Susceptibility

Copper Acyl Salicylate Has Potential as an Anti-Cryptococcus Antifungal Agent

Adepemi O. Ogundeji, Boitumelo F. Porotloane, Carolina H. Pohl, Pravin S. Kendrekar, Olihile M. Sebolai
DOI: 10.1128/AAC.02345-17

ABSTRACT

The in vitro antifungal activity of aspirin against cryptococcal cells has been reported. However, the unwanted effects of aspirin may limit its clinical application. Conceivably, a derivative of aspirin could overcome this challenge. Toward this end, this study considered the usage of an aspirinate-metal complex, namely, copper acyl salicylate (CAS), as an anti-Cryptococcus antifungal agent. Additionally, the study examined the effects of this compound on macrophage function. The in vitro susceptibility results revealed that cryptococcal cells were vulnerable (in a dose-dependent manner) to CAS, which might have effected growth inhibition by damaging cryptococcal cell membranes. Interestingly, when CAS was used in combination with fluconazole or amphotericin B, synergism was observed. Furthermore, CAS did not negatively affect the growth or metabolic activity of macrophages; rather, it sensitized those immune cells to produce interferon gamma and interleukin 6, which, in turn, might have aided in the phagocytosis of cryptococcal cells. Compared to our aspirin data, CAS was noted to be more effective in killing cryptococcal cells (based on susceptibility results) and less toxic toward macrophages (based on growth inhibition results). Taking these findings together, it is reasonable to conclude that CAS may be a better anti-Cryptococcus drug that could deliver better therapeutic outcomes, compared to aspirin.

INTRODUCTION

Although it has become easy to diagnose cryptococcal infections, even in resource-poor settings (1–4), treatment has largely remained difficult, due to current antifungal drugs being too expensive for some regions of the world. Importantly, the usage of some antifungal drugs is limited by their unintended side effects (5). A classic example is that of amphotericin B (administered to treat disseminated cryptococcal infections in patients with HIV), which is also known to target host renal tubules (5, 6). Given the risks associated with this drug and considering that, in South Africa, 13 of every 100 people in the total population are living with HIV (7), it is reasonable to extrapolate that a significant proportion of this population group is in danger of dying from this opportunistic fungus. Because of these challenges, there is an urgency to find better management strategies to control cryptococcal infections.

Our group has actively sought to identify and to demonstrate “new” applications of “old” drugs, typically those that are FDA approved and are currently purposed to treat noninfectious conditions such as inflammation, as anti-Cryptococcus drugs. In one of our recent studies, we successfully demonstrated that the prototypical anti-inflammatory drug aspirin can be repurposed as an anti-Cryptococcus antifungal agent (8). However, the usage of aspirin in clinical settings can often lead to gastrointestinal toxicity (9). In order to derive the maximum therapeutic benefits from aspirin, Sorenson considered preparing an aspirinate-copper complex (a complex in which copper is bound to four aspirin ligands) as an alternative to aspirin (10). Copper complexes are traditionally known to be more effective and less toxic than their individual parent compounds (11, 12). To illustrate this point, Sorenson noted that the aspirinate-copper complex was 30 times more effective than aspirin as an anti-inflammatory agent. Moreover, the aspirinate-copper complex was pharmacologically more active than aspirin in laboratory animals (10). Based on those findings, it became the aim of the current study to determine whether an aspirinate-metal complex, i.e., copper acyl salicylate (CAS), could also exert antimicrobial effects on cryptococcal cells and, importantly, whether it would yield better results than those reported by Ogundeji and coworkers for aspirin (8).

RESULTS AND DISCUSSION

CAS has antifungal activity and acts in synergy with conventional drugs.Table 1 summarizes the effects of CAS on the growth of the 10 clinical and 2 reference Cryptococcus strains tested. From Table 1, it can be observed that, as the concentration of CAS was increased, there was a corresponding reduction in growth of the cells. The greatest growth reduction was achieved at 844 μg/ml, which is equivalent to 1 mM. At that concentration, CAS effected ≥50% growth reduction, compared to the drug-free control. Interestingly, we previously reported that aspirin (at the same drug concentration [1 mM]) also effected ≥50% growth reduction (8). When the antimicrobial activity of CAS was directly compared with that of aspirin against all 10 strains, however, CAS was noted to be more effective in reducing growth (data not shown). This determination is important, as it confirms what is expected of a metal complex, that is, it should display strong biological activities, compared to its individual parent compounds (10, 11).

View this table:
TABLE 1

Effects of CAS on growth of C. neoformans and C. gattii

In addition to negatively affecting growth, drug treatment also influenced the ultrastructural appearance of the cells and their size (Fig. 1). Compared to treated cells (3.91 ± 0.06 μm in cell diameter), the nontreated cells (4.14 ± 0.05 μm in cell diameter) were observed to be bigger (P < 0.05). Furthermore, the nontreated cells appeared to be covered with an extracellular matrix, which could possibly be the capsule. Drug treatment seemed to reduce this covering, which might leave cells “naked” and vulnerable to adverse external conditions (i.e., the effects of other drugs when combined therapy is administered or even macrophage action). To ascertain whether cells might have lost their capsules in the presence of CAS, the amount of shed glucuronoxylomannan (GXM) in the growth medium was measured (Fig. 2). Here, it was noted that treated cells shed significantly (P < 0.01) more GXM than nontreated cells.

FIG 1
FIG 1

Scanning electron micrographs, showing the effects of CAS on the ultrastructure and size of cells. CAS-treated cells showed more extracellular matrix (which might be the capsule) on their cell wall surfaces, compared to nontreated cells. The treated cells were significantly (P < 0.05) smaller in diameter (3.91 ± 0.06 μm) than nontreated cells (4.14 ± 0.05 μm).

FIG 2
FIG 2

Quantitative ELISA results, showing the levels of GXM shed by nontreated cells and treated cells. The results indicate that cells shed their capsules significantly (P < 0.01) more in the presence of CAS than in its absence.

Tables 2 and 3 summarize the combined effects of CAS and amphotericin B and those of CAS and fluconazole on strain LMPE 046 (most sensitive to CAS), LMPE 052 (most resistant to CAS), LMPE 109 (Cryptococcus gattii reference strain), and LMPE 150 (Cryptococcus neoformans reference strain). When the data were analyzed, it was pleasing to note that certain drug combinations (shown in shaded cells) led to synergistic effects that translated into corresponding growth reduction of ≥50%. Importantly here, the concerned concentration of a particular drug (CAS, amphotericin B, or fluconazole) used in combined therapy was lower than the concerned concentration of that particular drug used alone. The significance of the latter finding is that, when CAS is used in combined therapy, it should (i) minimize the unintended risks associated with using amphotericin B or (ii) enhance the effectiveness of fluconazole, which is known to be fungistatic.

View this table:
TABLE 2

Combined effects of CAS and amphotericin B on selected Cryptococcus strainsa

View this table:
TABLE 3

Combined effects of CAS and fluconazole on selected Cryptococcus strainsa

CAS subjects cells to oxidative stress.Fig. 3A shows the effect of CAS on reactive oxygen species (ROS) production. The CAS-treated cells were shown to accumulate significant amounts of ROS (P < 0.01), compared to nontreated cells. In addition to being produced as part of normal oxygen metabolism of respiring cells (13), any impairment in the shuttling of electrons and the generation of membrane potential can lead to ROS. This impairment can, among other things, be induced by exposure of cells to certain drugs, as was the case with aspirin (8). In the latter study, it was shown that aspirin exposure led to loss of membrane potential. It is our assumption that, like aspirin, CAS might have led to loss of membrane potential and thus the observed ROS accumulation. Given the unwanted effects of ROS on cellular macromolecules (14), cells can signal an adaptive response under conditions of stress (15, 16). To investigate the latter, we sought to determine whether ROS may serve as a stimulant that activates a stress response pathway, by assaying the phosphorylation of p38 mitogen-activated protein kinase (MAPK). Figure 3B shows a significant increase (P < 0.01) in the levels of phosphorylation of p38 MAPK in CAS-treated cells, compared to levels recorded for nontreated cells. Based on the results in Fig. 3A and B, it is reasonable to conclude that CAS seems to have subjected cells to oxidative stress, as noted from the elevated levels of p38 MAPK and excessive accumulation of ROS. Therefore, it is our argument that ROS might have targeted the cell walls, as seen in Fig. 1, and compromised their integrity; importantly, this loss of integrity might have led to growth reduction, as noted in Table 1.

FIG 3
FIG 3

(A) ROS assay results, showing the effects of CAS on treated cells. Treatment of cells with CAS led to a significant (P < 0.01) increase in ROS accumulation, compared to nontreated cells. (B) p38 MAPK assay results, showing the effects of CAS on p38 phosphorylation levels. The p38 phosphorylation levels of CAS-treated cells were significantly (P < 0.01) higher than those of nontreated cells.

CAS improves the functioning of murine macrophages.When the effects of CAS on macrophages were considered, it was noted that CAS (at 844 μg/ml) led to a 6% reduction in metabolic activity (Fig. 4) of treated macrophages, compared to nontreated macrophages. This finding (6%) further implied that far more than 844 μg/ml (1 mM) CAS would need to be added to the cultivation medium of macrophages in order to yield a lethal dose, at which 50% of metabolic activity would be negatively affected. However, a 50% reduction was achieved at a concentration that was 10 times (i.e., 8,440 μg/ml) the determined MIC (Fig. 4). When the therapeutic index (TI) was calculated, it was determined that the therapeutic range was wide enough, as a concentration 10 times the MIC was required to yield a 50% lethal dose. In their review article, Tamargo et al. (17) reported that a drug with a TI of ≥10 is generally considered a safe drug. Thus, our findings are in line with those generally accepted in the literature.

FIG 4
FIG 4

Effect of CAS on macrophage metabolic activity, expressed as the percent change in metabolic activity. At the MIC (844 μg/ml), CAS was nontoxic to macrophages, as it did not yield a 50% reduction in metabolic activity. At 10 times the MIC (8,440 μg/ml), however, a 50% reduction in metabolic activity was achieved. LD50, 50% lethal dose.

We also determined whether CAS would chemically sensitize macrophages and thus enhance their phagocytic capability. The data shown in Fig. 5 revealed that the presence of CAS significantly enhanced the efficiency of macrophages to internalize cryptococcal cells, by 65%, compared to nontreated cells. Furthermore, non-CAS-treated macrophages produced significantly less interferon gamma (IFN-γ) than did CAS-treated macrophages (P < 0.05) (Fig. 6A). Similarly, non-CAS-treated macrophages produced significantly less interleukin 6 (IL-6) than did CAS-treated macrophages (P < 0.05) (Fig. 6B). Taken together, the findings presented in Fig. 5 and 6 suggest that CAS might have improved the functioning of macrophages, as those immune cells were able to produce more cytokines (Fig. 6), which might have allowed them to better recognize (perhaps due to the loss of the extracellular covering, as seen in Fig. 1) and to internalize more cryptococcal cells (Fig. 5).

FIG 5
FIG 5

Effect of CAS on macrophage (MØ) phagocytic capability. The addition of CAS significantly (P < 0.01) enhanced the phagocytic capability of macrophages, compared to that of nontreated macrophages.

FIG 6
FIG 6

Effects of CAS on macrophage (MØ) immunological responses. The addition of CAS led to significantly higher levels of proinflammatory cytokines, compared to nontreated macrophages, i.e., 74% increase in the level of IFN-γ (A) and 88% increase in the level of IL-6 (B) in the presence of CAS.

In conclusion, the manifestation of disseminated cryptococcal infections in HIV-infected individuals is a serious problem, particularly for people living in sub-Saharan Africa. Issues involving current antifungal drugs (amphotericin B and its side effects and fluconazole being fungistatic) only worsen the matter. Therefore, we have proposed a number of alternative drugs that may be considered to manage cryptococcal infections (8, 18). The current study looked specifically at the effects of a metal complex, CAS, and was informed by our initial work on aspirin. It was interesting to note that CAS was more effective than aspirin in controlling the growth of cryptococcal cells; this is a quality that is to be expected for a metal complex, and our findings are in line with the observations of Sorenson and coworkers with respect to aspirinate metal complexes. Although CAS may be more attractive than aspirin, it is not known at this point whether it will also produce less gastrointestinal toxicity. Therefore, studies are now required to examine this aspect.

For our in vitro susceptibility assays, we followed the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, which is significant because the work can be reproduced elsewhere and provides insight into the effectiveness of the test drug used. Moreover, the results give an indication of whether the drug may decrease the microbial burden in laboratory animals. We are currently seeking ethics approval to test the latter. We were able to establish that, like aspirin, CAS may inhibit the growth of cells by disrupting membrane function. Membranes are crucial for nonfermenting microbes such as Cryptococcus, which are entirely dependent on oxygen metabolism for energy to support their growth. Therefore, it will be interesting to see whether laboratory animals (which, like humans, respire) are adversely affected by CAS treatment. To date, we have established (in vitro) only that a mammalian cell line was not negatively affected by CAS and not that exposure to CAS was beneficial.

MATERIALS AND METHODS

Cells, cultivation, and standardization.A total of 10 clinical fungal strains (obtained from Universitas Academic Hospital, Bloemfontein, South Africa) were tested. The strains included 5 C. neoformans strains (LMPE 028 [C. neoformans var. neoformans], LMPE 030 [C. neoformans var. neoformans], LMPE 043 [C. neoformans var. neoformans], LMPE 046 [C. neoformans var. neoformans], and LMPE 047 [C. neoformans var. neoformans]) and 5 C. gattii strains (LMPE 045 [C. neoformans var. gattii], LMPE 048 [C. neoformans var. gattii], LMPE 052 [C. neoformans var. gattii], LMPE 054 [C. neoformans var. gattii], and LMPE 070 [C. neoformans var. gattii]). Moreover, reference strains for C. neoformans (strain H99 [LMPE 150]) and C. gattii (strain R265 [LMPE 109]) were included for comparison purposes. The strains were streaked on yeast-malt-extract (YM) agar (3,000 μg/ml yeast extract, 3,000 μg/ml malt extract, 5,000 μg/ml peptone, 10,000 μg/ml glucose, and 16,000 μg/ml agar) (Merck, South Africa) and incubated for 2 days at 30°C. Following this, five colonies were selected and suspended in 10 ml of distilled water. Next, standardized inocula (0.5 × 105 and 2.5 × 105 CFU/ml) were prepared as described by EUCAST (19). In addition, a murine macrophage cell line (RAW 264.7 [ATCC TIB-71], a kind donation by Peter Masoko and Raymond Makola, University of Limpopo, South Africa) was used. The cell line was initially obtained from ATCC. The cells were grown in 5% CO2 at 37°C in RPMI 1640 medium (Sigma-Aldrich, South Africa), supplemented with 10% fetal bovine serum (Biochrom, Germany), an antibiotic cocktail of penicillin (20 U/ml; Sigma-Aldrich) and streptomycin (20 g/ml; Sigma-Aldrich), and 2 mM l-glutamine (Sigma-Aldrich), until they reached 80% confluence. The cells were standardized to a final concentration of 1 × 105 cells/ml before use and then were seeded into the wells of a sterile, disposable, 96-well, flat-bottom, microtiter plate (Greiner Bio-One, Germany).

Drugs.CAS (a kind donation by Pravin Kendrekar, Health Sciences Department, Central University of Technology), fluconazole (Sigma-Aldrich), and amphotericin B (Sigma-Aldrich) were obtained as standard powders. CAS was dissolved in absolute ethanol (Merck) and fluconazole was dissolved in distilled water, while dimethyl sulfoxide (Merck) was used for amphotericin B. These compounds were further diluted using RPMI 1640 medium to reach the desired final concentrations in the wells of the microtiter plate; therefore, the final concentrations of drug diluents in which the stock solutions were prepared never exceeded 1%. For comparative purposes, a concentration gradient of 0.01 mM (8.44 μg/ml), 0.1 mM (84.4 μg/ml), and 1 mM (844 μg/ml), similar to that we used for aspirin (8), was used in the current study.

Susceptibility assays.The in vitro susceptibility assays were performed according to EUCAST guidelines (19). In brief, wells of sterile, 96-well, flat-bottom, microtiter plates were seeded with 100 μl of standardized cryptococcal cells. The cells were immediately treated with 100 μl of the test drug (CAS, fluconazole, or amphotericin B) at twice the desired final concentration, as stated above. Nontreated cells were included as controls. The plates were incubated for 48 h at 37°C before the optical density at 562 nm (OD562) of the wells was read using a spectrophotometer (Biochrom EZ Read 800 Research). At the end, the percent growth reduction was calculated as follows: (OD562 of treated cells/OD562 of nontreated cells) × 100%. Concentrations that led to ≥50% reductions in growth were used in the checkerboard assays. In anticipation of the checkerboard assays, the concentrations of amphotericin B and fluconazole that led to ≥50% reductions in growth were 1 μg/ml and 8 μg/ml, respectively; these figures were based on those we reported for aspirin (8). The current study and the aforementioned aspirin study were performed with the same strains, in the same laboratory, and during the same period.

Checkerboard assays.For the assays, LMPE 046 (most sensitive to CAS), LMPE 052 (most resistant to CAS), LMPE 109 (C. gattii reference strain), and LMPE 150 (C. neoformans reference strain) cells were used. Standardized cryptococcal cells (in 100 μl of RPMI 1640 medium) were seeded into the wells of a sterile microtiter plate and immediately were treated with CAS paired with amphotericin B (50 μl and 50 μl) or CAS paired with fluconazole (50 μl and 50 μl). The plate was incubated at 37°C for 48 h. At the end of the incubation period, OD562 readings were obtained, and then the fractional inhibitory concentration index (FICI) was calculated. The FICI (that is, the sum of the fractional inhibitory concentration [FIC] values) was defined as FICA + FICB, where FICA is MIC of drug A in combination/MIC of drug A alone and FICB is MIC of drug B in combination/MIC of drug B alone (8). FICI values were determined to establish whether there was synergism (FICI of ≤0.5), no interaction (FICI of >0.5 to 4), or antagonism (FICI of >4). The effect of CAS (at the determined MIC) on the ultrastructure of cryptococcal cells, the mode of action of CAS, and CAS-macrophage interactions were examined using the strain that showed the greatest sensitivity.

Effects of CAS on cellular ultrastructure.To perform scanning electron microscopy (SEM), 48-h-old cells (prepared as detailed for the in vitro susceptibility assays), i.e., nontreated cells and CAS-treated cells (844 μg/ml), were considered. The chosen cells were prepared for SEM as previously described by van Wyk and Wingfield (20). In brief, cells were chemically fixed with sodium phosphate-buffered 3% glutardialdehyde (Merck) and sodium phosphate-buffered 3% osmium tetroxide (Merck), following dehydration in a graded ethanol (Merck) series. Then the cells were dried, mounted on stubs, and coated with gold using an SEM coating system (Bio-Rad Microscience Division) (20). Cells were examined using a Shimadzu Superscan SSX 550 scanning electron microscope (Shimadzu, Japan). To determine the diameter of cells, 100 cells for each experimental condition (randomly selected from different locations acquired from different stubs) were measured.

To complement the experiment described above, the effect of CAS in causing cells to shed their capsule (GXM) was determined. In brief, treated and nontreated cells were prepared as detailed for the in vitro susceptibility assays and grown for 48 h. However, to determine the changes in the amounts of shed GXM, the supernatant was aspirated (at different time points, i.e., 0 h, 12 h, 24 h, and 48 h) from the wells. The supernatant was then transferred to the wells of an enzyme-linked immunosorbent assay (ELISA) microtiter plate (IMMY, USA) specific for GXM quantification. The plate was treated according to the guidelines provided by IMMY. The OD450 was measured using a spectrophotometer.

Effects of CAS on ROS and phospho-p38 MAPK.These assays were performed as detailed previously by Ogundeji et al. (8). For both assays, cells were prepared as detailed for the in vitro susceptibility assay. At the end of the 48-h incubation period, the plate was gently agitated to resuspend the cells. For ROS analysis, 90 μl of medium containing cells (separately collected from wells with nontreated cells and wells with CAS-treated cells) was aspirated and transferred to designated wells in a sterile, black, 96-well, flat-bottom, microtiter plate (Greiner Bio-One). The cells were reacted with 10 μl of the fluorescent dye 2′,7-dichlorofluorescin diacetate (DCFHDA) (1 mg/ml; Sigma-Aldrich) and incubated for 30 min in the dark at room temperature. The induced fluorescence was measured (excitation wavelength, 485 nm; emission wavelength, 535 nm) using a Fluoroskan Ascent FL microplate reader (Thermo-Scientific, USA). For phospho-p38 MAPK analysis, 100 μl of medium containing cells (separately collected from wells with nontreated cells and wells with CAS-treated cells) was aspirated and transferred to sterile, 1.5-ml Eppendorf tubes (Merck). Following this, the lysate was harvested from the cells according to a protocol and with materials provided in the phospho-p38 MAPK ELISA kit (Sigma-Aldrich). The lysate was then transferred to a sterile microtiter plate specific for phospho-p38 MAPK assays, and the lysate plate was treated according to the manufacturer's protocol. The plate was read at 450 nm using a Biochrom EZ spectrophotometer.

Cytotoxic effects of CAS on murine macrophages.The macrophage assay was performed as previously detailed by Ogundeji et al. (8). Standardized macrophages (1 × 105 cells/ml) were resuspended in fresh medium following overnight seeding at 37°C in a 5% CO2 incubator (Thermo-Scientific). The cells were immediately treated with 100 μl of CAS (1:1 [vol/vol]) to reach a final drug concentration of 844 μg/ml (MIC), 4,220 μg/ml (5 times the MIC), or 8,440 μg/ml (10 times the MIC). Nontreated macrophages were included as a control. The plate was incubated for 48 h at 37°C in a 5% CO2 incubator.

To determine CAS effects on metabolic activity, cells were reacted with 54 μl of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) (Sigma-Aldrich) in the presence of 1 mM menadione (Sigma-Aldrich). Following that, the plate was incubated in the 5% CO2 incubator. The OD492 of the wells was measured 3 h after initiation of the tetrazolium reaction, using a Biochrom spectrophotometer. The TI of CAS was calculated by dividing the 50% lethal dose by the MIC obtained, to determine whether the range was narrow or wide (17).

The phagocytic function of macrophages, i.e., the ability to internalize cryptococcal cells in the presence or absence of CAS, was measured using a phagocytosis stain, pHrodo Green Zymosan A BioParticles (Life Technologies, USA). In brief, 1 μl of this stain was added to 999 μl of standardized cryptococcal cells in RPMI 1640 medium, and the mixture was incubated for 1 h at 37°C while being slowly agitated. After 1 h, cryptococcal cells were washed twice with phosphate-buffered saline, centrifuged, and resuspended in 1,000 μl of fresh medium that contained twice the desired final concentration of CAS (844 μg/ml). A coculture was prepared by adding 100 μl of the stained cryptococcal cells to seeded macrophages (1:1 [vol/vol]) at an effector/target ratio of 1:1. The plate was incubated for 6 h at 37°C in a 5% CO2 incubator. After the incubation period, the induced fluorescence was measured (excitation wavelength, 492 nm; emission wavelength, 538 nm) using a Fluoroskan Ascent FL microplate reader.

At the end, the immunological response of macrophages to cryptococcal cells in the presence or absence of CAS was also measured. Here, cryptococcal cells were suspended in fresh RPMI 1640 medium that contained twice the desired final concentration of CAS (844 μg/ml) or in medium alone (without CAS). A coculture was prepared by adding 100 μl of the cryptococcal cells to seeded macrophages (1:1 [vol/vol]) at an effector/target ratio of 1:1. The plate was incubated for 6 h at 37°C in a 5% CO2 incubator. After the incubation period, the supernatants were collected and kept for cytokine ELISAs, i.e., IFN-γ and IL-6. Following this, 100 μl of the supernatant (separately obtained from cocultures with or without CAS) was transferred to wells of a sterile microtiter plate specific for IFN-γ or IL-6 (BioLegend, USA). The supernatants were then treated according to the respective manufacturer's protocol. The OD450 of the plates was measured using a Biochrom EZ spectrophotometer.

Statistical analysis.All data represent mean values of three biological replicates for each strain studied, unless stated otherwise. Where appropriate, standard deviations (SDs) and Student's t tests were calculated to determine the statistical significance of data for the different experimental conditions. P values of ≤0.05 were regarded as statistically significant.

ACKNOWLEDGMENTS

We are grateful for the services and assistance offered by Pieter van Wyk and Hanlie Globler in SEM work. We are also grateful for the input of the reviewers.

This work was supported by the National Research Foundation of South Africa (grant UID 87903) and the University of the Free State.

We have no conflicts of interest to declare.

FOOTNOTES

    • Received 15 November 2017.
    • Returned for modification 3 February 2018.
    • Accepted 7 May 2018.
    • Accepted manuscript posted online 14 May 2018.

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KEYWORDS

copper acyl salicylate
Cryptococcus
macrophages
membrane damage
synergy

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