Scytovirin was produced and purified as described previously (McFeeters et al., 2007b, 2013) using recombinant expression in Origami^TM E. coli cells and subsequent metal chelation chromatography and HPLC purification. Purified Scytovirin was lyophilized and resuspended in phosphate buffered saline (PBS).

Cryptococcal strains used were: (serotype A) H99S (Janbon et al., 2014), KN99a, KN99α, and Botswana clinical isolates B5, B15, B18, and B45 (Bisson et al., 2008); (serotype D) 24067, JEC21, B3501, B3502, Cap67, and clinical isolates AD3-80, AD5-33, AD7-17, AD11-23, AD11-75, AD11-77, AD12-15, AD12-22, and AD12-27 (Dromer et al., 2007; Desnos-Ollivier et al., 2015). Two clinical strains of C. gattii were also tested: R265 and R272 (Ngamskulrungroj et al., 2011). All strains were grown from frozen stock in Yeast Peptone Dextrose (YPD) broth to mid-log phase (36–42 h) at 37°C and then washed 3x with PBS.

The minimum fungicidal concentration was determined using a modified version of the CLSI M27-A2 protocol (CLSI, 2002). For each strain, a total of 2 × 10³ cells were resuspended in 900 μl of RPMI + 0.1 M MOPS (pH = 7.0) and aliquoted into sterile 5 mL culture tubes. The initial inoculum for each strain was plated on YPD to determine CFU. Different concentrations of Scytovirin in PBS were added in 100 μl to reach final concentrations of 0, 0.5, 1.0, 1.3, 1.5, 2.6, 5, 10, and 20 μM. The tubes were incubated at 37°C for 72 h, shaking at 150 rpm. After 72 h, serial dilutions of each tube were plated on YPD. In addition, 100 μl of an appropriate dilution was plated on YPD to determine CFU. We reasoned that C. neoformans would be maintained in a more aerobic environment by shaking (not settling to the bottom of a plate well) and provide a better indicator of inhibition. Also, measuring CFU provided a better indication of live cells. The concentration of Scytovirin that resulted in greater than 95% inhibition was considered the MFC (Supplementary Figure 1). It was confirmed that Scytovirin inhibited C. neoformans in a 96-well plate format and absorbance readings correlated with broth macrodilution, although typically resulted in lower MIC values. That is a 1 μM MFC in the reported fungal kill assay correlated to an approximate 300 μM MIC measured from 96-well plates.

Capsule size in vitro was measured as described (Zaragoza et al., 2003). Briefly, 2 × 10⁵ cells/mL of strains H99S (serotype A), 24067 and B3502 (serotype D) were added to DMEM in the presence of 0, 1, and 10 μM Scytovirin and incubated at 37°C + 5% CO₂ for 18 h. Cells were collected, suspended in 5 μl PBS and added to a microscope slide with India Ink. Cells were imaged on a Zeiss Axio Scope A1 inverted microscope with a 100X objective. For each strain, pictures of 50 cells were taken. The diameter of the cell body and capsule were measured using Zeiss Axiovision software. Capsule radial length was calculated by subtracting the cell body diameter from the diameter of the entire cell plus capsule and dividing by 2. Cells were also imaged before capsule induction as a control to ensure that capsule production was induced. The experiment was repeated three times.

To determine if the strains differed in ability to release capsular glucuronoxylomanan (GXM) into the medium in the presence of Scytovirin, capsule production was induced in DMEM as before. The next day, the supernatant was collected and the concentration of GXM in the media was measured by capture ELISA as previously described (Casadevall et al., 1992). Experiments were repeated in triplicate. The following antibodies were used: goat anti-mouse unlabeled IgM, anti-GXM Ab 2D10, anti-GXM ab 18B7, and labeled goat anti-mouse IgG1-AP.

To determine if Scytovirin affected melanin production or growth in the presence of cell wall stressors, Scytovirin (0, 1, 10, and 20 μM) was added to L-Dopa plates and YPD plates containing 0.5% Congo red. Log-phase cultures of serotype A strain H99S, and serotype D strains B3501, B3502, Cap67, AD-3-80, and AD11-23 were washed 3X with PBS, diluted to 1 × 10⁶ cells/mL and serial dilutions were spotted onto L-Dopa and 0.5% Congo red plates. Plates were incubated at 37°C for 2–7 days and the growth of different spot dilutions quantified.

Serotype D strains 24067, B3502, and Cap67, and serotype A strain H99S cells were labeled with the cell wall stain uvitex2B (Polysciences Inc., 1 μg/mL), 200 μg/mL AlexaFluor 568-conjugated Scytovirin, and 10 μg/mL AlexaFluor 488-conjugated IgM capsular antibody 12A1. Emissions from 410 to 480 nm (uvitex 2B), 495–525 nm (AlexaFluor 488), and 578–603 nm (AlexaFluor 568) were visualized using a Zeiss laser scanning confocal microscope. Images were processed with ImageJ (Abramoff et al., 2004).

The assay was performed in triplicate as previously described (Vaaje-Kolstad et al., 2005). Briefly, aliquots of Scytovirin (5 mg/mL) and chitin (MP Biochemicals, 20 mg/mL) were combined in PBS, pH 7.4, to produce 0.5 mL reaction mixtures containing 100 μg Scytovirin and 1 mg chitin. Reaction mixtures as well as control tubes containing only 100 μg Scytovirin or 1 mg chitin were incubated with continuous vortexing at 25°C for a given period of time. At defined time points between 5 min and 20 h, chitin was pelleted by centrifugation and the concentration of Scytovirin in the supernatant was determined by UV absorbance at 280 nm. The spectrophotometer was blanked using supernatant from a chitin only control tube. Additionally, supernatant samples were taken at each time point and analyzed by SDS-PAGE.

Cryptococcus neoformans strain 24067 was used to determine potential synergistic interactions of Scytovirin with AMB, 5FC, and FLC. Interactions were evaluated using broth microdilution checkerboard assays and FIC indexing as well as response surface modeling (Greco et al., 1995). The FIC index was defined as follows: (MFC of drug A, tested in combination)/(MFC of drug A, tested alone) + (MFC of drug B, tested in combination)/(MFC of drug B, tested alone). Since FIC indices calculated at the 95% MFC cutoff varied significantly, a 50% inhibition level, or MFC-2 (Te Dorsthorst et al., 2002), was used. For surface response modeling, the entire dataset was modeled using the response surface approach (Greco et al., 1995; Te Dorsthorst et al., 2002) and the interaction coefficient ICα determined with 95% confidence bounds. Data were processed using MATLAB R2016a (The MathWorks, Inc.). An 8 mg/mL stock solution of AMB (Alfa Aesar) was prepared in DMSO. Stock solutions of 20 mg/mL 5FC (Alfa Aesar) and FLC (Acros Organics) were prepared in distilled deionized H₂O. Subsequent dilutions were made in RPMI/MOPS pH 7.0. In each experiment, final Scytovirin concentrations ranged from 100 μg/mL to 7.6 × 10^-4 μg/mL (10.3 μM to 78 pM). Final concentrations of AMB tested ranged from 8 μg/mL to 6.1 × 10^-5 μg/mL, while concentrations of both 5FC and FLC ranged from 10 μg/mL to 7.6 × 10^-5 μg/mL. C. neoformans 24067 cells were grown from frozen stock in YPD broth for 36–42 h at 37°C and then washed 3x and re-suspended in PBS before dilution into RPMI/MOPS. Tests were performed in black walled 96-well microplates with clear, flat bottoms. In each well, 50 μL of tested antifungal and 50 μL Scytovirin were combined at four times the final concentration in abscissa and ordinate dimensions, respectively. Hundred microliters of 4,000 cells/mL inoculum in RPMI/MOPS was added to each well. Plates were incubated at 37°C for 40 h before 20 μL of PrestoBlue (ThermoFisher) was added to each well and allowed to incubate for 8 h. Fluorescence excitation was at 560 nm and emission was read at 590 nm using a SpectraMax M2 microplate reader and SoftMax Pro software (VWR). Drug-free control plates were used as positive growth controls. Plates with RPMI/MOPS only or C. neoformans inoculum with excess AMB, which displayed no detectable fluorescence difference, were used as negative growth controls.

Capsule diameter was analyzed using the nonparametric Wilcoxon Rank Sums test while a multivariate analysis of variance with simple effects was used to test for GXM release. For all tests, p-values <0.05 were considered significant.

Scytovirin binds high mannose moieties and has high specificity for Man4 (Adams et al., 2004; McFeeters et al., 2007b, 2013). Cryptococci produce highly mannosylated proteins and utilize mannose as a building block for their distinctive capsules. Therefore, Scytovirin was tested for inhibitory activity. Initially, cryptococcal susceptibility to Scytovirin was tested on the C. neoformans laboratory strains H99S (serotype A) and 24067 (serotype D). Scytovirin more potently inhibited 24067 cells (MFC of 1 μM) and more weakly inhibited H99S cells (MFC between 50 and 20 μM). To better gauge the ability of Scytovirin to inhibit pathogenic cryptococci, 23 strains covering 3 serotypes were tested, see Table 1. Having MFC values of 500 nM, the most potently inhibited serotype D strains were JEC21, B3502, and the clinical isolate AD12-27. The MFC of most other serotype D strains ranged from 1 to 5 μM, with one ranging to 20 μM. Representative C. gattii strains R265 and R272 were inhibited by Scytovirin, but the likely parent strain (R272) was more susceptible with an MFC of 500 nM. In general, serotype A strains were less susceptible. Botswana clinical isolates B5, B15, and B45 were most susceptible with MFC values of 20 μM. While their MFCs were greater than 20 μM, strains B18, H99S, and KN99a showed greater than 90% growth inhibition and KN99α greater than 75% growth inhibition in the presence of 20 μM Scytovirin.

Cryptococcus neoformans were exposed to fluorescently labeled Scytovirin and localization was determined by fluorescence confocal microscopy. Fluorescently labeled capsular antibodies and cell wall stain aided in localization. From visualization at variable time intervals, it was observed that Scytovirin quickly assimilated on the cell wall. No fluorescence overlap of Scytovirin was detected with any of the capsular antibodies tested (Figure 1). To determine whether susceptibility was correlated with Scytovirin binding to the cell wall, additional experiments were performed with strains of varying MFC values. It was found that in all cases (B3501, B3502, cap67, and H99S) Scytovirin binding was localized to the cell wall, irrespective of serotype.

To determine if the inhibitory activity of Scytovirin affected production of the capsule, strains 24067, B3502 (serotype D), and H99S (serotype A) were induced for capsule production in the absence or presence of two concentrations of Scytovirin. Interestingly, increasing concentrations of Scytovirin were associated with significant increases in capsule diameter for strain 24067, significant decreases in capsule diameter for strain B3502, and a small, but significant, increase in diameter for strain H99S (only at 1 μM) (Figure 2A). The largest changes were a 50% increase at 10 μM Scytovirin for strain 24067, a 35% decrease at 1 μM Scytovirin for strain B3502, and a 15% increase at 1 μM Scytovirin for strain H99S. The same trend is found when Scytovirin concentrations closest to each strains respective MFC were compared (1 μM is equal to the MFC for 24067, 1 μM or twice the MFC for B3502, and 10 μM or approximately one half the MFC for H99S).

To determine if Scytovirin affected release of capsular polysaccharides, strains 24067, B3502, and H99S were induced for capsule production in the presence or absence of Scytovirin as above. After overnight culture growth, the supernatant was collected and used to measure amounts of released polysaccharides. For both serotype D strains, Scytovirin concentrations 10-fold or greater than the MFC significantly inhibited release of capsular polysaccharides, which was attributed to fungicidal activity. Near the MFC, capsule release showed a potentially modest increase for 24067 but a significant decrease for B3502. No change in capsule release was observed for H99S (Figure 2B). Strain H99S was less susceptible to Scytovirin inhibition and in the absence of Scytovirin released 5- and 100-fold more capsule than strains 24067 and B3502, respectively, potentially mitigating any effects.

Scytovirin did not have any effect on cell wall integrity in the presence of 0.5% Congo red with control plates showing the same degree of growth as those with cell wall disrupting dye present for all strains tested (data not shown). Since Congo red binds β-glucans and interferes with the construction of the cell wall (Ram and Klis, 2006), this data suggests that Scytovirin does not bind β-glucans, as expected. Additionally, Scytovirin did not have any effect on melanization after 7 days incubation at 37°C with the same degree of melanization (darkening of conlonies) observed with and without Scytovirin present for all strains tested.

Biochemical characterization and structural studies have indicated similarity of Scytovirin to chitin binding proteins. With the strong accumulation of Scytovirin in the cryptococcal cell wall, the ability of Scytovirin to bind chitin was determined. Scytovirin did not show any interaction with chitin, Figure 3. Even over the course of 20 h, no binding or adsorption to chitin was observed, in agreement with previous chitin microcolumn data (Bokesch et al., 2003).

Using 24067 cells, the combinatorial effects of Scytovirin with the commonly used antifungals AMB, 5FC, and FLC were investigated. When the MFC at 95% inhibition (MFC-0) was used for the calculation, results varied significantly. This was in part due to the steep response of fungal inhibition of AMB, declining from no inhibition to complete inhibition within a factor of 4 in terms of AMB concentration, or with just three wells on the plate. Therefore, FIC indices were calculated at 50% inhibition (MFC-2), yielding more consistent results (Te Dorsthorst et al., 2002). The FIC index using MFC-2 was 0.3 for the combination of AMB and Scytovirin (Supplementary Figure 3). Similarly, FIC indexing values for 5FC and FLC in combination with Scytovirin were 0.56 and 0.75, respectively. To better evaluate synergy, ICα values from response surface modeling were also determined using a standard checkerboard arrangement of orthogonal increasing inhibitor concentrations. Data from the entire 96-well plate were used to create a 3-dimensional response surface map With ICα determined from fitting the response surface map to the surface equation relating inhibitor concentrations and the effect at those concentrations (Te Dorsthorst et al., 2002). Positive ICα values indicate synergy, negative values indicate antagonism, and values close to zero indicate no interaction. AMB presented the strongest synergistic interaction with Scytovirin, having an ICα of 2.27. 5FC and FLC both indicated lesser degrees of synergy than AMB, having ICα values of 0.72 and 0.60, respectively.