Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

Cryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.


C. neoformans growth is inhibited by DC-derived lysosomal fractions. Previous studies have
shown that C. neoformans growth can be inhibited by DCs 21,35 as well as by DC-derived lysosomal extract 36 .
Our previous studies showed that cathepsin B, which is found in the DC lysosome, can also significantly inhibit the growth of the fungus 26 . In order to identify other proteins within the lysosome that can inhibit the growth of C. neoformans, DC-derived lysosomal extract was first fractionated by molecular weight, and fractions were verified by SDS-PAGE (Fig. 1a). Protein concentration was measured in each fraction, and those containing at least 50 μg/ml protein (the protein concentration that was antifungal for DC lysosomal extract 36 )-fractions 1, 7, 8, and 10-were used in anti-cryptococcal assays. C. neoformans strain H99 was incubated at 1 × 10 5 cells/ml with either individual fractions in phosphate buffer (pH 5.5), total extract in phosphate buffer, or in phosphate buffer alone for 24 h at 37 °C, 5% CO 2 . All fractions that were tested (fractions 1, 7, 8, and 10) showed significant killing (p < 0.0001) of the cryptococcal growth when compared to the C. neoformans inoculum (Fig. 1b). These data show that multiple fractions of DC lysosomal extract have anti-cryptococcal activity, and therefore, there are multiple compounds within the lysosomal extract that can kill C. neoformans or inhibit cryptococcal growth. (a) Crude DC-derived lysosomal extracts were separated by molecular weight using a GELFrEE 8100 system into 12 different fractions and further resolved by standard SDS-PAGE. Proteins in the 12.5% gels were stained using BioSafe Coomassie Stain and visualized using a Gel Doc XR + system. (b) C. neoformans serotype A strain H99 yeast cells (1 × 10 5 cells/ml) were incubated in phosphate buffer (pH 5.5) with whole lysosomal extract or fractions of lysosomal extract for 24 h at 37 °C, 5% CO 2 , following which CFUs in the wells were determined. Data shown are means ± standard errors of the means (SEM) of the results of three independent experiments (n = 3), with each condition performed in duplicate. Two-tailed t-tests comparing H99 inoculum to each treatment were performed with asterisks (*) indicating significantly lower CFUs of treatment compared to the H99 inoculum (fraction 8: p = 0.0001, all others: p < 0.0001). www.nature.com/scientificreports/ In order to further identify individual proteins within the lysosomal extract with anti-cryptococcal activity, the fractions were analyzed by mass spectrometry to identify the contents of each fraction. Analysis revealed over 3000 different proteins present within the lysosomal fractions (Supplementary Table S1).

DC lysosomal proteins have anti-cryptococcal activity.
In order to determine the anti-cryptococcal potential of DC lysosomal proteins, ten different lysosomal proteins were tested for anti-cryptococcal activity. From the > 3000 proteins identified by mass spectrometry, ten test proteins were selected based on potential antimicrobial activity, such as those that are known antimicrobial peptides or cysteine proteases [41][42][43][44] . Concentrations of individual proteins used in our anti-cryptococcal assay were based upon either prior published concentrations [45][46][47][48] or were based on our previous studies with cathepsin B 26,[45][46][47][48] , where concentrations of 50 and 10 µg/ml were used. Specific protein concentrations used in this study are provided in the methods section. C. neoformans strain H99 was incubated at 1 × 10 5 cells/ml in a 50 μl volume separately with each protein in sterile 0.1 mM phosphate buffer, pH 5.5 (phosphate buffer) or in phosphate buffer alone for 24 h at 37 °C, 5% CO 2 , followed by diluting then plating on YPD agar to quantify CFUs. Percent cryptococcal inhibition was defined as CFUs of the experimental condition (C. neoformans incubated with test protein) divided by the CFUs of the control condition (C. neoformans incubated with phosphate buffer alone). We observed that incubation of C. neoformans with five of the lysosomal proteins (Coronin, HNE, MMP25, MPO, NOSTRIN) led to a significant (p < 0.0001) reduction of CFUs when compared to the C. neoformans grown in phosphate buffer alone which is shown as a high percent inhibition (Fig. 2a), while the remaining five proteins either did not inhibit fungal growth with low percent inhibition (cystatin B, striatin) or significantly enhanced fungal growth (calmodulin, p < 0.0001; calnexin, p < 0.0001; S100A6, p = 0.0034), as shown by negative percent inhibition (Fig. 2b).

Cryptococcal inhibition is dose-dependent.
To further elucidate the effective anti-cryptococcal concentration of each lysosomal protein, similar assays with reduced protein concentration were conducted on the five proteins that showed cryptococcal inhibition. Each of the five proteins were diluted to 10% and 1% of original concentration and incubated under the same conditions. Of those tested (coronin, HNE, MMP25, MPO, and NOSTRIN), only coronin (p < 0.0001), HNE (p < 0.0001), and MMP25 (p = 0.0008) significantly inhibited cryptococcal growth at the 10% protein concentration, but did not inhibit growth at the 1% concentration (Fig. 2c). Surprisingly, MMP25 significantly enhanced fungal growth (p = 0.0010) at 1% of the original concentration when compared to growth in phosphate buffer alone. While MPO was not able to reduce the growth of H99 at 10% concentration, when tested at 1%, it did have the capacity to significantly inhibit (p = 0.0213) the growth of the fungus. NOSTRIN was not effective at either of the reduced concentrations.
Minimum inhibitory concentration (MIC) of anti-cryptococcal lysosomal proteins. Next www.nature.com/scientificreports/ (for coronin), 18 µg/ml to 0.0176 µg/ml (for MPO), and 2 µg/ml to 0.0020 µg/ml (for NOSTRIN). Proteins were assessed in phosphate buffer (pH 5.5) as in our antifungal assays above as well as a more physiological relevant condition in RPMI-MOPS (pH 7.0), which is the media typically used in fungal MIC assays. Assays were completed over 48 h at 35 °C as per Clinical and Laboratory Standards Institute (CLSI) [49][50][51][52] . Results for these assays are summarized in Table 1. When testing in lysosomal-like conditions (pH 5.5) HNE, MMP25, and NOSTRIN retained similar anti-cryptococcal efficacy. In addition, these three compounds also retained some ability to inhibit yeast growth at pH 7.0. MPO was unable to inhibit cryptococcal growth at the highest tested concentration of 18 µg/ml under both conditions and did not replicate its previous effectiveness (Fig. 2a). MIC values for coronin were not able to be determined in these assays. Due to the nephrotoxicity associated with high doses of Amphotericin B (AmpB), examining methods to increase efficacy and lower dosage has become an area of intense research [53][54][55] . One method is the use of a combination of compounds that, by themselves, display anti-cryptococcal properties, such as current guidelines that advise treating cryptococcal meningitis using combination therapy with AmpB and flucytosine 56 . Therefore, we tested our anti-cryptococcal lysosomal proteins in combination with AmpB against the fully virulent C. neoformans strain H99 using a checkerboard method and categorized data based on the fractional inhibitory concentration indices (FICI) 57 . While still retaining their previously stated anti-cryptococcal capacity, none of the lysosomal proteins showed any synergistic effects when used in combination with AmpB (Supplemental Table S2). Each protein/AmpB combination scored between 1.0 and 1.8 placing them comfortably within the indifference category (0.5-4.0) and provided neither antagonistic nor synergistic results.
Time-kill kinetics of lysosomal proteins against C. neoformans. MIC assays are useful starting points for understanding antimicrobial properties of unknown compounds, since they have been standardized by various laboratories and agencies. The time-kill assay studies the kinetic rates of inhibition over a period of time rather than examining only the end result. While they give great quantitative results and the ability to explore fungicidal rates, their methods are not as widely uniform and they can become time consuming the more time points and concentration one examines 58 . For this, we used a modified version of the method proposed in a paper by Klepser et. al which has since been cited over 200 times since its publication 59 . Cryptococcal cells were incubated with 2X MIC values (listed in Table 1 or 2X Table 1 or 2X highest concentration tested if none could be determined) in an acidic phosphate buffer (pH 5.5) for 48 h. As conditions change within the test chamber, properties of the proteins may also be altered, therefore, pH was monitored throughout the study, but we did not observe any changes in pH over the course of the study. We measured the percent inhibition of cryptococcal growth over time using the five anti-cryptococcal lysosomal proteins (Fig. 3). Three of the five (NOSTRIN, HNE, and MPO) displayed sustained inhibition starting from as early as three hours post inoculation. Meanwhile, at the 3 h time point, coronin and MMP25 exhibited intermediate and no inhibition, respectively. However, by 24 h, the latter two proteins showed inhibition and caught up to the other three by 48 h.

Cytotoxicity of DC lysosomal proteins.
To test the relative cytotoxicity of these anti-cryptococcal lysosomal proteins on mammalian cells, a murine macrophage cell line (J774A.1) was used in conjunction with the Vybrant Cytotoxicity Assay Kit (G6PD assay) (Molecular Probes, Eugene, OR). Mammalian cells were incubated with the highest concentration of anti-cryptococcal protein (stated in methods section) in cell culture media or in cell culture media alone for 24 h at 37 °C, 5% CO 2, then cytotoxicity assay was performed at near physiological pH (7.5). After fluorescence was normalized to control wells (J774A.1 macrophages in cell culture media alone), percent cytotoxicity was defined as fluorescence of experimental wells (macrophages and proteins) divided by positive control wells (macrophages and cell-lysis buffer). A compound is defined as "cytotoxic" if there is a reduction of more than 30% of viable cells when compared to control wells 60 . All proteins showed low cytotoxicity (< 20%) at the highest concentration tested for cryptococcal inhibition assays (Fig. 4).

Discussion
The primary function of the DC lysosome is to break down many different biomolecules including pathogens that may cause harm to a host in order to generate peptide fragments for antigen presentation 39,61 . This rapid degradation by the lysosome favors presentation through the MHC II pathway rather than cross presentation via MHC I 62 . Studies have shown that it can kill a wide variety of pathogens regardless of whether they are fungal, bacterial, or viral [63][64][65][66][67] . Previous studies have shown that C. neoformans traffics to the lysosomal compartment Table 1. Minimum Inhibitory Concentrations (MIC) of Anti-cryptococcal Lysosomal Proteins in Phosphate Buffer (pH 5.5) or RPMI-MOPS (pH 7.0). a ODs of the protein were higher than yeast growth. b Growth present in the highest tested concentration. www.nature.com/scientificreports/ of DCs and is killed by its components 35,36 . It was also shown that contents of the DC lysosomal extract have activity against the fungus in vitro 26,36 . Furthermore, the DC lysosomal component cathepsin B can inhibit or kill the pathogen 26 . Due to the problems associated with current anti-cryptococcal drug therapies, finding new sources of treatment has become a major area of research.   www.nature.com/scientificreports/ To combat these ever-changing problems, most current research focuses on either discovering novel antifungal drugs or repurposing older drugs [68][69][70][71] . Our research focuses on identifying DC lysosomal components that are anti-cryptococcal and determining how these can be used to develop immune-based therapies to combat cryptococcosis. In accordance with our previous study on DC lysosomal proteins and their anti-cryptococcal effects, our current study has shown there are multiple DC lysosomal compounds that can inhibit and/or kill C. neoformans in an acidic buffer (pH 5.5) in a dose dependent manner. Three (HNE, MMP25, MPO) of the five compounds that showed anti-cryptococcal activity are known proteases that have been shown to have antimicrobial activity [72][73][74] . The other two are both associated with antimicrobial activity by other means: coronin is an actin binding protein that is involved with phagocytosis in leukocytes 75 and NOSTRIN modulates the release of the oxidative stressor nitric oxide 76 . Several of these proteins still displayed inhibition at reduced concentrations which shows dose-dependent activity. Contrary to these results, however, reduced concentrations of MMP25 showed significant increase in fungal growth and may be a cause for concern if used as a therapeutic.
In order to verify that the concentrations used in these studies were in the range of minimum inhibitory concentration (MIC) concentrations, we tested the five antifungal proteins (Fig. 2) (coronin, HNE, MPO, MMP25, and NOSTRIN) in MIC assays in accordance with CLSI guidelines [49][50][51][52] . Three proteins (HNE, MMP25, and NOSTRIN) showed promising results, as not only did they recapitulate their effects in low pH, but also displayed some anti-cryptococcal activity at the higher pH, albeit at a lower efficiency. This was expected as pH changes can affect the activity of many lysosomal proteins, notably cathepsins 77 . Despite the lower activity, these results point to a future of promising therapeutics for systemic cryptococcosis, since some of these proteins can also be antifungal at neutral pH. Two proteins had different MIC results compared to our original lysosomal assays. However, differences in methodology between standard MIC assays (conducted in non-CO 2 conditions and at 35 °C, as is standard MIC procedures in the field) and our antifungal assays (conducted at 37 °C, 5% CO 2 ), may have contributed to these discrepancies.
Currently, there are only a few classes of antifungal compounds, all of which can become toxic to the host at high concentrations 53,54 . Despite being the drug of choice when treating cryptococcal meningitis, AmpB does have these same concerns, especially targeting the kidneys, causing nephrotoxicity. We sought ways to reduce standard treatment concentrations by investigating synergistic activity that may exist between it and our anticryptococcal lysosomal proteins. However, none of the compounds showed any synergy when combined with AmpB at any of concentrations tested. These results are not too surprising owing to the fact that the H99 strain, while fully virulent, is still susceptible to AmpB at low doses and other compounds may not show synergistic effects. These results may differ and show synergistic effects when testing against a cryptococcal strain that is resistant to AmpB. Furthermore, synergistic combinations tend to target both pathogenic processes and cell growth to completely inhibit a fungal infection 78 . More pre-clinical research in this area is needed as these studies were conducted in vitro. We also wanted to further understand the kinetics of these anti-cryptococcal lysosomal proteins, so a time-kill assay was conducted to evaluate growth over 48 h, the same as a standard MIC assay. Three (HNE, MPO, NOSTRIN) of the five proteins showed sustained inhibition quickly, within 3 h post-incubation. However, by the time point of our typical lysosomal assays (24 h), the remaining lysosomal proteins all showed inhibition of cryptococcal growth, which was sustained through the 48 h experiment.
Despite previous reports of antimicrobial activity against bacterial pathogens [79][80][81][82] and/or interference with virulence pathways (such as the calcium-binding pathway 44,83 ), several proteins tested in this study (calmodulin, calnexin, cystatin B, S100A6, and striatin) showed either no cryptococcal inhibition or enhanced fungal growth. We hypothesized that this enhanced growth may be attributed to secretion of nutrients or trace metals that may enhance cryptococcal growth and we are currently analyzing the contents of media in these conditions. Additional studies are also underway in our laboratory to examine the mechanism(s) by which these components enhance cryptococcal growth. Cystatin B is a cysteine protease that inhibits cathepsin B by tightly binding to it 84 . While cystatin B did not show any efficacy in our studies, previous work has shown that when cathepsin B and other inhibitors (calpain and CA-074) are used in conjunction with cathepsin B, they lead to increased inhibition C. neoformans growth when compared to cathepsin B treatment alone 26 . Striatin proteins have a diverse range of functions in both filamentous fungi and mammals including the signaling of motor functions and cell division [85][86][87] . This family and the closely related pyristriatins that have select antimicrobial activity against Gram positive bacteria and some filamentous fungi as well as yeasts 80 . When tested against our yeast, C. neoformans, striatin showed no change in the cryptococcal growth. The S100 series of proteins are known antimicrobial peptides and are also known as alarmins, and these proteins have a range of antimicrobial activity 88,89 . S100A8 and S100A9 form a heterodimer named calprotectin, which has anti-cryptococcal activity by the depletion of zinc from the fungus [90][91][92] . However, the related protein S100A6, which has similar zinc-binding potential, significantly enhanced the cryptococcal growth 93 . The final two proteins tested, calmodulin and calnexin, also significantly enhanced cryptococcal growth. Calmodulin is a calcium binding protein that activates calcineurin and is implicated in increased pathogen defense 44,94 . The membrane associated protein calnexin is also a signaling protein that functions as a chaperone but has been also associated with increased adaptive immunity 95 . DCs that express higher than normal levels of calnexin were able to induce T cell function greater than unmodified DCs 96 . It is currently unknown why these proteins had either limited or opposite effects than previously stated literature would suggest or by what means they increased cryptococcal growth.
To our knowledge, the cytotoxicity of DC lysosomal proteins has not been tested in mammalian cells. Our findings indicated that of the proteins tested, none were cytotoxic at the concentrations needed for anti-cryptococcal activity. This shows promise that these compounds may be used further in the treatment for cryptococcosis. However, the physiological relevance and bioavailability of these compounds in a live model remains to be seen. Our laboratory is currently in the process of testing these lysosomal components in a murine model of cryptococcosis. Furthermore, the complex interactions of these proteins with other proteins residing within the lysosome has not been investigated. None of these proteins are usually present alone or act upon pathogens www.nature.com/scientificreports/ individually, and we recognize that there are many more proteins and protein combinations that may add to the complexity of these interactions. To help us understand these problems, we are actively working on elucidating the mechanism(s) of action for these anti-cryptococcal proteins, studying interaction of multiple lysosomal proteins, and identifying the mechanism(s) of enhanced cryptococcal growth in our lab. The present study has investigated the anti-cryptococcal activity of specific proteins of the DC lysosome. Five different fractions showed similar anti-cryptococcal activity to the entire extract. Mass spectrometry revealed many different compounds present within this extract that could potentially inhibit cryptococcal growth. Our results show that not only are there several that indeed inhibit cryptococcal growth, but these are also not cytotoxic to mammalian cells at these concentrations. From these findings, novel immune-based anti-cryptococcal treatments may be developed from immune-based proteins that could ease the burden brought on by this deadly disease.

Methods
Strains and media. Cryptococcus neoformans strain H99 (serotype A, mating type α) was recovered from 15% glycerol stocks stored at − 80 °C and were cultured for 18 h at 30 °C with shaking in yeast extract-peptonedextrose (YPD) broth (BD Difco; Franklin Lakes, NJ) and collected by centrifugation. Organisms were washed three times with sterile phosphate-buffered saline (PBS), and viable yeast cells were quantified using trypan blue dye exclusion in a hemocytometer. Cryptococcal cells were resuspended in appropriate medium at the concentration needed for each experiment.
Mice. Female

Generation of bone marrow-derived dendritic cells (BMDCs).
BMDC culture was performed as previously described 26,36 . Briefly, bone marrow was flushed from the femurs and tibiae of mice using sterile HBSS. Bone marrow cells were washed, counted, and plated in complete medium (RPMI-1640 supplemented with 10% FBS, 2 mM l-glutamine, 100 U penicillin/ml, 100 μg streptomycin/ml, and 50 mM 2-mercaptoethanol) supplemented with 20 ng/ml recombinant murine GM-CSF (Peprotech, Rocky Hill, NJ) at a concentration of 2 × 10 5 cells/ml in 10 ml (2 × 10 6 cells/plate). Cells were incubated at 37 °C, 5% CO 2 for a total of eight days. At day 3 of incubation, 10 ml complete medium + GM-CSF was added to each plate. At day 6 of incubation, half of the medium was removed, and 10 ml fresh complete medium + GM-CSF was added. BMDCs were harvested at day 8, and DCs were purified by negative selection using anti-F4/80 microbeads (Miltenyi Biotec, Auburn, CA) (to remove contaminating macrophages) followed by positive selection using magnetically labeled anti-CD11c microbeads according to the manufacturer's protocol (Miltenyi Biotec). Dendritic cell purity following this procedure was > 90% by flow cytometry.
Generation of lysosomal extract from BMDCs. DC lysosomal extracts were obtained as previously described using the lysosome isolation kit (Sigma-Aldrich, St. Louis, MO) 26,36 . Briefly, DCs were lysed using 1X extraction buffer (Sigma-Aldrich) followed by homogenization with a PowerGen 700 homogenizer (Fisher Scientific, Pittsburgh, PA) using the 7 × 110 mm homogenizer tip passed through the cells 15-20 times to disrupt 75-80% of the cells. The homogenized cells were then centrifuged at 1000 × g for 10 min to remove intact cells and cellular debris. The first supernatant was removed and centrifuged at 20,000×g for 20 min to pellet lysosomes. Lysosome purity was verified by flow cytometry for lysosomal marker LAMP1 (BD Biosciences, San Jose, CA), which showed > 90% pure lysosomes from this procedure. The pellet containing lysosomes was resuspended and sonicated for 20 s at 40% power on a Model 300 VT Ultrasonic Homogenizer (BioLogics, Inc., Manassas, VA), resulting in lysosome extract (600 mg/ml).

Fractionation and SDS-PAGE of lysosomal extract.
Twelve molecular weight protein fractions of crude lysosomal extract were separated from crude lysosomal extract using GELFREE 8100 System (Expedeon, San Diego, CA). The system uses HEPES running buffer and Tris Acetate sample buffer (Expedeon), and the system was run according to manufacturer's instructions as previously described 97 . Briefly, lysosomal extract was mixed with sample buffer and loaded into the 8% Tris-Acetate cartridge (Expedeon). Following loading of the sample, the instrument was paused for sample collection at predefined intervals. This process was repeated until all 12 fractions were collected. After the collection of fractions, standard SDS-PAGE was performed. For this, a 12.5% Precast Gel (Bio-Rad, Hercules, CA) was loaded with a Precision Protein Plus ladder (Bio-Rad) and each of the 12 samples. Gels were run for 55 min at 200 V in Tris/glycine/SDS running buffer (Bio-Rad). Protein bands in the gel were stained using BioSafe Coomassie Stain (Bio-Rad), and a Gel Doc XR + system (Bio-Rad) was used for visualization.
Anti-cryptococcal assays with lysosomal fractions. Anti-cryptococcal assays were performed as previously described 26,36 . Briefly, C. neoformans was resuspended in phosphate buffer (0.1 mM potassium phosphate monobasic supplemented with 2% RPMI-1640, pH 5.5) at concentration of 1 × 10 5 cells/ml and added to triplicate wells of a 96-well plate in a 50 μl volume. Lysosomal fractions were added to individual triplicate well sets at www.nature.com/scientificreports/ 50 μl volume for a final concentration of 50 μg/ml. Negative control wells included 50 μl C. neoformans with 50 μl of phosphate buffer to equal the volume of experimental wells. Positive control wells contained 50 μl C. neoformans and 50 μl DC lysosomal extract (at 50 ug/ml). Plates were incubated for 24 h at 37 °C, 5% CO 2 . Following incubation, cryptococcal cells in the plates were diluted in PBS and plated onto yeast extract-peptone-dextrose plates supplemented with 1 microgram/ml (ug) chloramphenicol (YPD agar). The plates were incubated at 30 °C for 2 days, and then CFUs were enumerated. Killing was defined as significantly reduced CFUs compared to the inoculum, and inhibition was defined as significantly reduced CFUs compared to Cryptococcus grown alone in phosphate buffer. Each assay was conducted in three independent experiments (n = 3), with each condition performed in duplicate. scan strategy, survey scan followed by acquisition of data-dependent CID spectra of the seven most intense ions in the survey scan above a set threshold. Methionine oxidation and cysteine carbamidomethylation were considered as variable modifications for all searches. Scaffold 4.0 (Proteome Software, Portland, OR, USA) was used to conduct an X! Tandem subset search of the Mascot data followed by cross-correlation of the results of both searches. The Scaffold confidence levels for acceptance of peptide assignments and protein identifications were 95 and 99%, respectively. (Supplementary Table S1).

Identification of lysosomal proteins by HPLC-ESI-MS/MS.
Anti-cryptococcal assays using DC lysosomal proteins. Anti-cryptococcal assays were performed as described above. Proteins were chosen based on previously-described antimicrobial activity, cysteine protease activity (similar to cathepsin B) 26 or calcium interference, which affects calcineurin, a known virulence factor for C. neoformans 44 . All proteins were derived from commercial sources and were > 95% pure. Each protein was prepared at a concentration determined by either published studies or from our previous studies with lysosomal proteins 26,[45][46][47][48] (50 μl) to equal 100 μl in each well, and plates were incubated at 37 °C, 5% CO 2 for 24 h. After incubation, contents of wells were diluted and plated on YPD agar to quantify CFUs. Each protein was tested in three independent experiments (n = 3), with each condition performed in triplicate. Additional anti-cryptococcal assays were performed to test dose-dependent activity of the DC lysosomal proteins. For these, proteins used were diluted from working concentrations to 1:10 and 1:100 with phosphate buffer for 10% and 1% concentrations, respectively, and the anti-cryptococcal assay was conducted as described above. Each anti-fungal assay was conducted in three independent experiments (n = 3), with each condition performed in triplicate. Percent inhibition was defined as 100% (positive inhibition control) minus the experimental condition CFUs divided by the negative inhibition CFUs × 100%. Negative percent inhibition is the result of higher CFUs in the experimental compared to negative inhibition control. www.nature.com/scientificreports/ measured in vitro using a checkerboard method as previously described 57 . Assays were conducted in either phosphate buffer (pH 5.5) or RPMI-MOPS (pH 7.0) (both described above) in 100 µl total. A 96-well plate was used with rows A-G and columns 1-9 used to create the checkerboard titrations between AmpB and a lysosomal protein. Off-scale MICs were considered to be the highest or lowest concentration tested in the assay. Each checkerboard assay was conducted in two independent experiments (n = 2) for each lysosomal protein and for each buffer.

Minimum inhibitory concentration (MIC) assays of anti-cryptococcal
Time-kill assay. Rate of cryptococcal killing by lysosomal protein was measured using a time-kill assay as previously described 59 . C. neoformans strain H99 was tested at a concentration of 1 × 10 5 cells/ml with lysosomal proteins at 2X their respective MICs (coronin: 25 μg/ml, HNE: 3.125 μg/ml, MPO: 18 μg/ml, MMP25: 0.78 μg/ ml, NOSTRIN: 0.25 μg/ml) in 1000 μl of phosphate buffer (pH 5.5). Solutions were incubated for 48 h at 35 °C with 50 μl aliquots being taken at hours 0-8, 12, 24, 36, and 48 post inoculation. Aliquots were diluted and plated on YPD agar for 48 h at 30 °C to enumerate CFUs. Each time-kill assay was conducted in two independent experiments (n = 2) for each protein along with fungus only growth control. Percent inhibition was calculated as described above with any negative inhibition values displayed as 0% inhibition.
Cytotoxicity assay. The macrophage cell line J774A.1 (TIB-67; ATCC, Manassas, VA) was used for cytotoxicity assays of the present study. J774A.1 cells were grown in cell culture medium (DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10% NCTC-109, 1% non-essential amino acids, 100 U penicillin/ml, and 100 μg streptomycin/ml) in CytoOne T75 tissue culture flasks (USA Scientific, Ocala, FL) at 37 °C, 5% CO 2 . J774A.1 cells were passaged according to manufacturer's directions. For detection of cytotoxicity of DC lysosomal proteins, the Vybrant Cytotoxicity Assay Kit (Molecular Probes, Eugene, OR) was used according to manufacturer's instruction at near physiological pH (7.5). Briefly, J774A.1 cells were added to a 96-well plate in triplicate at a concentration of 10 × 10 5 cells/ml in 25 μl. Lysosomal proteins were prepared similarly to the anticryptococcal assay except cell culture media was used for dilutions instead of phosphate buffer. Each protein was used at 25 μl per well. Controls included no-cell, untreated cells, and fully lysed cells. All wells were prepared in triplicate. Plates were incubated for 24 h at 37 °C, 5% CO 2 . After incubation, 50 μl of reaction mixture was added to all wells. Fluorescent readings were taken on a Synergy HTX multi-mode plate reader (BioTek, Winooski, VT) with filters for 530/25 (excitation) and 590/20 (emission). Each cytotoxicity assay was conducted in three independent experiments (n = 3), with each condition performed in triplicate.
Statistical analysis. Unpaired two-tailed t-tests were used to compare CFUs using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). Significant differences were defined as p < 0.05 and α = 0.05 unless noted otherwise.

Data availability
The mass spectrometry datasets generated during and/or analyzed during the current study are included in this published article (Supplementary Table S1). www.nature.com/scientificreports/