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. 2020 Jan 17;9:451.
doi: 10.3389/fcimb.2019.00451. eCollection 2019.

Association of Plasmodium berghei With the Apical Domain of Hepatocytes Is Necessary for the Parasite's Liver Stage Development

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Free PMC article

Association of Plasmodium berghei With the Apical Domain of Hepatocytes Is Necessary for the Parasite's Liver Stage Development

Lakshmi Balasubramanian et al. Front Cell Infect Microbiol. .
Free PMC article

Abstract

Plasmodium parasites undergo a dramatic transformation during the liver stage of their life cycle, amplifying over 10,000-fold inside infected hepatocytes within a few days. Such a rapid growth requires large-scale interactions with, and manipulations of, host cell functions. Whereas hepatocyte polarity is well-known to be critical for liver function, little is presently known about its involvement during the liver stage of Plasmodium development. Apical domains of hepatocytes are critical components of their polarity machinery and constitute the bile canalicular network, which is central to liver function. Here, we employed high resolution 3-D imaging and advanced image analysis of Plasmodium-infected liver tissues to show that the parasite associates preferentially with the apical domain of hepatocytes and induces alterations in the organization of these regions, resulting in localized changes in the bile canalicular architecture in the liver tissue. Pharmacological perturbation of the bile canalicular network by modulation of AMPK activity reduces the parasite's association with bile canaliculi and arrests the parasite development. Our findings using Plasmodium-infected liver tissues reveal a host-Plasmodium interaction at the level of liver tissue organization. We demonstrate for the first time a role for bile canaliculi, a central component of the hepatocyte polarity machinery, during the liver stage of Plasmodium development.

Keywords: 3D image analysis; bile canaliculi; hepatocyte polarity; liver tissue; plasmodium liver stage; ultrastructure.

Figures

Figure 1
Figure 1
Plasmodium liver stage development in vivo in 3d. (A) 3D imaging and analysis of Plasmodium berghei development during the liver stage of infection. One hundred μm liver sections from P. berghei infected mice were sectioned and stained for PVM using an anti-UIS4 antibody. Nuclei and cell membranes are visualized by DAPI and Phalloidin staining, respectively. Imaging was carried out using confocal microscopy at a resolution of 0.3 μm voxel. (B) Representative images from P. berghei-infected liver sections collected 24, 33, and 48 hpi, showing the PVM and nucleus in x-y, x-z, and y-z views. The scale bar is 10 μm. (C) Quantification of volume changes in the parasitophorous vacuole during P. berghei development in vivo. Images from B were segmented using an intensity thresholding method. (D) Quantification of volume changes in the parasitophorous vacuole during P. berghei development in vivo. Images from B were segmented using an automated geometric flow based segmentation method. (E) Quantification of volume changes at the indicated time points post infection in the infected and nearby uninfected hepatocytes. ***denote a p < 0.001 by Student's t-test. (F) Proportion of host cell volume occupied by the parasite during liver stage development in vivo. For (C–F), a representative image of the model for the segmented parasite (red) and the host hepatocyte (gray) in 3d, for the time points indicated, is shown along the x-axis. The scale bar is 10 μm. (G) Ultrastructure of P. berghei-infected cell at 48 hpi in vivo. The scale bar is 10 μm. (H) Selected regions from a P. berghei infected hepatocyte in vivo showing close association of the PVM with late endosome (i), lysosome (ii), autophagosome (iii), and a lipid droplet (iv). (I) Selected regions from a P. berghei infected hepatocyte in vivo showing close association of the PVM with ER (v) and a mitochondrion (vi). Distance distribution of PVM with ER (purple) and mitochondria (pink) (vii). Data is from at least 5 infected cells. (viii) shows a representative tomogram reconstruction of a close interaction of the PVM (cyan) with a mitochondrion (pink) and ER (purple). For (H,I), P denotes the parasite and the arrowheads point to the apposition of PVM with the indicated organelles. The scale bar is 200 nm.
Figure 2
Figure 2
Association of P. berghei with hepatocyte apical domains during liver stage development. (A) Serial EM sections showing the PVM in close proximity to the apical domains of the hepatocyte plasma membrane. The sections are 70 nm apart, with the boxed region highlighting the juxtaposition of the PVM with the apical region. The arrow points to the PVM, with the arrowhead showing the apical domain (AD). P indicates the parasite. (B) Quantification of the distance between the PVM and apical (pink) or basolateral (green) regions of the hepatocyte plasma membrane. Data is combined from distance measurements from at least five infected cells. (ii) highlights a region from (i) showing spatial proximity of the PVM to apical domains. (C) A section of a tomogram of the PVM associated with an apical region (left) and the corresponding reconstruction (right). The PVM is in cyan, the plasma membrane of the infected hepatocyte in green, and the plasma membrane of the adjacent non-infected hepatocyte in red. (D) Representative confocal images of infected liver sections at 24, 33, and 48 hpi, showing PVM (red), bile canaliculi (green), cell boundaries (gray), and nuclei (blue). The UIS4 and CD13 antibodies were used to mark the PVM and apical domains, respectively. The scale bar is 10 μm. (E) Quantification of surface voxels of the apical domain (CD13) on the PVM at different time points post infection. The inset figure shows the segmented model of apical domains (green) in proximity with the PVM (red). The scale bar is 10 μm. (F) Quantification of the total intensity of CD13 (apical domain/bile canaliculi marker) on the PVM at different time points post infection. The inset shows a schematic of the pipeline used for quantitative analysis. (G) Quantification of the alteration in hepatocyte polarity during P. berghei infection in vivo. Surface voxels of the apical domain on the infected hepatocyte surface in comparison with an uninfected hepatocyte at different time points post infection are shown. n.s and *denote p values that are non-significant and < 0.01 respectively, based on Student's t-test. The inset shows a model of 3D segmentation of an infected hepatocyte (gray) containing a parasite (red), with the apical domains (green).
Figure 3
Figure 3
Infected mice show higher levels of bile acid levels in the gallbladder. Total bile acid levels in gallbladders of P. berghei-infected and uninfected mice at 48 hpi. Result representative of two biological replicates. ***denotes p < 0.001 from Student's t-test.
Figure 4
Figure 4
Pharmacological modulation of hepatocyte polarity in vivo arrests P. berghei development in liver tissues. (A) Representative images of maximum intensity projections of liver sections from untreated and salicylate-treated mice, stained with the apical domain marker CD13. One hundred μm thick liver sections were imaged with a voxel size of 0.5 μm3. The scale bar is 10 μm. The top panel shows the raw image, the middle panel shows segmentation based on thresholding and the lower panel shows segmentation using an automated flow based method. (B) Quantification of total length of bile canaliculi (BC) between untreated and Salicylate treated mice. The top and bottom panels show quantifications from threshold based and flow based segmentation, respectively. (C) PVM volume changes upon salicylate treatment at 24 and 33 hpi. n.s. denotes the differences between treated and untreated conditions that are not statistically significant at these time points. (D) A comparison of surface voxels of bile canaliculi (BC) on hepatocytes between uninfected and infected hepatocytes, with and without salicylate treatment. The inset shows a representative segmented model of infected hepatocyte (gray) and bile canaliculi (green). The scale bar is 10 μm. n.s denotes the differences are not statistically significant. (E) Surface voxels of bile canaliculi on the PVM between Salicylate treated and untreated conditions. The inset shows a representative segmented model of PVM (red) within an infected hepatocyte (gray) with surrounding bile canaliculi (green). The scale bar is 10 μm. (A–E) Are representative of two biological replicates, n.s. denotes the differences are not statistically significant, ** and ***denote p < 0.01 and < 0.001, respectively. Statistical significance is assessed using Student's t-test.
Figure 5
Figure 5
Schematic of P. berghei interacting with the apical domains of hepatocytes during liver stage development in the liver tissue. Plasmodium berghei (green) development within hepatocytes (gray) requires interaction of the parasite vacuole membrane with the hepatocyte apical domain, or bile canaliculi (BC). When hepatocyte polarity is altered by salicylate treatment, the interaction is reduced and the parasite growth is arrested (bottom panels).

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