Medical Mycology April 2004, 42, 149 /158

Cryptococcus neoformans var. gattii can exploit Acanthamoeba castellanii for growth
STEPHANIE D. MALLIARIS*, JUDITH N. STEENBERGEN$ & ARTURO CASADEVALL*,$ *Division of Infectious Diseases, Bronx NY, USA; $Microbiology and Immunology, Albert Einstein College of Medicine, Bronx NY, USA

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It has recently been proposed that the origin and maintenance of virulence in certain environmental fungi is influenced by their interactions with non-vertebrate hosts such as amoebae and nematodes. In prior studies we have shown that the interactions of the soil amoebae Acanthamoeba castellanii with Cryptococcus neoformans varieties neoformans and grubii resemble those with macrophages. Here we extend those studies to C. neoformans variety gattii and describe quantitative differences in the type and outcome of the interactions observed relative to the other varieties. C. neoformans var. gattii proliferated in the presence of A. castellanii but the interaction was primarily extracellular with a paucity of phagocytic events. Experiments with acapsular cells coated with polysaccharide suggest that differences in the capsule structure may be responsible for the different interactions between cells of varieties neoformans , grubii , and gattii with amoebae. The ability of C. neoformans var. gattii to exploit amoebae indicates that despite major biological differences between C. neoformans varieties, all retain the ability to be pathogenic for A. castellanii . Keywords C. neoformans var. gattii , environmental, phagocytosis, virulence

Introduction
Cryptococcus neoformans is an environmental fungus that is pathogenic to humans. Cryptococcus neoformans isolates have been grouped into three varieties known as grubii (serotype A), neoformans (serotype D), and gattii (serotypes B and C), on the basis of phenotypic, serological, genetic, biochemical, and epidemiological criteria [1]. C. neoformans variety neoformans and variety grubii are found worldwide, often in soils contaminated with avian excreta [2]. In contrast, C. neoformans var. gattii is generally found in tropical and subtropical regions, particularly in Australia, but also in South America and southern California [3,4]. In the 1990s, several species of eucalyptus trees were identified as natural habitats of C. neoformans var. gattii serotype B. Recently, serotype C has been isolated

Received 1 July 2003; Accepted 8 August 2003 Correspondence: A. Casadevall, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA. Email: casadeva@aecom.yu.edu

from almond tree (Terminalia catappa ) detritus in Columbia [5]. The locales of these variety gattii isolates indicate that this variety inhabits a different ecologic niche than varieties neoformans and grubii [6,7]. There are some notable differences in the clinical manifestations of cryptococcosis caused by the different varieties of C. neoformans . Clinical disease with C. neoformans var. gattii predominantly occurs in immunologically normal hosts and typically presents with pneumonia and to a lesser extent, meningitis [8]. In contrast to disease caused by varieties neoformans and grubii , mass lesions are relatively common in clinical infections with variety gattii isolates. Cryptococcomas in brain parenchyma are associated with seizures, cranial nerve palsies, and hydrocephalus, and may require surgical resection. C. neoformans var. gattii has a high rate of recurrence or persistence and prolonged antifungal therapy is required. The AIDS epidemic has not significantly altered the incidence of C. neoformans var. gattii disease [8,9]. In contrast, clinical infections with C. neoformans var. neoformans commonly occur in immunocompromized individuals
DOI: 10.1080/13693786310001616500

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and the incidence has greatly increased with the continuing AIDS epidemic, particularly in underdeveloped countries [8,10]. C. neoformans var. neoformans clinically manifests as meningitis and less frequently with pneumonia. Based on differences between the two varieties, such as natural habitat and disease manifestation, it has been suggested that C. neoformans var. neoformans and C. neoformans var. gattii be classified as different species [11]. There are virulence factors, however, that are shared between the two varieties, such as the presence of a polysaccharide capsule, melanin synthesis, and the ability to grow at physiological temperatures (reviewed in [12]). Melanin synthesis and the polysaccharide capsule are expressed in both the natural environment and the host. Recently, it was reported that C. neoformans var. neoformans is pathogenic to the soil amoeba, Acanthamoeba castellanii [13]. A. castellanii is a free living soil amoeba that can act as a host for several intracellular pathogens, including bacteria, such as Legionella pneumophila , and fungi [13 /15]. Despite the ecological differences between the varieties of C. neoformans , we hypothesized that C. neoformans var. gattii would also interact with A. castellanii , since C. neoformans var. gattii is associated with macrophages in clinical infection. The interactions of C. neoformans var. gattii with A. castellanii were similar to those seen with C. neoformans var. neoformans with macrophages [16]. Although C. neoformans var. gattii and A. castellanii have not been associated environmentally, amoebae are ubiquitous in many ecosystems and A. castellanii has been established as a model system for exploring the interactions between amoebae and various microorganisms including C. neoformans [13]. The results of this study demonstrate that C. neoformans var. gattii replicates in the presence of amoebae and further support the view that fungal virulence is maintained by interactions between the fungi and environmental predators.

C. C. neoformans var. neoformans 3501, serotype D, was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA), and C. neoformans var. grubii H99, serotype A, was a gift from J. Perfect (Durham, NC, USA). Cap67 is an acapsular mutant of strain 3501 obtained from E. Jacobson (Richmond, VA, USA) [17]. All C. neoformans strains were maintained at /808C. Cultures were initiated by inoculating 25 ml Sabouraud (SAB) dextrose broth (Difco; Becton Dickinson, Sparks, MD, USA) with a loop of the / 808C stock and grown at 308C at 150 r.p.m. for 48 h. Acanthamoeba castellanii strain 30324 (ATCC) was maintained in 750-ml tissue culture flasks (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ, USA) with 25 ml peptone yeast extract (PYG) glucose (ATCC medium 354) broth at 288C. Amoebae cultures were maintained by transferring to fresh medium every 5 / 7 days. The supernatant from the old flask was poured off and replaced with 10 ml phosphate-buffered saline (PBS). After tapping the flask firmly 10 times, the PBS was removed and spun down at 320 g for 10 min. The supernatant was poured off, the pellet suspended in 10 ml PBS, and 500 ml of the washed amoebae was added to 25 ml of fresh PYG in a new tissue culture flask [18,19].

Fungal killing assay

Acanthamoeba castellanii were collected by centrifugation at 320 g for 10 min, washed twice with PBS, suspended in PBS, counted with a hemacytometer and diluted in PBS to the appropriate density. A. castellanii cells were added to wells of a 96-well tissue culture plate (Falcon; Becton Dickinson Labware) at 1 )/104 cells per well and allowed to adhere for 1 h at 288C. C. neoformans cells were collected, washed three times with PBS, and counted with a hemacytometer. To achieve an initial 1:1 effector-to-target ratio, 1 )/104 washed fungal cells were added to the wells containing A. castellanii and the plates were incubated at 288C. At 0, 24 and 48 h, the contents of selected wells were pulled through 27 gauge syringe needles five to seven times to lyse the amoebae, and then all contents were removed from the wells using a pipette. The contents were serially diluted, plated onto SAB agar, and incubated at 308C for fungal colony-forming unit (c.f.u.) determination. Viability of A. castellanii under these conditions was determined by trypan blue exclusion assay at the same time intervals. Fifty microliters of a 1:20 dilution of trypan blue stain was added to the wells and the percentage of dead amoebae was determined by counting the number of amoeboid cells unable to exclude the dye per 100 amoebae counted.
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Materials and methods

Strains and culture conditions
Cryptococcus neoformans var. gattii strains 106.97, 107.97, 1339 were obtained from T. Mitchell (Durham, NC, USA). Strain I6 was obtained from U. Banerjee (New Delhi, India). Serotype assignment was confirmed using a Crypto Check Kit (Iatron Laboratories, Tokyo, Japan) according to the manufacturers instructions. Strains I6 and 1339 were determined to be serotype B and strains 106.97 and 107.97 were serotype

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Control for trypan blue consisted of wells containing A. castellanii without fungi.

Phagocytosis of cap67 with added GXM

To evaluate the potential of capsular polysaccharide from the three varieties to influence phagocytosis we used the phenomenon of polysaccharide attachment to a non-encapsulated strain followed by the use of such strains in phagocytosis assays. To prepare cap67 cells with an attached capsule, several encapsulated strains were first grown in SAB medium for 10 days and the cell free culture supernatant was collected. This supernatant was then added to 1 )/106 non-encapsulated cap67 cells, the suspension was incubated at room temperature for 5 h, cells were then washed the with PBS, suspended in PBS and used in amoebae phagocytosis experiments as described above in assays with amoebae:yeast ratio of 1:15. Staining for GXM with specific antibody followed by immunofluorescence revealed strong attachment of polysaccharide to cap67 in all cases.

Phagocytosis/attachment assay
In a 96-well tissue culture plate, 1 )/104 A. castellanii and C. neoformans cells (1:1 effector-to-target ratio) were co-incubated at 288C for 2 h. The plates were spun at 140 g for 6 min before the supernatant was removed with a pipette. The cells were fixed by adding 100 ml per well of ice-cold methanol for 30 min at 48C. After washing twice with PBS, 100 ml of a 1:20 dilution of Giemsa stain (Sigma, St Louis, MO, USA) was added for 15 min, and then the plate was washed with PBS. The plate was examined with a microscope at )/400 magnification and the phagocytosis index was measured by counting the number of amoeba cells with one or more internalized C. neoformans cells per 100 amoeboid cells. The same assay is used to determine the attachment index, which was defined as the number of cells with one or more C. neoformans attached to the exterior of the amoeba per 100 amoeboid cells. Attached and ingested C. neoformans could be differentiated by the presence of the latter in a phagocytic vacuole in the amoeba cytoplasm. To confirm internalization of the C. neoformans , fluorescence microscopy was used. A. castellanii cells were collected, washed, and suspended in PBS at 5 )/ 105 cells per ml. A volume of 300 ml was added to eightwell tissue culture slides (Falcon; Becton Dickinson Labware) and the slides were incubated for 90 min at 288C to allow the cells to adhere. C. neoformans var. gattii strains were then collected, washed three times, added to the amoebae (3 )/105 cells in 200 ml PBS) and incubated for 24 h at 288C. Post incubation, the medium was removed and the slides were washed with PBS, fixed with ice-cold methanol at 48C for 30 min, and washed again. The slides were then incubated with Superblock Blocking Buffer (Pierce, Rockford, IL, USA) overnight at 48C to reduce nonspecific antibody binding. Monoclonal antibody 18B7, which binds to the polysaccharide capsule of C. neoformans [20], was conjugated to fluorescein isothiocyanate (FITC; Molecular Probes; Eugene, OR) by incubation in a 5 mg/ml solution of FITC dissolved in 50 mmol/l borate buffer (pH 8.0) for 2 h at 378C [21]. The FITC-conjugated 18B7 was added at 50 mg/ml, and the slides were incubated for 2 h at room temperature. The slides were then washed and coverslips were mounted using GelMount (Biomedia, Foster City, CA, USA). The slides were viewed with an Olympus AX70 microscope (Melville, NY, USA) equipped with a FITC filter.
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Capsule size measurement

Cryptococcus neoformans and A. castellanii cells were grown and washed as described above. A volume of 2 ml of a 5 )/105 cells/ml suspension of A. castellanii cells was added to each well of a six-well tissue culture plate (Falcon; Becton Dickinson Labware), and the plate was incubated for 1 h. Cells from each C. neoformans strain evaluated were suspended at 1 )/ 106 cells/ml, added to each well to achieve an E:T ratio of 1:1, and the plates were incubated at 288C. At 0, 24 and 48 h, contents of the wells were agitated with a pipette, aspirated, and then placed in 15-ml Polypropylene Conicle Tubes (Falcon; Becton Dickinson Labware). The tubes were centrifuged at 350 g and the pellet was suspended in a small amount of PBS. One drop of India ink (Becton Dickinson) was added to a 10-ml volume of cell suspension on a microscope slide and covered by a coverslip. The slides were examined using the )/100 objective on an Olympus AX70 microscope. Digital photographs of the cells were examined using Photoshop (Microsoft, Seattle, WA, USA) and average diameter measurements of the entire encapsulated fungal cell, as well as the cell body, were calculated. These measurements were used to determine the percent of the whole cell that the capsule represents, calculated two ways: using width and then using volume. The budding index of the fungal cells was also determined by counting the percentage of cells with a bud attached (a bud is defined as a cell with a visible daughter cell still attached).

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Supernatant toxicity assay

To examine the effect of the C. neoformans var. gattii supernatant on A. castellanii , 0.4-mm translucent cell culture inserts (Falcon; Becton Dickinson Labware) were used so that C. neoformans could be incubated near, but not in contact, with A. castellanii while maintaining supernatant continuity. For these experiments, A. castellanii cells were washed and suspended at 1 )/105 cells/ml. A volume of 500 ml of this suspension was added to each well in a 24-well tissue culture plate (Falcon; Becton Dickinson Labware), cell culture inserts were placed in appropriate wells, and the plates were incubated at 288C for 1 h. C. neoformans were washed, suspended, and added at a 10:1 C. neoformans : A. castellanii ratio to the inserts or wells accordingly. At 0, 24 and 48 h, inserts were removed, and trypan blue exclusion was used to determine the viability of the A. castellanii in the different conditions.
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Results
Cryptococcus neoformans var. gattii replicates in the presence of A. castellanii . C. neoformans var. gattii replicated in PBS in the presence of A. castellanii (Fig. 1). In contrast, no significant C. neoformans var. gattii growth occurred in PBS without amoebae. Strains 1339, 106.97, and 107.97 grew most rapidly during the initial 24 h with c.f.u. increases of 323, 577 and 460% respectively. Strain I6 grew slower, increasing 140% at 24 h and 391% at 48 h. Serotype C strains had significantly higher rates of growth than the serotype B strains (Fig. 1). The growth of C. neoformans var. gattii in the presence of amoebae was compared to C. neoformans var. neoformans and var. grubii (Fig. 2). Strains of each variety proliferated to comparable CFU levels when incubated in PBS with A. castellanii .

Amoeba viability
The growth of C. neoformans in PBS with A. castellanii suggested that this fungus used the amoeba cells for nutrition. Consequently, we measured the viability of A. castellanii in the presence and absence of C. neoformans . More A. castellanii cells were killed in the presence of C. neoformans var. gattii than in PBS (P B/0.001) (Fig. 3A). Over the 48-h time interval, the number of dead amoebae in PBS rose from B/1% at 0 h to 11% at 48 h, whereas in the presence of C. neoformans var. gattii , /25% of amoebae were dead at 48 h (Fig. 3B). Survival of A. castellanii was significantly reduced in the presence of C. neoformans var. gattii compared to incubation in PBS alone (P B/0.001 for all strains of C. neoformans var. gattii tested). The percentage of amoeba cells killed by C. neoformans var. gattii and C. neoformans var. neoformans was similar (Fig. 3B).

Transmission electron microscopy

Acanthamoeba castellanii was collected, washed, counted, and suspended at a density of 5 )/105 cells/ ml in PBS and placed in microcentrifuge tubes (USA Scientific, Ocala, FL, USA). C. neoformans var. gattii cells were then washed, counted, and 1.5 )/106 cells were added to the tubes at a 3:1 effector-to-target ratio. The amoeba-yeast suspension was then incubated for 2 h at 288C. The cells were collected and fixed with 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer at room temperature. The grids were prepared as described [22] and viewed with a model 102 electron microscope (Siemens, Iselin, NJ, USA).

Scanning electron microscopy

Acanthamoeba castellanii and C. neoformans were prepared in the same manner as described for transmission electron microscopy. Scanning electron microscopy was performed as described [23]. Briefly, samples were incubated in 2.5% glutaraldehyde in 0.1 mol/l cacodylate for 1 h at room temperature, applied to poly-L-lysine coated coverslips, and serially dehydrated in alcohol. The samples were dried (Samdri-790; Tousimis, Rockville, MD, USA), coated with gold-palladium (Desk-1; Denton Vacuum, Cherry Hill, NJ, USA), and viewed using a JEOL JAM-6400 electron microscope (Tokyo, Japan).

Phagocytosis and attachment of C. neoformans by A. castellanii

Acanthamoeba castellanii ingested C. neoformans var. gattii cells, but the efficiency of this process was significantly lower than that observed in our earlier studies with variety neoformans and variety grubii [13]. The phagocytic index after incubation of A. castellanii with C. neoformans for 2 h ranged from 3.6% (1339) to 6% (107.97) (Fig. 4A). When compared with strains of C. neoformans var. neoformans and var. grubii , fewer phagocytic events were observed with yeast cells of var. gattii . (Fig. 4B). The phagocytic indices for C. neoformans var. gattii strains 1339 and 106.97 were 2.8 and 2.4% respectively, while for var. neoformans strain 3501 and var. grubii strain H99 the phagocytic indices were
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Statistical analysis
Statistical analysis was performed using the Students t test. Both statistical analysis and graphs were compiled using Microsoft Excel 2000 (Redmont, WA, USA).

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Fig. 1 Growth of four strains of Cryptococcus neoformans var. gattii in the presence and absence of Acanthamoeba castellanii . Panels A, B, C, and D denote data for strains I6, 1339, 106.97, and 107.97 respectively. Solid, hatched, and open bars denote average c.f.u. at 0, 24 and 48 h, respectively. The error bars represent 1 standard deviation. There are signicant differences (P B/0.001) for all four strains grown in the presence of A. castellani between 0 and 24 h. The asterisk represents signicant difference in growth between this serotype B strain and both serotype C strains (P B/0.001). Values shown are the average of ve measurements.

37.8 and 18% respectively */representing approximately a 10-fold difference in phagocytic efficiency between the two varieties (P B/0.001). The attachment index for strains of var. gattii was significantly higher than for strains of var. neoformans and grubii , consistent with attachment without ingestion for var. gattii . The attachment index for var. neoformans and grubii was lower presumably because those events were frequently followed by phagocytosis (Fig. 5). To investigate possible explanations for the different levels of phagocytosis observed, we measured yeast cell capsule size before and after incubation with amoebae. For three of four strains tested, the percent of the fungal cell that is comprised of capsule increased significantly when incubated with A. castellanii for 24 h (data not shown). Next, we examined the toxicity of the var. gattii supernatant, and whether or not heat-killed var. gattii is toxic to amoebae. The var. gattii supernatant was not more toxic to the amoebae than either plain PBS or supernatant from var. neoformans strain 3501 and var. grubii strain H99. In fact, more amoebae were viable after incubation in the supernatant of serotype C
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strains (106.97 and 107.97) than PBS (P 0/0.049 and 0.015) (data not shown). When heat-killed C. neoformans var. gattii cells were incubated with A. castellanii , there was no increase in amoebae toxicity relative to that observed in PBS alone (data not shown). To examine the effect of the variety gattii polysaccharide on amoebae relative to that from the other varieties, we exploited the phenomenon that soluble capsular polysaccharide will attach to acapsular cap67 cells to form a small capsule and then performed a phagocytosis assay [24,25]. The results indicated the same trend as the previous phagocytosis assay */the cap67 cells with capsules derived from H99 and 3501 polysaccharide were phagocytosed at a significantly higher rate than those with capsules derived from 1339 and 106.97 polysaccharide (P 0/0.001 to P 0/0.003 for all) (Fig. 6).

Interactions between A. castellanii and C. neoformans var. gattii

Transmission electron micrographs show amoebal pseudopods extending towards and surrounding the

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Fig. 2 Comparison of the growth of Cryptococcus neoformans varieties gattii strains 106.97 and 1339, grubii strain H99 and neoformans strain 3501 in the presence and absence of Acanthamoeba castellanii . Panels A, B, C, and D denote data for strains 106.97, 1339, H99, and 3501 respectively. Solid, hatched, and open bars denote average c.f.u. at 0, 24 and 48 h, respectively. Note that the experiment shown in Fig. 2 uses some of the same strains as employed in the experiment shown in Fig. 1 but these experiments were done independently. Growth in presence of A. castellanii is signicantly greater than growth in PBS alone for all strains (P B/0.001). The error bars represent 1 standard deviation. Values shown are the average of ve measurements.

C. neoformans var. gattii cells (Fig. 7). Some amoebae interacted with more than one C. neoformans var. gattii . Similarly, scanning electron micrographs showed pseudopods of amoebae interacting with yeast cells (Fig. 8). Internalized cells in A. castellanii were relatively rare, but the immunofluorescence assays confirmed that some A. castellanii cells internalize C. neoformans var. gattii cells (Fig. 9).

Discussion
Understanding how virulence of environmental fungi is maintained is an important problem in medical mycology. C. neoformans var. gattii has not been as widely studied as C. neoformans var. neoformans or C. neoformans var. grubii , but all three varieties are believed to have several virulence factors in common such as melanin synthesis, the capsule polysaccharide, and the ability to grow at mammalian temperatures. Recent studies have shown that environmental predators, such as amoebae and nematodes, may exert selection pressures on environmental fungal species such as C. neoformans , and that such predation may

select for adaptations in the fungus that allow virulence in mammalian hosts [26]. We have established that A. castellanii is a model host for examining fungal/host interactions [13]. Our prior work has demonstrated that C. neoformans var. neoformans can exploit A. castellanii for growth, resulting in amoebal death and fungal growth [13]. The method by which this occurs was shown to be similar to C. neoformans infection of mammalian macrophages [16]. In contrast, the nonenvironmental yeast Saccharomyces cerevisiae and Candida albicans were rapidly killed by A. castellanii . The invertebrate nematode Caenorhabditis elegans has also been used to examine environmental interactions of C. neoformans [27]. Following ingestion of C. neoformans by C. elegans, C. neoformans is able to replicate and even kill the nematode [27]. Given the precedents suggesting a relationship between C. neoformans virulence and interactions with other hosts such as amoebae and nematodes, we investigated whether the interactions of A. castellanii with C. neoformans var. gattii were similar to those described earlier for C. neoformans var. neoformans and C. neoformans var. grubii . Currently, there are two
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Fig. 3 Killing of Acanthamoeba castellanii by Cryptococcus neoformans . Percent of amoebae that are unable to exclude trypan blue stain after incubation with and without strains of C. neoformans are shown. (A) Four strains of C. neoformans var. gattii compared to PBS. (B) Two strains of C. neoformans var. gattii (1339 and 106.97) compared to C. neoformans var. neoformans strain 3501, C. neoformans var. grubii strain H99, and PBS. Solid, hatched, and open bars denote average c.f.u. at 0, 24 and 48 h, respectively. The error bars represent 1 standard deviation. Values shown are the average of ve measurements.

known environmental niches of C. neoformans var. gattii . Serotype C has been associated with almond trees, and serotype B is found in several species of eucalyptus tree; specifically, in tree hollows of those trees where moisture may be present [5,7]. Clinical isolates, however, are sometimes recovered in areas where these tree species are not endemic, suggesting that other niches may exist [28,29]. It is known that amoebae are ubiquitous in the biosphere, and therefore there is a high likelihood that amoebae and C. neoformans var. gattii coexist in the same habitat. In this regard, amoebae are abundant in an array of environments, including many types of soil and freshwater [30 /32]. Our results show that C. neoformans var. gattii , like C. neoformans var. neoformans and C. neoformans var. grubii , can exploit A. castellanii for growth. Coincubation of the amoebae with cells of each variety resulted in increased death amoebae cells and fungal growth. However, there were interesting differences in the outcome of the interaction between cells of the three C. neoformans varieties and A. castellanii . The phagocytic indices resulting from the interactions of C.
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Fig. 4 Phagocytosis of Cryptococcus neoformans by Acanthamoeba castellanii . (A) Phagocytic index for four strains of C. neoformans var. gattii , I6, 1339, 106.97, and 107.97. (B) Phagocytic index for two strains of C. neoformans var. gattii (1339 and 106.97) relative to C. neoformans var. neoformans strain 3501 and C. neoformans var. grubii strain H99. The phagocytic index was determined by counting the total number of uorescent fungal cells per 100 amoebae. For each experimental condition the number of repetitions was ve. Error bars represent 1 standard deviation.

neoformans var. gattii with A. castellanii were significantly lower than those observed after incubation of amoebae with either C. neoformans var. neoformans or C. neoformans var. grubii cells. The observed phago-

Fig. 5 Phagocytic (solid bar) and attachment (hatched bar) index of fungal cells by Acanthamoeba castellanii after incubation for 2 h. For each experimental condition the number of repetitions was ve. Error bars represent 1 standard deviation.

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Fig. 6 Phagocytosis of cap67 cells incubated with supernatants from various strains of Cryptococcus neoformans by Acanthamoeba castellanii . For each experimental condition the number of repetitions was ve. Error bars represent 1 standard deviation.

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cytic effect does not appear to involve a difference in capsule growth after co-incubation with amoeba since strains from each variety were shown to manifest an increase in capsule size after exposure to amoebae. Although the mechanism for this effect is uncertain, the experiment with cap67 cells suggests that it is related to structural differences in the capsular polysaccharide of variety gattii . In this regard, significantly fewer phagocytic events were observed with cap67 coated with polysaccharide of variety gattii as compared to cap67 coated with polysaccharide from varieties neoformans and grubii . The capsular glucuronoxylomannan of C. neoformans var. gattii is more highly substituted than those of varieties grubii or neoformans , a structural difference that may translate into reduced affinity for amoeba phagocytic receptors [33]. The lower phagocytic index for C. neoformans var. gattii suggests that phagocytosis of variety gattii by A. castellanii is not essential for fungal growth or death of the amoeba, since no significant difference was seen in the percentage of dead amoebae after incubation with any of the three varieties of C. neoformans . Transmis-

Fig. 8 Scanning electron micrographs of Cryptococcus neoformans var. gattii interacting with Acanthamoeba castellanii after 24 h incubation. (A) Strain I6: A. castellanii beginning to envelope C. neoformans var. gattii . (B) Strain107.97: pseudopods of an amoeba beginning to encircle C. neoformans var. gattii . (C) Strain 107.97: multiple C. neoformans var. gattii adhering to the amoeba cell wall. Scale bars represent 10 mm.

sion and scanning electron micrographs showed C. neoformans var. gattii adhering to amoeba cells, suggesting that attachment of fungal cells to the

Fig. 7 Transmission electron micrographs of Cryptococcus neoformans var. gattii interacting with Acanthamoeba castellanii . (A) Strain 107.97: pseudopods beginning to engulf fungal cell. (B) Strain 107.97: three fungal cells adhering to one amoeba. (C) Strain I6: ve fungal cells adhering to one amoeba. Scale bars represent 5 mm

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Fig. 9 (A) Immunouorescent and (B) corresponding brighteld microscopy of an Acanthamoeba castellanii after incubation in PYG medium for 24 h with Cryptococcus neoformans var. gattii strain I6 (serotype B). Panel A shows two fungal cells within an amoeba. Original magnication used the )/100 objective.

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amoeba was sufficient to kill A. castellanii . Fungal toxicity to amoeba cells appears to require direct contact since no increase in amoeba death was apparent when fungal cells were separated from amoebae by cell culture inserts. Consistent with this notion, fungal conditioned medium did not mediate toxicity to amoeba cells. In summary, our results show that C. neoformans var. gattii proliferates in the presence of A. castellanii in an interaction where ingestion of the fungal cell by amoebae is a relatively rare phenomenon. In contrast, the interaction of C. neoformans var. neoformans and C. neoformans var. grubii with amoebae results in phagocytosis, intracellular growth, and death of the host cell. Our findings suggest biological differences in the interaction of the C. neoformans varieties with a potential environmental predator, which imply that observations made with strains of varieties neoformans and grubii cannot necessarily be extrapolated to strains of variety gattii .

Acknowledgments
A.C. is supported by National Institutes of Health Awards AI33774, AI13342, and HL59842. The authors thank J. D. Nosanchuk for his technical and editorial assistance.

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