30th Annual Meeting of the German Society for Parasitology

Prof. Dr. apl.

Prof. Dr.

Physiological jump in erythrocyte redox potential during Plasmodium falciparum development occurs independent of the sickle cell trait

Parasite-Host Interaction

Molecular Parasitology

Abstract text 1. Introduction The redox state of the host-parasite unit has been hypothesized to play a central role for the fitness of the intraerythrocytic blood stages of the human malaria parasite Plasmodium falciparum. In particular, hemoglobinopathies have been suggested to cause a more oxidizing environment, thereby protecting from severe malaria. 2. Objectives We wanted to determine the redox state of the host-parasite unit during P. falciparum blood-stage development in wild-type (hemoglobin AA) or sickle trait (hemoglobin AS) erythrocytes. 3. Materials &amp; Methods We used parasite-encoded targeted variants of the redox-sensitive green-fluorescent protein 2 (roGFP2) for stage-dependent confocal microscopy redox measurements in wild-type or sickle trait erythrocytes. 4. Results Our roGFP2 single-cell measurements revealed a reducing steady-state redox potential of −304 ± 11 mV for the erythrocyte cytosol during ring-stage development and a rather sudden oxidation to −278 ± 12 mV during trophozoite-stage development around 28 h post invasion. There was no significant difference between wild-type or sickle trait erythrocytes regarding the stage dependence and the detected increase of the redox potential during the intraerythrocytic life cycle. The steady-state redox potential of the parasite cytosol, between −304 and −313 mV, was highly reducing throughout the life cycle. The redox potential in the parasitophorous vacuole at the interface between the secretory pathway and the erythrocyte was −284 ± 10 mV and remained stable during trophozoite-stage development with implications for the export of disulfide-containing proteins. 5. Conclusion Non-invasive roGFP2 measurements revealed a much more reducing steady-state redox potential of the erythrocyte and parasite cytosol than deduced from invasive glutathione measurements. Parasite-dependent modifications of its host cell result in a physiological +30 mV jump in erythrocyte redox potential during trophozoite-stage development that can be explained by a drastic efflux of reduced glutathione. Most important, there was no difference between the stage-dependent steady-state redox potentials of infected wild-type or sickle trait erythrocytes in contrast to the often iterated hypothesis that so-called oxidative stress protects from malaria [1]. [1] Haag M et al. 2022 Redox Biol 58: 102536.

Talk

Role of endothelial cells and miRNA in the pathogenesis of human malaria caused by Plasmodium falciparum infection

Abstract text Plasmodium falciparum is known to cause severe complications to the human host due to cytoadhesion of infected erythrocytes (IEs) to host endothelial cells (ECs). In recent years, evidence has accumulated that cytoadhesion is not the only explanation for the complications associated with malaria. Indirect cell-cell communication can occur through secretion of extracellular vesicles (EVs) into the circulation, containing proteins, DNA, mRNA and microRNAs (miRNAs). Secreted EVs can be taken up by recipient cells, and EVs miRNAs can alter the gene expression in these cells. EVs derived from IEs are known to contain both parasite proteins and human miRNAs. miRNAs can block mRNA by directing degradation through a protein complex or by destabilization of the messenger. Altered miRNA profiles have been described for many types of infections. The miRNA profiles of human ECs during malaria pathogenesis have not yet been studied. We postulate that dysfunction of ECs may play an important role in the prognosis of severe malaria complications. Certain miRNA candidates might control these events by affecting gene expression within the ECs. Methods: In this study, we investigated if miRNAs play a role in pathogenesis during P. falciparum infection. First, we analysed the miRNA expression profile in the IEs as well as their secreted vesicles using NGS sequencing. Second, we characterised the mRNA and miRNA expression profiles of human brain ECs exposed to co-incubation with P. falciparum IEs. Results: NGS and bioinformatic analysis showed that 20 miRNA candidates were differentially expressed in the vesicles secreted from the IEs compared to the non-infected ones. In addition, 8 mature microRNA candidates were differentially downregulated in the vesicles secreted from the IEs compared to the non-infected ones. Upon co-incubation of human brain ECs with P. falciparum under low shear stress 8 mature microRNA candidates were differentially expressed. Conclusion: Our experiments showed that the microRNA profiles of the human erythrocytes and ECs are affected during P. falciparum infection. Mapping these changes might give us more clues about the inflammatory pathways that are turned on during the severe outcomes of the infection.

Human brain endothelial cell activation during Plasmodium falciparum infection

Abstract text Introduction: Sequestration of Plasmodium falciparum in human microvessels occurs due to an interaction between a parasite ligand and host endothelial cell (EC) receptor in a process called cytoadhesion. Cytoadhesion results in microvascular obstruction, impaired perfusion, hypoxia and finally organ failure. PfEMP1 (Plasmodium falciparum erythrocyte membrane protein-1) is encoded by approximately 60 var genes per parasite genome and is considered the most important surface antigen of P. falciparum-infected erythrocytes (IEs). Objectives: Characterization of the tropism of PfEMP1 for different EC receptors and the subsequent immune response triggered by cytoadhesion. Materials &amp; methods: In this study, we enriched P. falciparum isolates IT4 over resting or TNF-α-activated human brain microvascular endothelial cells (HBMECs). Furthermore, we stimulated HBMECs with enriched parasites for 4 hours and 8 hours separately and characterized the HBMECs transcriptomic changes. Moreover, in order to simulate the in vivo condition we stimulated HBMECs with IEs under defined shear stress. mRNA and miRNA profiles of stimulated HBMECs were characterized by NGS. Results: Transcriptome analysis indicated that 101 differentially expressed genes (DEGs) were identified in HBMECs after 4 hours of stimulation and 51 DEGs were identified in HBMECs after 8 hours of stimulation. Reactome analysis of DEGs revealed the enrichment in antigen presentation and interferon gamma signal pathway. Interestingly, we found the mRNA expression level of oligoadenylate synthase (OAS) gene family increased significantly after 4 hours of stimulation. Conclusion: By comparing the transcriptomes of HBMECs stimulated for 4 h and 8 h, we observed upregulation of immunoregulatory genes within the first 4 h of stimulation. However, these immune response declines by reaching 8 h of coincubation. Besides, shear stress stimulation may affect the mRNA and miRNA profiles of HBMECs.

Reverse genetics reveals new genes involved in host cell modification by the malaria parasite P. falciparum

Abstract text Following invasion of human erythroyctes, the malaria parasite P. falciaprum massively changes the biochemical and biophysical properties of the host cell. This process directly and indirectly underpins the pathology associated with malaria infection, and is driven by a large number of proteins encoded by the parasite, and which are transported to the host cell. To understand to role specific exported proteins play in host cell modification, we initiated a medium-scale reverse genetic analysis of a number of genes of interest (GOI) we believed may be involved in this process. Over the course of 6 years we generated 76 genetically modified parasite lines using a number of techniques. In contrast to previous studies, we find that a large majority of the GOI (14/18) are resistant to both direct inactivation, or introduction of the GFP coding sequence. Use of a smaller tag, 3xHA, allowed us to generate 5 further parasite lines which also included a glmS ribozyme to allow modulation of protein levels. Additionally, we obtained 2 knockout lines. A detailed analysis of three parasite lines revealed involvement of the GOI/POI in a number of host cell modification processes and uncovered novel mutant phenotypes never before reported. Further analysis of one specific parasite line lacking an exported J-domain protein highlighted a potential role for residual human HSP70 proteins in host cell modification processes, the first time this has been experimentally demonstrated. Taken together, our data highlight the importance of exported proteins for parasite growth, survival and pathology in the human host and host cell, and underpin the complex nature of the host parasite interaction at a molecular level.

The role of host Rab GTPases during the intracellular liver stage of Plasmodium berghei

Abstract text 1. Introduction The liver stage of Plasmodium berghei is marked by extensive growth of a few sporozoites to produce thousands of progeny and establish the blood-stage infection. To ensure the fast growth, the parasite needs to acquire nutrients from within the host cells. Although late endosomes and the Golgi apparatus have been shown to associate with the parasitophorous vacuole in which the parasite resides, we hypothesize that additional host membrane compartments contribute to a successful liver stage infection. 2. Objectives In our study, we wanted to identify membrane trafficking pathways that interact with the parasitophorous vacuole. To this end, we investigated the localization of host Rab GTPases during the infection. Rab GTPases are key regulators of specific trafficking events, which might be altered by the parasite. Other intracellular parasites have been shown to target specific Rab proteins to ensure their survival. 3. Materials &amp; Methods We infected hepatoma cells expressing tagged Rab GTPases and infected them with P. berghei. Through microscopy, we observed the localization of the different Rab proteins in infected cells at various time points during the infection. With additional immunofluorescence staining we determined the arrangement of additional host and parasite structures. 4. Results Our initial screening of different Rab proteins showed association of Rab-labelled membranes with the parasite. Amongst predicted candidates like late endosomes, additional specific membrane compartments were observed accumulating in close proximity to the parasitophorous vacuole. Intriguingly, we identified a host Rab protein that not only accumulates near the parasite but is present on the parasitophorous vacuole. It showed clear colocalization with the parasitophorous vacuole membrane marker UIS4 throughout the development of the parasite. 5. Conclusions Our data demonstrate that specific host membranes can be found in close proximity to the parasite-containing vacuole during infection. The presence of a host Rab on the membrane surrounding the parasite suggests this particular candidate is relevant to the infection. The localization of this Rab and of the neighboring host vesicles suggests some interplay between host Rab GTPases and the growing parasites.

Session XV
Parasite-Host-Interactions III – Protozoa 1

Appointment

Chairs

Programme

Session XVParasite-Host-Interactions III – Protozoa 1

Appointment

Chairs

Programme

Session XV
Parasite-Host-Interactions III – Protozoa 1