Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

Moonah, Shannon; Sanders, Natalie G.; Persichetti, Jason K.; Sullivan, David J.

doi:10.1128/ec.00088-14

Cited by 14 publications

(11 citation statements)

References 38 publications

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“…Our results allowed us to observe a remarkable reduction of lysis in A549 cells by adding PEG of 3,350 Da, whereas the damage to the cell membrane appeared to occur through the action of a pore-forming protein present in the A. dowii venom and F1 fraction. These results are consistent with the fact that PEG can inhibit the hemolysis produced by pore-forming proteins [68].…”

Section: Discussionsupporting

confidence: 90%

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Ramírez-Carreto

Pérez-García

Salazar-García

et al. 2019

J. Venom. Anim. Toxins incl. Trop. Dis

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Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU 50 ) was determined in hemolysis assays using erythrocytes from sheep (HU 50 = 10.7 ± 0.2 μg), goat (HU 50 = 13.2 ± 0.3 μg), rabbit (HU 50 = 34.7 ± 0.5 μg), and human (HU 50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC 50 ) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.

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Section: Discussionsupporting

confidence: 90%

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Ramírez-Carreto

Pérez-García

Salazar-García

et al. 2019

J. Venom. Anim. Toxins incl. Trop. Dis

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“…The erythrocyte suspension (0.1 mL) was incubated with 50 μg · mL −1 of Hcp‐Cj for 4 hrs at 37°C. The samples were then centrifuged at 1000 × g for 5 min and optical density of the supernatant at 550 nm was recorded as an indicator of hemolysis degree . Cytotoxicity of purified Hcp‐Cj protein against HepG2 (liver cancer cells) was evaluated using sulforhodamine B (SRB) assay .…”

Section: Methodsmentioning

confidence: 99%

Structural basis for the pathogenesis of Campylobacter jejuni Hcp1, a structural and effector protein of the Type VI Secretion System

et al. 2018

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The Type VI Secretion System (T6SS) provides enhanced virulence to Campylobacter jejuni and has been associated with a high incidence of bloody diarrhea. The hemolysin-coregulated protein (Hcp)-the hallmark of the T6SS-can act as a structural and effector protein. Unlike other T6SS-harboring bacteria, which possess multiple Hcp proteins each performing different functions, C. jejuni possesses only one Hcp protein, and its structural and functional role has not been elucidated previously. Here, we report the structure and functional studies of Hcp from C. jejuni. We found similarities between the hexameric ring structure of Hcp-Cj and that of Hcp3 from Pseudomonas aeruginosa. Through functional studies, we showed two roles for Hcp-Cj that is, in cytotoxicity toward HepG2 cells and in biofilm formation in C. jejuni. In structure-based mutational analyses, we showed that an Arg-to-Ala mutation at position 30 within the extended loop results in a significant decrease in cytotoxicity, suggesting a role for this loop in binding to the host cell. However, this mutation does not affect its biofilm formation function. Collectively, this study supports the dual role of Hcp-Cj as a structural and effector protein in C. jejuni.

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“…These determinations have both increased in throughput and become more economical with the introduction of a 96‐well format (Richards et al ., ) and the development of a fluorescence‐based assay for measuring the transport of substrates (van Schalkwyk et al ., ). Thus far, 19 Plasmodium transporters have been expressed and characterised in this system, including PfHT1 (Woodrow et al ., ; Woodrow, Burchmore & Krishna, ), PfENT1 (Carter et al ., ; Parker et al ., ), PfENT4 (Frame et al ., ), the P i :Na + symporter PfPiT (Saliba et al ., ), PfAQP (Hansen et al ., ; Promeneur et al ., ), PfCRT (Martin et al ., ; Summers et al ., ; Richards et al ., ; van Schalkwyk et al ., ), the formate‐lactate channel PfFNT (Marchetti et al ., ; Hapuarachchi et al ., ), PfCAX (Rotmann et al ., ), the cationic amino acid transporter PbNPT1 (Rajendran et al ., ), and PfHLYIII (Moonah et al ., ). Nevertheless, not all attempts to express Plasmodium transporters in the Xenopus oocyte have been successful (Henry et al ., ; Cobbold, Llinas & Kirk, ) and in a few cases the signal‐to‐background ratio was very modest, such that it prevented direct and robust measurements of the transporter's activity [e.g.…”

Section: The Plasmodium Transportomementioning

confidence: 97%

The transportome of the malaria parasite

Martin

2019

Biological Reviews

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Membrane transport proteins, also known as transporters, control the movement of ions, nutrients, metabolites, and waste products across the membranes of a cell and are central to its biology. Proteins of this type also serve as drug targets and are key players in the phenomenon of drug resistance. The malaria parasite has a relatively reduced transportome, with only approximately 2.5% of its genes encoding transporters. Even so, assigning functions and physiological roles to these proteins, and ascertaining their contributions to drug action and drug resistance, has been very challenging. This review presents a detailed critique and synthesis of the disruption phenotypes, protein subcellular localisations, protein functions (observed or predicted), and links to antimalarial drug resistance for each of the parasite's transporter genes. The breadth and depth of the gene disruption data are particularly impressive, with at least one phenotype determined in the parasite's asexual blood stage for each transporter gene, and multiple phenotypes available for 76% of the genes. Analysis of the curated data set revealed there to be relatively little redundancy in the Plasmodium transportome; almost two‐thirds of the parasite's transporter genes are essential or required for normal growth in the asexual blood stage of the parasite, and this proportion increased to 78% when the disruption phenotypes available for the other parasite life stages were included in the analysis. These observations, together with the finding that 22% of the transportome is implicated in the parasite's resistance to existing antimalarials and/or drugs within the development pipeline, indicate that transporters are likely to serve, or are already serving, as drug targets. Integration of the different biological and bioinformatic data sets also enabled the selection of candidates for transport processes known to be essential for parasite survival, but for which the underlying proteins have thus far remained undiscovered. These include potential transporters of pantothenate, isoleucine, or isopentenyl diphosphate, as well as putative anion‐selective channels that may serve as the pore component of the parasite's ‘new permeation pathways’. Other novel insights into the parasite's biology included the identification of transporters for the potential development of antimalarial treatments, transmission‐blocking drugs, prophylactics, and genetically attenuated vaccines. The syntheses presented herein set a foundation for elucidating the functions and physiological roles of key members of the Plasmodium transportome and, ultimately, to explore and realise their potential as therapeutic targets.

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Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

Cited by 14 publications

References 38 publications

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Structural basis for the pathogenesis of Campylobacter jejuni Hcp1, a structural and effector protein of the Type VI Secretion System

The transportome of the malaria parasite

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