30th Annual Meeting of the German Society for Parasitology

Prof.

Prof. Dr.

A proteomic view on the egress-related vesicles of malaria gametocytes [Exit-WS]

Abstract text Introduction Gametocyte egress from the host erythrocyte follows an inside-out mode during which the membrane of the parasitophorous vacuole ruptures prior to the erythrocyte membrane. Membrane rupture requires the exocytosis of specialized secretory vesicles of the parasites; i.e. the osmiophilic bodies (OBs) involved in rupture of the parasitophorous vacuole membrane, and vesicles, termed g-exonemes, that harbor the perforin-like protein PPLP2 needed for erythrocyte lysis. While several OB proteins are known, like G377 and MDV1/Peg3, the protein composition of the g-exonemes remains unclear. Material/Method We employed BioID analyses to unveil the interactomes of OBs and g-exonemes. BioID is a proximity labelling application for detecting putative protein-protein interactions. The method uses a promiscuous biotin ligase (BirA), fused to a bait protein that can be induced to biotinylate interacting and proximal proteins during a defined labelling period by biotin supplementation. TurboID represents an optimized version of the BioID, which uses a biotin ligase with higher activity and therefore decreases the labelling period dramatically. Biotinylated proteins can be isolated, analyzed via mass spectrometry (MS) and investigated as candidate interactors with the bait protein or as constituents within a subcellular domain. Results Here, we used high-resolution imaging and BioID methods to study the two types of egress vesicles in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the g-exonemes and that the two types of vesicles exhibit distinct proteomes. In addition to known egress-related proteins, our analyses revealed novel markers of OBs and g-exonemes, including components involved in protein processing and vesicle trafficking. Discussion Our data provide insight into the immense molecular machinery required for the inside-out egress of P. falciparum gametocytes.

Molecular Parasitology

Parasite-Host Interaction

Talk

Intracellular Leishmania major parasites drive host cell death and cell-to-cell transfer depending on the parasite proliferation rate (Exit WS)

Abstract text Introduction: The virulence of the intracellular pathogens relies largely on the ability to survive and replicate within phagocytes, but also to be released and transferred into new host cells. Recent findings suggest that release of Leishmania parasites from infected host cells is strictly regulated, with the mechanisms involved still to be determined. Objectives: To study Leishmania major (L. major) transmission from infected monocytes to newly recruited cells in the context of infected host cell death. Materials &amp; methods: We quantified L. major transfer and uptake of host cell material together with the parasite into adoptively transferred cells in vivo using intravital 2-photon microscopy analysis and flow cytometry analysis of CD11c-YFP reporter mice. Also, we visualized cell death by live cell imaging, and by intravital 2-photon imaging of a fluorescence resonance energy transfer-based (FRET-based) cell-death-biosensor. Results: We observed increased original host cell material uptake in infected as compared to uninfected cells. This suggests that original host cells can be phagocytosed by new cells together with the parasites. Also, infected host cells show signs of cell death before parasite transfer in vitro and show more Caspase-3 activity, indicating apoptosis, compared to uninfected cells in vivo. Lastly, we detected higher L. major proliferation in TUNEL+ compared to TUNEL- murine intraperitoneal macrophages using a photoconversion-based proliferation biosensor in vitro. In addition, our data indicate that high parasite proliferation is associated to shorter infection time of the infected host in vitro. Conclusion: These data suggest that a high L. major proliferation rate induces cell death and thereby drives the dissemination of the pathogen to new phagocytes.

Parasite Immunology

3D electron tomography of Plasmodium – Exit WS

Abstract text The life cycle of Plasmodium includes a short phase of sexual reproduction in the mosquito. Immediately after uptake of the blood meal gametocytes are activated and start to differentiate. Male gametocytes produce eight male flagellated microgametes that egress from the erythrocyte with the help of axonemal movement. Axonemes are microtubule-based cytoskeletal structures essential for ciliary beating and flagellar motility. Canonical axonemes consist of nine doublet microtubules which are linked with dynein motors and nexin linkers and surround two central microtubules. Axonemal microtubules can polymerize from basal bodies, these are extracellular parts of the bipartite microtubule organizing center (MTOC) that connect the axoneme through a nuclear pore with the spindle. Here, we investigate gamete formation in wild-type and mutant parasites. The mutants lack the merozoite thrombospondin-related anonymous protein (MTRAP) and do not egress from the infected cell. Cells were fixated at different time points and serial sections were imaged by transmission electron microscopy to generate several 3D volumes. 3D datasets were segmented by surface rendering to generate 3D models. The generated 3D models reveal that Plasmodium axonemes are formed by a zipping process, cause nuclear fractioning and can feature several aberrations. We imaged both the connection and apparent rupture of the bipartite MTOC into basal bodies and the spindle pole, suggesting a model of axoneme formation. Overall, our data reveal that an astonishing level of inaccuracy stills allows rapid axoneme assembly and efficient gamete motility and fertilization.

L. major hijacks hypoinflammatory monocytes to perpetuate infection in an interleukin-7 dependent feedback loop (Exit WS)

Abstract text Introduction: Leishmania major (L. major) infection of the skin is marked by large heterogeneity in both pathogen physiological states and immune cell populations recruited to the infection site. Our previous work suggested that pathogen proliferation and cell-to-cell transfer is preferentially occurring in distinct phagocyte subpopulations. However, the link between pathogen physiology with the activation of infected host cells remains poorly understood. Objectives: To investigate the dynamics of the recruitment, activation and deployment of cellular defence mechanisms of phagocytes recruited to the site of L. major infection in the context of differential pathogen proliferation rates. Materials &amp; Methods: Using an in vivo biosensor for L. major proliferation in combination with single cell RNAseq, we investigated transcriptional profiles of infected phagocytes isolated from infected skin dependently of the intracellular pathogen proliferation rate. Bone marrow chimeric mice, cell-specific depletion and cytokine blockade were used to characterize candidate cells and molecules with a possible impact on pathogen control using flow cytometry and multiparameter microscopy. Results: We observed that fast versus slow L. major proliferation is linked to clearly defined infected cell populations with differential gene expression profiles. In particular, we identifed a distinct monocyte subpopulation, termed Lo3, which harboured low proliferating L. major and exhibited a hypoinflammatory phenotype. Furthermore, our data suggest that Interleukin-7 (IL-7), which is produced in the tissue upon induction of the immune response, acts as a feedback mechanism which restricts the immune response via the Lo3 monocytes. In particular, depletion of Lo3 monocytes, transient inhibition of IL-7, or absence of the IL-7 receptor on phagocytes drastically enhanced L. major clearance via an enforced T cell response. Conclusion: L. major hijacks an immunoregulatory feedback mechanism which is mediated via IL-7 and hypoinflammatory monocytes which are specifically characterized by the infection of low proliferating pathogen. These cells seem to dampen the adaptive immune effector response and and perpetuate pathogen persistence in the tissue.

A transdifferentiated human macrophage-like cell line to elucidate the cell-to-cell spread of Leishmania major (Exit WS)

Abstract text Leishmania is the causative agent of the tropical neglected disease leishmaniasis and it infects macrophages as its definitive host cell. While the routes of initial infection are well understood, the mechanism of parasite spread to other cells remains vague, partially due to lack of genome editing methods in human macrophages. Hence, we set up an infection model in BLaER1 cells, which can be edited on a genomic level and transdifferentiated into a macrophage-like phenotype. To proof their suitability as infection model, we first examined the immunophenotype of BLaER1 cells and found it to be comparable with macrophages. We also confirmed their susceptibility for the infection with Leishmania promastigotes using confocal microscopy. The infection rate and cytokine responses were very similar when comparing infected BLaER1 and macrophages. Focusing on host cell death as a possible mechanism of the cell-to-cell spread of Leishmania, we induced pyroptosis in BLaER1 and used BLaER1 GSDMD knockout cells as a control. As expected, we found BLaER1 GSDMD knockout cells to be more resistant to pyroptosis induction than BLaER1 wild type cells based on the duration until IL-1ß release and cell death. Live cell imaging revealed that induction of pyroptosis results in release of parasites from host cells and it is clearly delayed in the BLaER1 GSDMD knockout cells. Finally, we quantified the rate of parasite transfer to new host cells in a co-incubation assay. Here, the transfer rate was significantly increased in a pyroptosis dependent manner. Taken together, these findings demonstrate the suitability of BLaER1 cells as a Leishmania infection model to study cell to cell spread on a cell biological level and suggests pyroptosis as a cell death mechanism that can contribute to the spread of the parasites to new host cells.

The novel conoidal methyltransferase PCKMT is responsible for the initiation of motility in concert with other conoidal proteins in Toxoplasma gondii (Exit WS)

Abstract text Toxoplasma gondii is an obligate intracellular parasite critically depending on invasion and egress from infected cells to ensure survival. A crucial structure for these processes is the apical complex which is highly conserved in most apicomplexans. Within this structure, the conoid, constructed by tubulin fibers, and preconoidal rings are crucial for parasite motility, invasion, and egress. Recently, we identify two proteins that localise to the preconoidal rings in two independent splitCas9 phenotypic screens: CGP and PCKMT. These proteins appear to be responsible for the initiation of motility after egress signalling. While CGP is an important structural protein for the stability of the preconoidal rings and localisation of many proteins at this structure, PCKMT, a putative lysine methyltransferase, is critical for the recruitment of formin 1 (FRM1) to the conoid. To further identify interacting proteins of CGP, FRM1 and PCKMT, we performed proximity labeling experiments using TurboID tags. These assays revealed that FRM1, CGP and PCKMT possibly interact directly with each other. Other important components for motility such as GAC and AKMT were also identified, together with additional, hypothetical identified proteins. AKMT is a methyltransferase that has been shown to affect the recruitment of GAC to the apical complex and therefore is required for motility. We hypothesise that the coordinated activation of methylation by AKMT and PCKMT is not only required for the recruitment of GAC and FRM-1 to the conoid, but a prerequisite for the initiation and maintenance of gliding motility. We currently compare the methylation of candidate proteins in presence and absence of AKMT and PCKMT respectively in an attempt to decipher the regulation of motility by methylation.

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