evaluation-of-extracellular-vesicle-function-during-malaria-infection

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

University of Fribourg

Malaria remains one of the greatest public health challenges worldwide, particularly in sub-Saharan Africa. The clinical outcome of individuals infected with Plasmodium falciparum parasites depends on many factors including host systemic inflammatory responses, parasite sequestration in tissues and vascular dysfunction. Production of pro-inflammatory cytokines and chemokines promotes endothelial activation as well as recruitment and infiltration of inflammatory cells, which in turn triggers further endothelial cell activation and parasite sequestration. Inflammatory responses are triggered in part by bioactive parasite products such as hemozoin and infected red blood cell-derived extracellular vesicles (iRBC-derived EVs). Here we demonstrate that such EVs contain functional miRNA-Argonaute 2 complexes that are derived from the host RBC. Moreover, we show that EVs are efficiently internalized by endothelial cells, where the miRNA-Argonaute 2 complexes modulate target gene expression and barrier properties. Altogether, these findings provide a mechanistic link between EVs and vascular dysfunction during malaria infection.

Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria.

Bacterial pathogens represent a constant threat to human health that was exacerbated in recent years by a dramatic increase of strains resistant to last resort antibiotics. The immune system of higher vertebrates generally evolved several efficient innate and adaptive mechanisms to fight ubiquitous bacterial pathogens. Among those mechanisms, immune proteases were recognized to contribute essentially to antibacterial immune defense. The effector serine proteases of the adaptive immune system, the granzymes, exert potent antimicrobial activity when they are delivered into the bacterial cytosol by prokaryotic membrane disrupting proteins, such as granulysin.In this chapter, we are detailing experimental protocols to study the synergistic cytotoxic effects of human granzymes and granulysin on extracellular as well as on intracellular bacterial pathogens in vitro. In addition, we provide a simple and fast-forward method to biochemically purify native cytotoxic effector molecules necessary to perform this kind of investigations.

Killing Bacteria with Cytotoxic Effector Proteins of Human Killer Immune Cells: Granzymes, Granulysin, and Perforin.

Growing attention is drawn toward the role of extracellular vesicles (EVs) in infectious diseases. EVs, which are small vesicles released by cells, are involved in cellular communication, immune regulation, and pathogenesis. EVs act as messenger carrying functional cargoes, including RNA, DNA, lipids and proteins from a donor cell to regulate the function of a recipient cell. In malaria, EVs play a key role in regulating the progression from the blood to the transmission stage by promoting the switch between asexual and sexual stages that are taken up by mosquitoes. In addition to their role in parasite communication, EVs modulate the immune system and regulate endothelial cell function.In this chapter, we describe protocols to isolate, purify and characterize EVs derived from Plasmodium falciparum infected red blood cell culture.

Production and Characterization of Extracellular Vesicles in Malaria.

Bone infections are difficult to treat and can lead to severe tissue destruction. Acute bone infections are usually caused by Staphylococcus aureus. Osteoclasts, which belong to the monocyte/macrophage lineage, are the key cells in bone infections. They are not well equipped for killing bacteria and may serve as a reservoir for bacterial pathogens. Silver has been known for centuries for its bactericidal activity. Here, we investigated the bactericidal effects of nano-silver particles in bacteria infected human osteoclasts. We found that nano-silver in per se non-toxic concentration enhanced the bactericidal activity in osteoclasts against intracellular Methicillin-resistant, virulent Staphylococcus aureus. The reduced bacterial survival in nano-silver pretreated cells correlated with increased reactive oxygen responses towards the invading pathogens. Overall, these results indicate that nano-silver compounds should be considered as an effective treatment and prevention option for bacterial bone and orthopedic implant infections.

Silver-nanoparticles increase bactericidal activity and radical oxygen responses against bacterial pathogens in human osteoclasts.

The parasite Plasmodium falciparum causes the most severe form of malaria. Cell communication between parasites is an important mechanism to control population density and differentiation. The infected red blood cells (iRBCs) release small extracellular vesicles (EVs) that transfer cargoes between cells. The EVs synchronize the differentiation of the asexual parasites into gametocytes to initiate the transmission to the mosquito. Beside their role in parasite communication, EVs regulate vascular function. So far, the exact cargoes responsible for cellular communication remain unknown. We isolated EVs from cultured iRBCs to determine their small RNA content. We identified several types of human and plasmodial regulatory RNAs. While the miRNAs and tRNA-derived fragments were the most abundant human RNAs, we also found Y-RNAs, vault RNAs, snoRNAs and piRNAs. Interestingly, we found about 120 plasmodial RNAs, including mRNAs coding for exported proteins and proteins involved in drug resistance, as well as non-coding RNAs, such as rRNAs, small nuclear (snRNAs) and tRNAs. These data show, that iRBC-EVs carry small regulatory RNAs. A role in cellular communication is possible since the RNAs were transferred to endothelial cells. Furthermore, the presence of Plasmodium RNAs, in EVs suggests that they may be used as biomarker to track and detect disease.

Article	Year
Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria. Nature communications\| PubMed ID: 27721445	2016
Killing Bacteria with Cytotoxic Effector Proteins of Human Killer Immune Cells: Granzymes, Granulysin, and Perforin. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27914086	2017
Production and Characterization of Extracellular Vesicles in Malaria. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28828673	2017
Silver-nanoparticles increase bactericidal activity and radical oxygen responses against bacterial pathogens in human osteoclasts. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 29155361	2017
Malaria infected red blood cells release small regulatory RNAs through extracellular vesicles. Scientific reports\| PubMed ID: 29343745	2018

Pierre-Yves Mantel

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