillus Fumigatus ◽

Host Cell ◽

Cell Invasion ◽

Host Cell Invasion ◽

Integrin Α5β1

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A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion

PLoS ONE ◽

10.1371/journal.pone.0161965 ◽

2016 ◽

Vol 11 (9) ◽

pp. e0161965 ◽

Cited By ~ 10

Author(s):

Daniel Andritschke ◽

Sabrina Dilling ◽

Mario Emmenlauer ◽

Tobias Welz ◽

Fabian Schmich ◽

...

Keyword(s):

Salmonella Typhimurium ◽

Host Cell ◽

Cell Invasion ◽

Host Cell Invasion ◽

Sirna Screen ◽

Genome Wide ◽

A Genome

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Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

Acta Parasitologica ◽

10.2478/s11686-010-0056-9 ◽

2010 ◽

Vol 55 (4) ◽

Cited By ~ 9

Author(s):

Adriana Aguado-Martínez ◽

Gema Álvarez-García ◽

Gereon Schares ◽

Verónica Risco-Castillo ◽

Aurora Fernández-García ◽

...

Keyword(s):

Monoclonal Antibody ◽

Host Cell ◽

Cell Invasion ◽

Neospora Caninum ◽

Parasitophorous Vacuole ◽

Membrane Surface ◽

Chronic Phase ◽

Host Cell Invasion ◽

Invasion Mechanisms ◽

The Matrix

AbstractNeospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

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1,3,4-Thiadiazoles Effectively Inhibit Proliferation of Toxoplasma gondii

Cells ◽

10.3390/cells10051053 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1053

Author(s):

Lidia Węglińska ◽

Adrian Bekier ◽

Katarzyna Dzitko ◽

Barbara Pacholczyk-Sienicka ◽

Łukasz Albrecht ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Direct Action ◽

Human Fibroblasts ◽

Imidazole Ring ◽

Host Cells ◽

In Vitro Assays ◽

Host Cell Invasion

Congenital and acquired toxoplasmosis caused by the food- and water-born parasite Toxoplasma gondii (T. gondii) is one of the most prevalent zoonotic infection of global importance. T. gondii is an obligate intracellular parasite with limited capacity for extracellular survival, thus a successful, efficient and robust host cell invasion process is crucial for its survival, proliferation and transmission. In this study, we screened a series of novel 1,3,4-thiadiazole-2-halophenylamines functionalized at the C5 position with the imidazole ring (1b–12b) for their effects on T. gondii host cell invasion and proliferation. To achieve this goal, these compounds were initially subjected to in vitro assays to assess their cytotoxicity on human fibroblasts and then antiparasitic efficacy. Results showed that all of them compare favorably to control drugs sulfadiazine and trimethoprim in terms of T. gondii growth inhibition (IC50) and selectivity toward the parasite, expressed as selectivity index (SI). Subsequently, the most potent of them with meta-fluoro 2b, meta-chloro 5b, meta-bromo 8b, meta-iodo 11b and para-iodo 12b substitution were tested for their efficacy in inhibition of tachyzoites invasion and subsequent proliferation by direct action on established intracellular infection. All the compounds significantly inhibited the parasite invasion and intracellular proliferation via direct action on both tachyzoites and parasitophorous vacuoles formation. The most effective was para-iodo derivative 12b that caused reduction in the percentage of infected host cells by 44% and number of tachyzoites per vacuole by 93% compared to non-treated host cells. Collectively, these studies indicate that 1,3,4-thiadiazoles 1b–12b, especially 12b with IC50 of 4.70 µg/mL and SI of 20.89, could be considered as early hit compounds for future design and synthesis of anti-Toxoplasma agents that effectively and selectively block the invasion and subsequent proliferation of T. gondii into host cells.

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