Macrolides Inhibit Capsule Formation of Highly Virulent Cryptococcus gattii and Promote Innate Immune Susceptibility

Cryptococcus gattii is a fungal pathogen, endemic in tropical and subtropical regions, the west coast of Canada, and the United States, that causes a potentially fatal infection in otherwise healthy individuals. Because the cryptococcal polysaccharide capsule is a leading virulence factor due to its resistance against innate immunity, the inhibition of capsule formation may be a promising new therapeutic strategy for C. gattii.

ABSTRACT

Cryptococcus gattii is a fungal pathogen, endemic in tropical and subtropical regions, the west coast of Canada, and the United States, that causes a potentially fatal infection in otherwise healthy individuals. Because the cryptococcal polysaccharide capsule is a leading virulence factor due to its resistance against innate immunity, the inhibition of capsule formation may be a promising new therapeutic strategy for C. gattii. Macrolides have numerous nonantibiotic effects, including immunomodulation of mammalian cells and suppression of bacterial (but not fungal) pathogenicity. Thus, we hypothesized that a macrolide would inhibit cryptococcal capsule formation and improve the host immune response. Coincubation with clarithromycin (CAM) and azithromycin significantly reduced the capsule thickness and the amount of capsular polysaccharide of both C. gattii and C. neoformans. CAM-treated C. gattii cells were significantly more susceptible to H₂O₂ oxidative stress and opsonophagocytic killing by murine neutrophils. In addition, more C. gattii cells were phagocytosed by murine macrophages, resulting in increased production of tumor necrosis factor alpha (TNF-α) by CAM exposure. After CAM exposure, dephosphorylation of Hog1, one of the mitogen-activated protein kinase (MAPK) signaling pathways of Cryptococcus, was observed in Western blot analysis. In addition, CAM exposure significantly reduced the mRNA expression of LAC1 and LAC2 (such mRNA expression is associated with cell wall integrity and melanin production). These results suggest that CAM may aid in inhibiting capsular formation via the MAPK signaling pathway and by suppressing virulent genes; thus, it may be a useful adjunctive agent for treatment of refractory C. gattii infection.

INTRODUCTION

Cryptococcus gattii is an endemic fungal pathogen that is seen not only in tropical and subtropical regions but also in the Pacific Northwest of Canada and the United States and sporadically in other parts of the United States and that infects otherwise healthy individuals, causing pulmonary and cerebral infections (1, 2). C. gattii occurs in apparently healthy individuals and in those with immune impairment, e.g., those with impairment due to diabetes and HIV. Its occurrence is more common in apparently healthy persons than that of C. neoformans. The fungi commonly enter the host via inhalation into the lungs, where they encounter neutrophils and resident phagocytes, including macrophages and dendritic cells, whose response has a pronounced impact on disease outcome. These phagocytic cells contain pattern recognition receptors that enable recognition of specific components of the cryptococcal cell wall and capsule, resulting in the induction of expression of several inflammatory cytokines to trigger adaptive T-cell immunity against fungi (3). However, Cryptococcus possesses several virulence factors, including a thick polysaccharide capsule, melanin production, and secretion of various enzymes, that aid in evading the immune system or enhancing its ability to develop within the phagocyte (4, 5).

Macrolides are antibiotics classically known as protein synthesis inhibitors with extensive broad antimicrobial effects on Gram-positive cocci and atypical pathogens. Moreover, several studies demonstrate the immunomodulatory effects of macrolides. The role of macrolides in regulating excessive inflammation is well characterized, and their role in activating innate immunity, including their ability to inhibit neutrophil migration (6), regulate monocyte differentiation into macrophages, and modulate macrophage function (7, 8), has been established. Extensive data support the observation that in mammalian cells, macrolides influence intracellular mitogen-activated protein kinase (MAPK), especially extracellular signal-regulated kinase 1 (ERK1)/2, and the NF-κB pathway downstream of ERK (9, 10). In addition, several studies have shown the inhibiting effect of macrolides on bacterial virulence factors. At sub-MIC levels, macrolides inhibit the synthesis of exotoxin A, elastase, phospholipase C, pyocyanin, etc., all of which are virulence factors associated with Pseudomonas aeruginosa infection (11–13). Tateda et al. (14) reported that azithromycin (AZM) interferes with autoinducer synthesis of Pseudomonas aeruginosa, leading to a reduction of virulence factor production. Subinhibitory concentrations of telithromycin were shown previously to suppress the capsular formation of Streptococcus pneumoniae (15). On the basis of that previous study, we speculated that macrolides may inhibit the virulence of C. gattii, particularly with respect to reduction of the polysaccharide capsule, and may be responsible for an altered innate immune response, especially that involving neutrophils and macrophages, which is consistent with effective host protection. This is the first study to have investigated the effects of macrolides on fungi and to have demonstrated a promising new treatment strategy to manage refractory fungal infection by inhibiting virulence factors.

RESULTS

Macrolide exposure reduced capsule formation of C. gattii.

To examine the effect of CAM for capsule formation, we measured the capsule thickness of 200 randomly selected C. gattii cells with or without CAM exposure. As shown in Fig. 1 and Table 1, capsule thickness was reduced significantly by CAM exposure in C. gattii strains PNG18, JP02, and R265 (untreated cells versus CAM-treated cells) (for strain PNG18, 1.93 ± 0.18 μm versus 0.60 ± 0.137 μm [P < 0.0001]; for strain JP02, 1.78 ± 0.15 μm versus 0.62 ± 0.66 μm [P < 0.0001]; for strain R265, 1.24 ± 0.59 μm versus 0.54 ±0.04 μm [P < 0.001]). No significant differences were observed in C. neoformans strain H99 and C. gattii strain JP01; however, in both, capsule thickness was prone to being reduced by CAM exposure. We also examined the inhibitory effect of capsule formation by using azithromycin (AZM), the other of the existing macrolide antibiotics, with the result that AZM also reduced the capsule formation of C. gattii R265 significantly (untreated cells versus AZM-treated cells) (for strain R265, 1.01 ± 0.52 μm versus 0.52 ± 0.01 μm [P < 0.01]). Furthermore, we performed the assay by using cells pretreated with MOPS (morpholinepropanesulfonic acid) buffer to induce the growth of the capsule (16). As shown in Table 1, a CAM-mediated reduction of capsule thickness was still observed in the cells with enlarged capsule (untreated cells versus CAM-treated cells with enlarged capsule) (for strain R265, 5.65 ± 0.44 μm versus 3.57 ± 0.44 μm [P < 0.0001]). The sizes of the cell bodies were not altered significantly by macrolides in either C. gattii or C. neoformans. Next, we measured the total amount of capsular polysaccharide (CPS) with or without macrolide exposure. Significant reductions in the amount of CPS in the CAM-treated group compared with the untreated controls were seen in both C. gattii and C. neoformans (Fig. 2); likewise, the amount of CPS in cells of AZM-treated C. gattii strain R265 was also reduced significantly. No significant differences in in vitro growth with or without CAM were observed (data not shown). Ultimately, macrolide treatments reduced CPS production and inhibited capsule growth in both C. gattii and C. neoformans; however, the size of cell body was not affected.

FIG 1.

CAM reduced the capsule thickness of C. gattii strains JP02 and R265. The cells were incubated with or without CAM for 24 h, and the capsule thickness of 200 randomly selected cells was measured. The bar in the first panel denotes 10 μm and applies also for the rest of the panels. Data are presented as means ± SEM. India ink staining, ×1,000.

TABLE 1.

The effects of macrolides on capsule thickness and cell body size^a

Species and strain name	Macrolide	Exposure	Capsule size (μm; mean ± SD)	P value	Body size (μm; mean ± SD)	P value
C. neoformans
H99	CAM	+	0.47 ± 0.07	NS	5.04 ± 0.81	NS
		−	0.66 ± 0.24		5.00 ± 0.75
C. gattii
JP01	CAM	+	0.55 ± 0.05	NS	6.07 ± 0.97	NS
		−	0.70 ± 0.08		6.45 ± 0.95
PNG18	CAM	+	0.60 ± 0.137	<0.0001	6.20 ± 0.51	NS
		−	1.93 ± 0.18		6.09 ± 0.70
R265	CAM	+	0.54 ± 0.04	<0.001	5.62 ± 0.68	NS
		−	1.24 ± 0.59		5.93 ± 0.45
JP02	CAM	+	0.62 ± 0.66	<0.0001	5.41 ± 0.53	NS
		−	1.78 ± 0.15		5.43 ± 0.27
R265	AZM	+	0.52 ± 0.01	<0.01	5.83 ± 0.60	NS
		−	1.01 ± 0.52		5.57 ± 0.36
R265 (+ MOPS)	CAM	+	3.57 ± 0.44	<0.0001	7.92 ± 1.25	NS
		−	5.65 ± 0.44		7.27 ± 1.20

^aCAM, clarithromycin; AZM, azithromycin; NS, not significant.

FIG 2.

The amounts of capsular polysaccharide of C. neoformans and C. gattii were reduced by macrolides. C. gattii strains were grown in mYNB at 30°C with or without macrolides for 24 h. The CPS fraction was extracted, and the total amount of polysaccharides was monitored using the phenol-H₂SO₄ method. Data are presented as means ± SEM (*, P < 0.05).

Susceptibility to oxidative stress and murine neutrophil killing in CAM-treated cells.

Previous studies reported that the size of the capsule is associated with resistance to oxidative stress and intracellular survival (5). In the present study, we proceeded on the assumption that cells with thin capsules and whose capsule thinness was dependent on the presence of CAM were more susceptible. C. gattii cells exposed to or not exposed to CAM were coincubated in the presence of 2 mM and 0.5 mM H₂O₂ against JP02 and R265, respectively. As indicated in Fig. 3A, the CAM-exposed cells showed significantly reduced H₂O₂ resistance to killing compared with the untreated controls. We next examined susceptibility to H₂O₂ by using cells with capsule growth induced in the presence of MOPS buffer. The cells with enlarged capsule had become more resistant against oxidative stress than the untreated control cells, indicating that the capsule size may play an important role in overcoming oxidative stress. In a parallel approach, we investigated whether CAM-exposed cells were susceptible to opsonophagocytic killing by neutrophils since oxidative stress is a major mechanism of neutrophil-mediated killing. As shown in Fig. 3B, resistance to neutrophil-mediated killing was significantly lower in CAM-treated C. gattii than in the untreated controls. In general, cells with smaller capsules mediated by CAM exposure become more susceptible to the innate immunity of neutrophils.

FIG 3.

CAM promotes the susceptibility of H₂O₂-induced oxidative stress and neutrophil opsonophagocytic killing. (A) C. gattii strains JP02 and R265, with or without CAM pretreatment, were exposed to 2 mM H₂O₂ for 3 h. C. gattii strain R265 was also treated with 50 mM MOPS buffer to enlarge capsule size and assayed to determine susceptibility to H₂O₂-induced oxidative stress. (B) Cells with or without CAM pretreatment were opsonized and incubated with murine neutrophils for 1 h. Data are presented as means ± SEM of results from three experiments (*, P < 0.05; **, P < 0.01).

Phagocytosis and inflammatory cytokine production resulting from macrophage repair in CAM-treated cells.

In previous investigations examining whether C. gattii might suppress cell-mediated immunity, polysaccharide capsule-mediated inhibition of phagocytosis by antigen-presenting cells reduced subsequent T-cell responses, dendritic cell maturation, and tumor necrosis factor alpha (TNF-α) production. We suspected that cells with smaller capsules mediated by CAM exposure might be more extensively phagocytosed and might show improved cytokine production. We compared the in vitro phagocytic rates of cryptococcal cells and levels of cytokine release from murine macrophage-like cell line J774 with and without CAM exposure. As shown in Fig. 4A, the phagocytic rates (means ± standard errors of the means [SEM]) of untreated cells of strains JP02 and R265 were 6.20% ± 2.34% and 8.50% ± 4.76%, respectively. Macrophages preexposed to CAM with both strains (for strain JP02, 22.03% ± 3.23% [P < 0.05]; for strain R265, 91.63% ± 0.76% [P < 0.001]) demonstrated a significant increase in phagocytic rate. Next, we measured the levels of TNF-α, one of the cytokines that are important for Th1-mediated immunity, released from macrophages after cell inoculation with or without CAM exposure. CAM-exposed cells showed a significant increase in TNF-α production compared with untreated cells (Fig. 4B). Our results indicate that the cells with smaller capsules mediated by CAM exposure were more extensively phagocytosed and that the level of cytokine released from macrophages for host protection was increased.

FIG 4.

CAM accelerates C. gattii phagocytosis by macrophages and release of TNF-α. (A) The ratio of macrophages phagocytosed in C. gattii. (B) The amount of TNF-α production from macrophages against C. gattii with or without CAM exposure. Data are presented as means ± SEM of results from three experiments (*, P < 0.05; ***, P < 0.001).

CAM influenced the antifungal susceptibility of C. gattii.

A recent study reported that physiological differences in C. neoformans, such as melanization or capsule enlargement, influenced antifungal susceptibility (17); therefore, we assessed amphotericin B (AMPH-B), fluconazole (FLCZ), voriconazole (VRCZ), and flucytosine (5-FC) susceptibilities in C. gattii with or without CAM exposure using Etest. As shown in Table 2, the MICs of FLCZ, VRCZ, and 5-FC were >2-fold lower in C. gattii treated with CAM, in contrast to the results seen with AMPH-B, indicating that capsule size reduction mediated by CAM exposure may improve the antifungal response to C. gattii.

TABLE 2.

The effects of CAM on antifungal susceptibilities^a

Antifungal	MIC (μg/ml)
	Strain JP02		Strain R265
	Untreated	CAM treated	Untreated	CAM treated
AMPH-B	0.125	0.125	0.125	0.125
FLCZ	2	0.5	1	0.125
VRCZ	0.094	0.016	0.012	0.002
5-FC	1	0.38	1.5	0.5

^aAMPH-B, amphotericin B; FLCZ, fluconazole; VRCZ, voriconazole; 5-FC, flucytosine.

The influences of CAM on the Hog1 MAPK signaling pathway and virulence gene expression.

We previously examined several factors reported to be related to pathway-associated capsule formation to examine which pathways are involved in CAM-mediated capsule size reduction. Bahn et al. demonstrated previously that the phosphorylation of the C. neoformans Hog1 MAPK signaling pathway was related to capsule enlargement resulting from extracellular stress (18). Another previous report indicated that dephosphorylation of Hog1 by 1 M NaCl induced capsular size reduction in C. neoformans (19). We tested the phosphorylation status of Hog1 during CAM exposure. In contrast to untreated cells, Hog1 was dephosphorylated at 2 and 4 h after CAM exposure (Fig. 5A). To further assess the remaining factors associated with capsule formation, we performed quantitative reverse transcription-PCR (RT-PCR) to estimate the levels of mRNA expression of CAP59, Mpk1, and LAC1/LAC2, genes whose expression is related to capsule formation or cell wall integrity (20). Significant reductions of LAC1 and LAC2 mRNA expression in the cells coincubated with CAM compared to yeast extract-peptone-dextrose (YPD) alone are shown in Fig. 5B. Overall, CAM treatment might aid in inhibiting capsule formation via suppression of Hog1 MAPK signaling and LAC1/LAC2 mRNA expression in C. gattii.

FIG 5.

CAM inhibited the phosphorylation of Hog1 and mRNA expression of some virulence genes of Cryptococcus associated with capsular formation in C. gattii R265. (A) C. gattii strain R265 was grown to the mid-exponential phase and treated with CAM in liquid YPD medium, and total protein extracts were prepared for Western blot analysis. The same blot was stripped and probed with polyclonal anti-Hog1 antibody as a loading control (Hog1). (B) mRNA expression of LAC1, LAC2, CAP59, and Mpk1 of R265 with CAM (black bar) or without CAM (white bar) exposure for 2 h. The fold change data were calculated relative to the cells without CAM and were normalized to the expression of the internal control, GPD1. Data are presented as means ± SEM of results from three experiments (*, <0.05 [versus untreated control]).

DISCUSSION

Current recommendations for managing C. gattii infections are based primarily on extrapolating data from clinical trials, including data from patients with C. neoformans infections, and on individual case series and expert opinion. The Infectious Diseases Society of America guidelines outline therapeutic approaches based on host status, infection site, complications of cryptococcosis, and limited therapeutic options in resource-limited settings. Overall, these guidelines recommend similar antifungal treatment regimens for C. gattii and C. neoformans (21). Despite the availability of antifungal therapies, C. gattii still has a mortality rate of close to 20%. Furthermore, C. gattii has higher pathogenicity than C. neoformans (22), and elevated FLCZ (and VRCZ) MICs with genotypes VGIIa and VGIIc have been reported previously from the Pacific Northwest (23, 24), emphasizing the importance of correct species identification and the necessity of improving and searching for alternative therapies.

In the present study, we demonstrated that CAM suppresses capsule enlargement and enhances the innate immune response, particularly with respect to phagocytosis of neutrophils and macrophages. We also showed that CAM-exposed C. gattii becomes more susceptible to antifungal agents such as FLCZ, VRCZ, and 5-FC. Capsule enlargement is a distinguishing feature of Cryptococcus spp. that is associated with virulence in mammalian hosts. It was shown previously by comparing encapsulated strains to acapsular mutants that the C. neoformans capsule suppresses host innate immunity (25). In addition, capsule enlargement inhibits complement-mediated phagocytosis (26) and killing via oxidative stress. Therefore, downregulation of capsule formation appears to be an attractive adjunct therapy for severe cryptococcosis.

Cryptococcus spp. is an intracellular pathogen for mammalian phagocytic cells, which indicates that it possesses mechanisms that avoid killing by microbicidal factors in the phagolysosome, including oxygen- and nitrogen-derived free radicals, antimicrobial peptides, and lytic enzymes. Recently, capsule enlargement was shown to play a critical role in avoiding killing, and cells with enlarged capsules were shown to be more resistant to stress factors, including oxidative killing (5). We assessed whether macrolide-mediated capsule size reduction was related to susceptibility to oxidative stress. As shown in Fig. 3, macrolide-treated C. gattii exhibited decreased resistance to H₂O₂ killing compared with untreated cells. Moreover, it is well known that neutrophils kill C. neoformans in part via producing fungicidal oxidants in the phagolysosome (27). We hypothesized that CAM-treated C. gattii was more susceptible to neutrophil-mediated killing; therefore, we performed opsonophagocytic killing using murine neutrophils, which resulted in a lower rate of intracellular survival in CAM-treated cells than in untreated cells.

In addition, the importance of a Th1 immune response-mediated cytokine, such as TNF-α, interleukin-12 (IL-12), and interferon gamma, produced by DC and macrophages to control the cryptococcal burden is evident from the high incidence of severe C. neoformans infections in human immunodeficiency virus-infected patients and from data obtained in numerous mouse models (28, 29). In our previous study, the rates of phagocytosis and production of Th1 cytokines such as TNF-α and IL-6 were reduced in C. gattii compared with C. neoformans due to differences in thickness and capsular structure (30). Thus, we evaluated whether CAM-mediated capsule size reduction promotes phagocyte and cytokine production by murine macrophages. As shown in Fig. 4A, the number of macrophages taking in C. gattii was significantly increased in CAM-treated cells, especially in the R265 strain. Furthermore, the level of TNF-α released from murine macrophages was higher in CAM-treated cells. Infection by C. gattii has a poor prognosis because it is partly mediated by the lower immune response of antigen-presenting cells due to the enlarged capsule size. CAM-mediated capsule size reduction might result in an efficient immune response for host protection.

To our knowledge, the present study was the first to evaluate the effects of macrolides against fungus. Macrolide exposure significantly reduced the capsule thickness of highly virulent C. gattii, and the amount of CPS was similarly reduced. Previous studies showed that bacteriostatic antibiotics, such as macrolides and clindamycin, reduce capsule size in S. pneumoniae by inhibiting protein synthesis (15). Furthermore, several previous studies demonstrated the effects of inhibiting macrolide-mediated bacterial virulence, especially in P. aeruginosa (11–13). Zaragoza et al. reported that a concentration of AMPH-B that was below the MIC reduced capsule formation and resulted in significantly greater phagocytosis of C. neoformans (31). Their study results may demonstrate a phenomenon similar to that represented by our present results since AMPH-B is classified a polyene macrolide, possessing a large macrocyclic lactone ring. Although few studies have been published on the subject, exploring the effects of macrolide on fungus is a promising new strategy for controlling the pathogenesis of fungal infection.

In the present study, CAM-treated cells with smaller capsules became more susceptible to azole antifungals (almost 2-fold reductions in FLCZ, VRCZ, and 5-FC MICs). It has been shown that both melanization and the size of the polysaccharide capsule are susceptibility-influencing factors (17). In our study, melanization was not related to this phenomenon since we grew the cells on RPMI agar, which indicates the importance of capsule size. Vitale et al. showed that, among isolates with enlarged capsules, FLCZ MICs were significantly higher than those seen with isolates grown under normal conditions (32). In contrast, the susceptibility of AMPH-B was not affected by CAM exposure. A previous study showed that AMPH-B killed the yeast not only breaking the cell membrane by direct binding of ergosterol but also by inducing the accumulation of reactive oxygen species (ROS) in the cells (33). Macrolides are able to inhibit the production of ROS from neutrophils (34), leading to the suspicion that the antifungal effect of AMPH-B might be compensated for by CAM-mediated inhibition of ROS production. Taken together, the susceptibilities to azoles and 5-FC are higher in fungi with a smaller capsule than in those with an enlarged capsule, but the mechanism of capsule size influencing antifungal susceptibility is still unclear; thus, additional studies are needed.

Extensive data have documented the immunomodulatory effects of macrolides on the interactions between MAPK signaling and transcription factor expression. For example, a previous study demonstrated that CAM treatment resulted in decreased levels of MUC5AC gene expression and ERK1/2 phosphorylation in P. aeruginosa-infected mouse lung homogenates, suggesting that it inhibits MUC5AC glycoprotein production via ERK inhibition (35). MAP kinases in fungal pathogens are also key in controlling adaptation to environmental stress, including changing pH, oxidative and osmotic stress, and nutrient limitations. Recent studies showed the essential role of these pathways in controlling virulence factors such as capsule biogenesis in C. neoformans or morphogenesis. The Hog1 MAPK signaling pathway is highly conserved in diverse fungi and negatively regulates CPS and melanin production in C. neoformans (18). Therefore, we examined whether CAM-mediated capsule size reduction influences the MAPK signaling pathway of C. gattii. Dephosphorylation of Hog1 occurred after CAM exposure (Fig. 5A), suggesting that CAM inhibits capsular formation via the Hog1 MAPK signaling pathway. Next, we performed quantitative RT-PCR to examine the levels of expression of genes related to capsular production or cell wall integrity, including CAP59, LAC1/LAC2, and Mpk1, as reported for previous studies (5, 36). Interestingly, CAM-treated cells showed significantly reduced mRNA expression of LAC1/LAC2 compared with untreated cells. Although it is difficult to clearly explain the relationship between downregulation of LAC1/LAC2 and capsule size reduction, one explanation could be that, due to LAC1/LAC2 transcriptional suppression, the unstable cell wall inhibits sequential polysaccharide capsule formation.

This study had some limitations. First, we did not evaluate whether CAM treatment in the presence or absence of existing antifungals would improve outcomes of C. gattii infection in the experimental model. However, in our unpublished data, the clearance of C. gattii treated with CAM was promoted in the lungs 24 h after intratracheal inoculation. In addition, acapsular mutants did not produce disease in a murine model (37), suggesting the clinical efficacy of CAM-mediated capsular size reduction. Second, we could not satisfactorily identify the target of CAM responsible for capsule size reduction. As shown in the present study, CAM might directly or indirectly affect multiple gene transductions related to capsule formation. This problem may be resolved in a future study comparing levels of gene expression using comprehensive analyses such as microarray or RNA sequencing.

In summary, we demonstrate that macrolides promote host protection by diminishing the capsule thickness of C. gattii. As the capsule is the primary virulence factor for resistance against host innate immunity, the present findings highlight the efficacy of the use of macrolides as an adjunctive therapy by regulating virulence against refractory C. gattii infection.

MATERIALS AND METHODS

Strains, culture, and CAM exposure.

C. gattii strain JP01 (genotype VGIIc) and strain JP02 (genotype VGIIc) are strains that were clinically isolated in Japan (38). C. gattii strain R265 (genotype VGIIa), a clinical isolate from an outbreak on Vancouver Island, was provided by K. J. Kwon-Chung (NIAID, NIH, Rockville, MD, USA) (39). C. gattii strain PNG18 (genotype VGI) was clinically isolated from a patient in Papua New Guinea (40).

These strains were routinely precultured in liquid yeast extract-peptone-dextrose (YPD) medium at 30°C with or without clarithromycin (Taisho Toyama Pharmaceutical Co., Ltd., Tokyo, Japan) for 24 h with shaking at 150 rpm. In the preliminary study, we measured the capsule size and the amount of polysaccharides using several concentrations (1, 10, and 100 μg/ml). We selected the CAM concentration of 100 μg/ml to reduce capsule size and to prepare the cells with small capsule since the largest reductions of both capsule size and the amount of capsular polysaccharides were observed with that concentration (data not shown). To obtain the capsular polysaccharide (CPS), cells were grown in modified yeast nitrogen broth (mYNB) (YNB without amino acids [BD] and supplemented with complete supplement mixture [Formedium] and 0.5% [wt/vol] d-glucose).

Isolation and quantification of CPS.

C. gattii strains were cultured in mYNB at 30°C with or without CAM for 24 h. The CPS fraction was extracted using a previously described method (41) with some modifications. Briefly, capsule components were extracted from the remaining cellular material by the use of dimethyl sulfoxide. After undergoing centrifugation, supernatants were collected and dialyzed against Milli-Q water. The total amount of polysaccharides was monitored using the phenol-H₂SO₄ method (42), and absorbance spectroscopy was performed at 490 nm.

H₂O₂ susceptibility test.

C. gattii cells cultured with or without CAM were washed twice and resuspended in water at a density of 2 × 10³ cells/ml. Hydrogen peroxide (H₂O₂) (2 mM for strain JP02 and 0.5 mM for strain R265) was added, and the reaction mixtures were incubated at 30°C with rotation for 48 h. Controls included tubes without H₂O₂ that were plated in parallel in potato dextrose agar plates and incubated at 30°C for 48 h.

Isolation of murine neutrophils.

Neutrophil-enriched peritoneal cavity (PEC) lavage fluid was collected as previously described (20). Briefly, phagocytes were obtained by lavage of the peritoneal cavity (8 ml/animal with phosphate-buffered saline [PBS] containing 20 mM EDTA) of mice treated 24 h and again 2 h prior to cell harvest by intraperitoneal administration of 10% casein–PBS (1 ml/dose). Cells collected from the PECs were enriched for neutrophil or monocyte cells separated using Ficoll density gradient centrifugation according to the manufacturer's protocol. The neutrophil-enriched PECs were counted by trypan blue staining and adjusted to a density of 7 × 10⁶ cells/ml.

Opsonophagocytic killing assay.

Opsonophagocytic killing assay was performed as previously described (43). Killing during incubation for 3 h at 30°C with rotation was assessed by combining 10² PBS-washed, mid-log-phase C. gattii cells (in 10 μl) (with or without CAM), complement source (in 20 μl), 10⁵ murine phagocytes (in 40 μl), and Hank's buffer with Ca⁺⁺ and Mg⁺⁺ (Gibco) plus 0.1% gelatin (130 μl). The complement source was the serum of baby rabbits aged 3 to 4 weeks (Pel-Freez Biologicals, Rogers, AR, USA). After stopping the reaction by incubating at 4°C, viable counts were determined in serial dilutions.

Phagocytosis of C. gattii by murine macrophages.

The phagocytic ratio of murine macrophage J774 cell lines was estimated as described previously (44). Briefly, cryptococcal cells (with or without CAM) labeled using calcofluor white M2R (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) (multiplicity of infection, 5) were added to each well and incubated for 3 h at 37°C in <5% CO₂. Cells were fixed and labeled with CellMask orange (Thermo Fisher Scientific, Inc. Waltham, MA, USA). After mounting was performed, labeled Cryptococcus internalization was analyzed using a Zeiss LSM 700 laser confocal microscope (Carl Zeiss AG, Oberkochen, Germany), and the images were processed using ZEN software (Zeiss Microscopy).

Western blot of Hog1 phosphorylation.

C. gattii cells were cultured overnight in YPD medium at 30°C with shaking at 150 rpm and were diluted to an optical density at 530 nm (OD₅₃₀) of 0.1 and recultured for 2 h. Yeast cultures with or without CAM were harvested at each time point (1, 2, 4, and 6 h) at 3,500 rpm for 10 min at 4°C and then washed once in iced DNase/RNase-free water. The cells were resuspended in 1 ml of iced water, transferred to glass beads, and centrifuged at 10,000 rpm for 10 min at 4°C. After the supernatant was discarded, the pellets were lyophilized and the cells were disrupted by bead beating for 10 min at 4°C. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA). For each sample, 50 μg of total protein was loaded on 12.5% SuperSep Ace (Wako, catalog no. 198-14941) and transferred to nitrocellulose. After blocking was performed, the membranes were probed with phospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal antibody (Cell Signaling Technology, catalog no. 4511S) (1:10,000 dilution) and Hog1-specific rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, catalog no. 25757) (1:4,000) by incubation for 1 h at room temperature for phosphorylated Hog1 determination and total Hog1 protein-independent phosphorylation. After washing the membranes 3 times each for 10 min each time in Tris-buffered saline containing Tween 20 (TTBS; 50 mM Tris–HCl [pH 7.6], 0.9% NaCl, 0.1% Tween 20 [Sigma-Aldrich, catalog no. P9416]), a secondary incubation was performed with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA, catalog no. 32430) (1:20,000 dilution) for 1 h at room temperature. After washing the membrane 3 times each for 10 min each time in TTBS, the green fluorescent protein (GFP) signal was detected using an ECL kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan, catalog no. 296–69901). Phosphorylated Hog1 protein and total Hog1 protein were detected as bands migrating at molecular masses of approximately 43 and 50 kDa, respectively.

RT-PCR.

C. gattii strain R265 cultured overnight in YPD medium at 30°C were diluted in YPD medium alone and YPD medium with 100 μg/ml of CAM, and RNA was isolated after 0.5, 1, 2, and 4 h of incubation with shaking at 150 rpm by using an RNeasy minikit (Qiagen) according to the instructions of the manufacturer. cDNA was reverse transcribed using a High-Capacity reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Next, approximately 25 ng of cDNA was used as a template in reactions with 0.5 μM forward and reverse primers and SYBR green (Applied Biosystems) according to the manufacturer's instructions. Reactions were performed using a StepOnePlus real-time PCR system (Applied Biosystems), and quantitative comparisons were conducted using the threshold cycle (ΔΔC_T) method. The primers used in the present study ware as follows (20): GPD1-F (5′-CATCGAGCATGGAGACCTTGAG-3′), GPD1-R (5′-AACCCTTGAAGCGACCATGTG-3′), LAC1-F (5′-ACCTTCATGGCAACGAGTTC-3′), LAC1-R (5′-ACAACCACAGCCAACTTTCC-3′), LAC2-F (5′-ACCCTTTACTTCGTGTCGTCCA-3′), LAC2R-R (5-TCCACCCTCCATCCAGAAAGTA-3′), Mpk1-F (5′-TGGATTTGTTGAGCAAGCTG-3′), Mpk1-R (5′-TCCTTACAGGAGGCATGGAG-3), CAP59-F (5′-CGGATGGACGTCATCAAGAG-3′), and CAP59-R (5′-GGGATAACGTCAAACGAGTTGTA-3′). The fold change values were calculated against the cells cultured in YPD without CAM exposure, and GPD1 was used as the internal control.

ELISA.

The TNF-α enzyme-linked immunosorbent assay (ELISA) kit that was used in this study was purchased from R&D Systems (Minneapolis, MN, USA). Cytokine levels were measured according to the manufacturer's instructions.

Antifungal susceptibility test.

The Etest (bioMérieux, St. Louis, MO, USA) was performed according to the manufacturer's instructions. The antifungal drugs flucytosine (5-FC), fluconazole (FLCZ), voriconazole (VRCZ), and amphotericin B (AMPH-B) were tested using RPMI 1640 containing 1.5% agar supplemented with 2% glucose and buffered with MOPS to pH 7. Etest gradient strips were placed on an inoculated agar plate by seeding the inoculum on the surface of the plate and were left to dry for 15 min. The plates were incubated at 37°C for 48 h. MIC readings were obtained at the point of intersection between the ellipse corresponding to growth inhibition and the Etest strip.

Statistical analysis.

All data were analyzed using Prism 6 GraphPad software and are presented as means ± SEM. Differences between results obtained with the treatment group and the controls were tested for significance using the Mann-Whitney U test.