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Paul Walther (2011-2017)

Leishmania major promastigote entry of the of the autophagic compartment and amastigote escape from the parasitophorous vacuole (joint project with Dr. Ger van Zandbergen)

To properly investigate Leishmania disease initiation and propagation, infective promastigote stages and amastigote stages as well as human host macrophages (MF) are to be the focus of investigation. During the first three years we setup a new lab and research group in the Paul Ehrlich Institute, we acquired additional funding for a Zeiss Live 7 high speed confocal imaging system and a Wohlwend High Pressure Freezing Unit 

(i) we visualized the L. major containing parasitophorous vacuoles in MF using state of the art high pressure freezing and electron tomography focusing on promastigote interactions with autophagy compartments and amastigote escape from the parasitophorous vacuoles (ii) we down regulated autophagy and lysosomal maturation in MF (iii) we established a new assay to express eGFP-LC3 and LAMP2-mCherry using lentiviral transduction of primary human MF for dynamic imaging. We successfully adapted our high pressure freezing preparation protocol, using transparent sapphire discs as specimen support and prepared the samples for STEM tomography. During the second 3-year funding period (i) we aim to dissect the molecular basis leading to promastigote entry of the autophagic pathway and amastigote escape in the cytoplasm. (ii) We will further develop high pressure freezing based electron microscopy and improve 3D electron microscopy. We will, therefore, investigate Leishmania infected LC3 and/or LAMP2 transduced macrophages by serial sectioning TEM as well as by FIB-SEM tomography to correlate Leishmania compartment proteins analyzed confocally, with the 3D-EM data. These methods allow for tomographic reconstruction of both compartment target proteins and infected MF. (iii) To increase our understanding of Leishmania propagation in human host cells, we will apply this technique for new target proteins coming from a mass spectrometry screen of isolated Leishmania compartments.




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