Plasmodium Secretion Induces Hepatocyte Lysosome Exocytosis and Promotes Parasite Entry

Abstract

The invasion of a suitable host hepatocyte by Plasmodium sporozoites is an essential step in malaria infection. We demonstrate that in infected hepatocytes, lysosomes are redistributed away from the nucleus, and surface exposure of lysosome-associated membrane protein 1 (LAMP1) is increased. Lysosome exocytosis in infected cells occurs independently of sporozoite traversal. Instead, a sporozoite-secreted factor is sufficient for the process. Knockdown of SNARE proteins involved in lysosome-plasma membrane fusion reduces lysosome exocytosis and Plasmodium infection. In contrast, promoting fusion between the lysosome and plasma membrane dramatically increases infection. Our work demonstrates parallels between Plasmodium sporozoite entry of hepatocytes and infection by the excavate pathogen Trypanosoma cruzi and raises the question of whether convergent evolution has shaped host cell invasion by divergent pathogens. 105 order to define a perinuclear region where lysosomes can be differentially counted. Using a mask 106 tool, all LAMP1 signal outside this nuclear/perinuclear isosurface was suppressed, allowing 107 addition of a new LAMP1 fluorescence channel corresponding exclusively to LAMP1 localized 108 in perinuclear area. After image processing, we obtained an unmasked LAMP1 signal 109 corresponding to total lysosomes and a masked LAMP1 signal localized to the perinuclear region, 110 corresponding to perinuclear lysosomes. Isospots were constructed based on these two classes of 111 LAMP1 signal, which allowed the quantification of total and perinuclear lysosomes per cell for 112 each image-stack. The surface segmentation function of Imaris was used to identify the cell 113 boundary using phalloidin-signal. Intensity based co-localization was performed by creating 114 region of interest (ROI) specific to the sporozoite structure in the CSP channel using the Imaris 115 isosurface module. Pearson's correlation coefficient for co-localization analysis of endocytic 116 vesicles and CSP was performed in the ROI using the Imaris co-localization module. 117 Generation of sporozoite supernatant 118 Salivary gland sporozoites were isolated using standard protocols. Sporozoites incubated with 20% 119FBS for 20 min at RT and spun at 13,0000 x g for 4 min at 4 ˚C. The supernatant is collected and 120 again spun at 13, 0000 x g for 4 min at 4 ˚C to ensure the preparation is free of intact sporozoites. 121Hepa1-6 cells were exposed to supernatants at different sporozoite to hepatocyte ratio for 90 min. 122Cells were washed and subjected to 3D immunofluorescence microscopy or flow cytometry as 123 described previously. 124 Statistical analysis 125 fashion. Immunofluorescent microscopy of Hepa1-6 cells infected with SPECT2 -P. yoelii 131 sporozoites. Infection was assessed after (a) 5 or (b) 30 min and processed for fluorescence 132 microscopy using DAPI (blue) for DNA, Phalloidin (white) for actin visualization, antibodies to 133 LAMP1 (red) for LE/lysosomes and CSP (green) for parasites. Isospots for LE/lysosomes and 134 isosu...

Plasmodium sporozoites on the move: Switching from cell traversal to productive invasion of hepatocytes

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Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality

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Protein regulation strategies of the mouse spleen in response to Babesia microti infection

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