Shotgun Label-Free Proteomic Analyses of the Oyster Parasite Perkinsusmarinus

Leibowitz, Márcia Pimenta; Tavares, Guilherme Campos; Pereira, Felipe Luiz; Perdigão, Cristiana; Azevedo, Vasco; Figueiredo, Henrique César Pereira

doi:10.14302/issn.2326-0793.jpgr-17-1571

Cited by 3 publications

(1 citation statement)

References 29 publications

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“…However, tetra-polyploidy pose a significant bottleneck for the assembly (El-Sayed et al, 2007;Bogema et al, 2020). Proteome studies identified that P. marinus possess 4,073 non-redundant hypothetical proteins, of which 36 and 27% are involved in metabolic and cellular processes, respectively (Marcia et al, 2017). Additionally, the rhoptry proteins such as serine-threonine kinases, protein phosphatases, proteosomes, and a virulent candidate merozoite surface protein 3, which are known to play a crucial role in parasite invasion and cell-cell communication during the invasion in P. falciparum were also identified in P. marinus.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

Yadavalli

Umeda

Waugh

et al. 2021

Front. Bioeng. Biotechnol.

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Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of pPmMOE[MOE1]:GFP and 25.0 × 106P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

Yadavalli

Umeda

Waugh

et al. 2021

Front. Bioeng. Biotechnol.

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Mutation of the Surface Layer Protein SlpB Has Pleiotropic Effects in the Probiotic Propionibacterium freudenreichii CIRM-BIA 129

et al. 2018

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Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese-ripening starter, and currently considered as an emerging probiotic. As an example, the P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties. Its consumption accordingly exerts healing effects in different animal models of colitis, suggesting a potent role in the context of inflammatory bowel diseases. This anti-inflammatory effect depends on surface layer proteins (SLPs). SLPs may be involved in key functions in probiotics, such as persistence within the gut, adhesion to host cells and mucus, or immunomodulation. Several SLPs coexist in P. freudenreichii CIRM-BIA 129 and mediate immunomodulation and adhesion. A mutant P. freudenreichii CIRM-BIA 129ΔslpB (CB129ΔslpB) strain was shown to exhibit decreased adhesion to intestinal epithelial cells. In the present study, we thoroughly analyzed the impact of this mutation on cellular properties. Firstly, we investigated alterations of surface properties in CB129ΔslpB. Surface extractable proteins, surface charges (ζ-potential) and surface hydrophobicity were affected by the mutation. Whole-cell proteomics, using high definition mass spectrometry, identified 1,288 quantifiable proteins in the wild-type strain, i.e., 53% of the theoretical proteome predicted according to P. freudenreichii CIRM-BIA 129 genome sequence. In the mutant strain, we detected 1,252 proteins, including 1,227 proteins in common with the wild-type strain. Comparative quantitative analysis revealed 97 proteins with significant differences between wild-type and mutant strains. These proteins are involved in various cellular process like signaling, metabolism, and DNA repair and replication. Finally, in silico analysis predicted that slpB gene is not part of an operon, thus not affecting the downstream genes after gene knockout. This study, in accordance with the various roles attributed in the literature to SLPs, revealed a pleiotropic effect of a single slpB mutation, in the probiotic P. freudenreichii. This suggests that SlpB may be at a central node of cellular processes and confirms that both nature and amount of SLPs, which are highly variable within the P. freudenreichii species, determine the probiotic abilities of strains.

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Colpodella sp. (ATCC 50594) Life Cycle: Myzocytosis and Possible Links to the Origin of Intracellular Parasitism

et al. 2021

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Colpodella species are free living bi-flagellated protists that prey on algae and bodonids in a process known as myzocytosis. Colpodella species are phylogenetically related to Apicomplexa. We investigated the life cycle of Colpodella sp. (ATCC 50594) to understand the timing, duration and the transition stages of Colpodella sp. (ATCC 50594). Sam-Yellowe’s trichrome stains for light microscopy, confocal and differential interference contrast (DIC) microscopy was performed to identify cell morphology and determine cross reactivity of Plasmodium species and Toxoplasma gondii specific antibodies against Colpodella sp. (ATCC 50594) proteins. The ultrastructure of Colpodella sp. (ATCC 50594) was investigated by transmission electron microscopy (TEM). The duration of Colpodella sp. (ATCC 50594) life cycle is thirty-six hours. Colpodella sp. (ATCC 50594) were most active between 20–28 h. Myzocytosis is initiated by attachment of the Colpodella sp. (ATCC 50594) pseudo-conoid to the cell surface of Parabodo caudatus, followed by an expansion of microtubules at the attachment site and aspiration of the prey’s cytoplasmic contents. A pre-cyst formed at the conclusion of feeding differentiates into a transient or resting cyst. Both DIC and TEM microscopy identified asynchronous and asymmetric mitosis in Colpodella sp. (ATCC 50594) cysts. Knowledge of the life cycle and stages of Colpodella sp. (ATCC 50594) will provide insights into the development of intracellular parasitism among the apicomplexa.

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Shotgun Label-Free Proteomic Analyses of the Oyster Parasite Perkinsusmarinus

Cited by 3 publications

References 29 publications

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

Mutation of the Surface Layer Protein SlpB Has Pleiotropic Effects in the Probiotic Propionibacterium freudenreichii CIRM-BIA 129

Colpodella sp. (ATCC 50594) Life Cycle: Myzocytosis and Possible Links to the Origin of Intracellular Parasitism

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