Article Figures & Data
Figures
- FIG 1
Model of DEC-AmB-LLs, liposomes loaded with sDectin-1, AmB, and rhodamine. AmB (blue oval structure) was intercalated into the lipid bilayer of 100-nm-diameter liposomes. sDectin-1 (DEC, green globular structure) was coupled to the lipid carrier DSPE-PEG. Both DSPE-PEG-DEC and red fluorescent DHPE-rhodamine (red star) were also inserted into the liposomal membrane. sDectin-1, rhodamine, AmB, and liposomal lipids were in a 1:2:11:100 mole ratio (Table S1). Two sDectin-1 monomers (two DSEP-PEG-DEC molecules) must float together in the membrane to bind strongly to cell wall beta-glucans (red sugar moieties). The two liposomal controls examined were BSA-AmB-LLs that contain equal microgram amounts of 65-kDa BSA in place of 22-kDa sDectin-1 (i.e., 0.33:2:11:100 mole ratio) and AmB-LLs lacking any protein coating (0:2:11:100 mole ratio). From these mole ratios, the surface area of an 100-nm-diameter liposome, and the published estimate of 5 × 106 lipid molecules per 106 nm2 of lipid bilayer (70), we estimated that there were approximately 3,000 rhodamine molecules in each liposome preparation and 1,500 sDectin-1 monomers in each DEC-AmB-LL. Note that for simplicity the proper ratios of these molecules are not shown.
- FIG 2
sDectin-1-coated DEC-AmB-LLs bound strongly to germinating conidia and germ tubes of A. fumigatus, while AmB-LLs and BSA-AmB-LLs did not. A. fumigatus conidia were germinated and grown for 8 to 10 h in VMM plus 1% glucose at 37°C in 24-well microtiter plates before being fixed and stained with fluorescent liposomes. (A) Rhodamine red fluorescent DEC-AmB-LLs bound swollen conidia (white arrows) and germ tubes of A. fumigatus. (B) Rhodamine red fluorescent AmB-LLs did not bind at detectable frequencies. No AmB-LLs were detected even when the red channel was enhanced as in this image. The smallest red dots in plate A represent individual 100-nm-diameter liposomes viewed based on their fluorescence (orange arrows). Large clusters of liposomes form the more brightly red stained areas. (C and D) Staining with DEC-AmB-LLs. (E and F) Staining with BSA-AmB-LLs. Conidia in panels C and E were photographed in the green fluroescent channel, while for those in panels D and F, green and red fluorescent channels were combined. Labeling was performed in LDB1 for 60 min. All three liposome preparations were diluted 1:100 such that liposomal sDectin-1 and BSA proteins were at final concentrations of 1 μg/100 μl. Germlings were viewed in the green channel alone for cytoplasmic fluorescent EGFP expression and in the red channel for rhodamine fluorescent liposomes. Cells in panels A and B were photographed at ×63 magnification under oil immersion in a compound fluorescence microscope, and red fluorescence was further enhanced in panel B to detect potentially individual liposomes. Cells in panels C through F were photographed at ×20 on an inverted fluorescence microscope.
- FIG 3
sDectin-1-coated DEC-AmB-LLs bound germinating conidia and hyphae of mature A. fumigatus cells, while untargeted AmB-LLs and BSA-AmB-LLs did not. A. fumigatus conidia were germinated and grown for 16 h in VMM plus 1% glucose at 37°C in 24-well microtiter plates before being fixed and stained with fluorescent liposomes. Cells were stained with rhodamine red fluorescent DEC-AmB-LLs diluted 1:100 such that sDectin-1 was at 1 μg/100 μl (A to D) and with the equivalent amount of red fluorescent AmB-LLs for 60 min (E and F). (A) DIC image alone. (B) Combined DIC and red fluorescence image. Panels A and B show that rhodamine fluorescent DEC-AmB-LLs bound to germinating conidia (white arrows) and hyphae. In panel B, the smallest red dots represent individual 100-nm liposomes (orange arrows). (C to F) Cytoplasmic EGFP and the red fluorescence of liposomes. Panels C and D show that nearly all conidia and most hyphae stained with DEC-AmB-LLs. Panels E and F show that AmB-LLs did not bind. Cells in panels A and B were photographed at ×63 under oil immersion, and those in panels C to F were photographed at ×20 on an inverted fluorescence microscope.
- FIG 4
sDectin-1-coated DEC-AmB-LLs bound 2 orders of magnitude more frequently to A. fumigatus than control AmB-LLs, and binding was inhibited by soluble beta-glucan. Samples of 4,500 A. fumigatus conidia were germinated and grown at 37°C for 36 h in VMM plus 1% glucose, fixed in formalin or examined live, and incubated for 1 h with a 1:50 dilution of liposomes in liposome dilution buffer LDB1. Unbound liposomes were washed out. Multiple fields of red fluorescent images were photographed at ×20 and red fluorescence enhanced equivalently for all images. Each photographic field contained approximately 25 swollen conidia and an extensive network of hyphae (not shown). (A, B, and C) Labeling of formalin-fixed cells. (D, E, and F) Labeling of live cells. (G, H, and I) Inhibition of DEC-AmB-LL labeling of fixed cells by 1 mg/ml of laminarin, a soluble beta-glucan versus 1 mg/ml of sucrose as a control. (A, D, and G) The numbers of red fluorescent liposomes and clusters of liposomes were counted, averaged per field, and plotted on a log10 scale. The numerical average is indicated above each bar and on the vertical axis. Standard errors are shown. Fold differences and P values are indicated for the performance of DEC-AmB-LLs relative to AmB-LLs. Examples of photographic fields of liposomes used to construct the adjacent bar graphs are shown in panels B, C, E, F, H, and I.
- FIG 5
DEC-AmB-LLs inhibited the growth A. fumigatus far more efficiently than AmB-LLs. Samples of 4,500 A. fumigatus conidia were germinated and grown in 96-well microtiter plates in VMM plus 1% glucose for 8 to 56 h at 37°C and treated at the same time with liposome preparations delivering the indicated concentrations of AmB to the growth media—for panels A through D, 3 μM AmB, a 1:300 fold dilution of all three liposome preparations; for panel E, 0.09 μM; for panel F, 0.18 μM; and for panel G, 0.9 to 3 μM)—or an equivalent amount of liposome dilution (Dil) buffer LDB2. Viability and growth were estimated using CellTiter-Blue reagent (A and C) or by measuring hyphal length (B and D) or by scoring percent germination (E, F, and G). Background fluorescence from wells with CellTiter-Blue reagent in the media but lacking cells and liposomes was subtracted. Standard errors are indicated. Fold differences and P values are indicated for comparisons of the performance of DEC-AmB-LLs to AmB-LLs. Inset photos in panels B and D show examples of the length of hyphae assayed for AmB-LL- and DEC-AmB-LL-treated samples. One unit of hyphal length in panels B and D equals 5 μm. Panels A and B and panel C and D compare the results from two biological replicate experiments with independently conjugated sDectin-1 and assembled liposomes.
Supplemental Material
TABLE S1
Liposome compositions. Comparison of the chemical composition of liposomes discussed in the article. Download Table S1, TIF file, 0.4 MB.
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FIG S1
The modified mouse sDectin-1 DNA MmsDectin1lyshis and protein MmsDectin-1. (A) The codon-optimized DNA sequence of MmsDECTIN1lyshis was cloned into pET-45B (NCBI BankIT submission 2173810; length, 577 bp). The vector pET-45b sequence is highlighted in red, with the start codon underlined. Cloning sites are in green, codons for Gly and Ser (G and S) flexible linker residues are in yellow, reactive Lys (K) residues are in purple, mouse sDecetin-1 is in light blue, the terminal Ala codon to put stop codons in frame is in yellow, and stop codons are in bold. (B) The modified mouse sDectin-1 protein being synthesized. The N terminus and His tag from the pET-45B vector are in red, Gly and Ser flexible linker residues are in yellow, reactive Lys residues are in purple, and mouse sDecetin-1 is in light blue. The final Ala residue/codon is to put stop codons and PacI site in frame. Length, 199 amino acids; molecular weight, 22,389.66 g/mol; theoretical pI, 7.74. Download FIG S1, TIF file, 0.10 MB.
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FIG S2
SDS-PAGE analysis of sDectin-1 in cell extracts and after affinity purification. sDectin-1 protein was produced in the BL21 strain of E. coli grown in Luria broth overnight from the pET-45B plasmid without IPTG induction. The protein was solubilized in GuHCl buffers, purified by nickel-nitrilotriacetic acid (Ni-NTA) resin, and examined by SDS-PAGE after GuHCl was removed by dialysis. Extraction of protein into buffers that also contained reducing agent 2-mercaptoethanol and Triton X-100 detergent greatly increased recovery from insoluble inclusion bodies (center lanes) relative to buffers without them (right lanes). Protein was examined on a 12% acrylamide gel stained with Coomassie blue. The approximate molecular weight of modified sDectin-1 (22 kDa) is indicated. Extraction of these cells with urea buffers even at 60°C yielded very little protein (not shown). Download FIG S2, TIF file, 0.3 MB.
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FIG S3
sDectin-coated liposomes, DEC-AmB-LLs, bound strongly to Candida albicans and Cryptococcus neoformans cells. (A, C, and E) Bright-field images of C. albicans strain Sc5314 and C. neoformans strain H99 labeled with DEC-AmB-LLs diluted 1:100 in LDB1; (B, D, and F) combined bright-field and red fluorescence images showing that rhodamine red fluorescent DEC-AmB-LLs bound strongly to these cells. Plain uncoated AmB-LLs and BSA-AmB-LLs did not bind detectably to these cells (not shown). Cells in panels A and B were photographed at ×63 under oil immersion, and those in panels C to F at ×20 on an inverted fluorescent microscope. Download FIG S3, TIF file, 0.8 MB.
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FIG S4
sDectin-1-coated DEC-AmB-LLs and BSA coated BSA-AmB-LLs were less toxic to HEK293 cells than uncoated AmB-LLs. Human embryonic kidney HEK293 cells grown to 30 to 40% cell density in RPMI lacking red indicator dye in 96-well microtiter plates. Cells were treated for 2 h with the AmB-loaded liposomes indicated or a deoxycholate micelle suspension of AmB (DOC), washed twice, and then incubated for an additional 16 h. All treatments delivered a final concentration of 30 or 15 μM AmB into the media. The 0 μM control wells received an amount of liposome dilution buffer LDB2 equivalent to the 30 μM treatment. CellTiter-Blue assays estimated cell viability and survival. Background fluorescence from wells with CellTiter-Blue reagent in the media but lacking cells and liposomes was subtracted. Standard errors are indicated. Percent difference and P values are indicated for comparisons of the performance of DEC-AmB-LLs to AmB-LLs. Download FIG S4, TIF file, 0.1 MB.
Copyright © 2019 Ambati et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
