Verbrugghe E et al. (2019), In vitro modeling of Batrachochytrium dendrobat...

XB-ART-56473

PLoS One 2019 Nov 14;1411:e0225224. doi: 10.1371/journal.pone.0225224.

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In vitro modeling of Batrachochytrium dendrobatidis infection of the amphibian skin.

Verbrugghe E , Van Rooij P , Favoreel H , Martel A , Pasmans F .

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The largest current disease-induced loss of vertebrate biodiversity is due to chytridiomycosis and despite the increasing understanding of the pathogenesis, knowledge unravelling the early host-pathogen interactions remains limited. Batrachochytrium dendrobatidis (Bd) zoospores attach to and invade the amphibian epidermis, with subsequent invasive growth in the host skin. Availability of an in vitro assay would facilitate in depth study of this interaction while reducing the number of experimental animals needed. We describe a fluorescent cell-based in vitro infection model that reproduces host-Bd interactions. Using primary keratinocytes from Litoria caerulea and the epithelial cell line A6 from Xenopus laevis, we reproduced different stages of host cell infection and intracellular growth of Bd, resulting in host cell death, a key event in chytridiomycosis. The presented in vitro models may facilitate future mechanistic studies of host susceptibility and pathogen virulence.

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Species referenced: Xenopus laevis
Genes referenced: pak1

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	Fig 1. Fluorescent staining of early stages of Litoria caerulea PAK infection by Bd. (A, E) Host cells and (B, F) Bd spores were visualised using a green and red cell tracker, respectively. (C, G) Nuclear content was stained with Hoechst and all pictures were merged in (D, H). (A-D) After 4 hours, initial contact was observed between host cells and Bd spores, as indicated by a white arrow (D). (E-H) 24 hours after inoculation, marked intracellular colonization was seen in Litoria caerulea host cells, as indicated by a white asterisk (H). Scale bar = 20 Î¼m.
	Fig 2. Confocal microscopy of Bd-infected host cells after 24 hours. Invasion of Bd inside (A-C) PAK of Litoria caerulea and (D-F) continuous A6 cells of Xenopus laevis was analyzed using confocal microscopy. (A, D) Host cells were stained with a green cell tracker and (B, E) nuclear content was stained with Hoechst. Both stainings were merged in (C, F). By scanning different layers within the cell via confocal microscopy, Bd spores were validated being intracellular, as indicated by a white asterisk. Scale bar = 20 Î¼m.
	Fig 3. Bd development in A6 cells. Shown is an overlay of the fluorescent signals of (A-G) Bd-infected A6 cells (green cell tracker), extracellular Bd (Calcofluor White (blue)) and extra-and intracellular Bd (Alexa Fluor 568 (red)) or (H) caspase-3 activation (red) and nuclear content (Hoechst (blue)). (A) Four hours after inoculation, formation of germ tubes (GT) was observed and (B) within 24 hours, these tubular structures penetrated the A6 cells (). (C) At day 1â2 p.i., new intracellular chytrid thalli () are formed and the cell content of the mother thallus (MT) is transferred into the new daughter thallus (DT). (D) At day 2â3 p.i., the emptied mother thallus evanesces, resulting in intracellular Bd bodies () that (E) develop intracellularly into sporangia at day 3â4 p.i. (F) Once the sporangia reach the stage of a mature zoosporangium (day 4â5 p.i.), they use a discharge tube () to release their contents into the A6 cells (G). (H) At day 5â6 p.i., caspase-3 activation was observed in A6 cells associated with Bd (white arrow). Scale bar = 20 Î¼m. Individual pictures of the different fluorescent channels can be found in S2 and S3 File.
	Fig 4. Epibiotic and endobiotic growth of Bd on and in A6 cells, 2 days p.i.. (A) Bd-exposed A6 cells were stained using a green cell tracker. (B) Bd was visualized using Alexa Fluor 568, resulting in red fluorescence of both intracellular and extracellular Bd. (C) The cell wall of extracellular Bd was coloured using Calcofluor White, showing blue fluorescence. The pictures were merged in (D). Two days after inoculation, both epibiotic and endobiotic growth were observed. Epibiotic growth can be described as development outside the host cell (squares with a full line), which stains Bd both blue and red. Endobiotic growth (squares with a dashed line) is visualized as a red staining of the intracellular chytrid thalli (*) at the end of the germ tube (GT). Scale bar = 20 Î¼m.

References [+] :

Bates, Amphibian chytridiomycosis outbreak dynamics are linked with host skin bacterial community structure. 2018, Pubmed